The crystal structure of the MICA–NKG2D complex shows that NKG2D binds to one MICA molecule as a homodimer. One of the NKG2D molecules binds mostly to the α1 domain of MICA, while the other binds mostly to the α2 domain (6). The contact between these two molecules creates a small pocket (roughly 6 Å wide × 6 Å thick × 14 Å long) (42).

The NKG2D homodimer overlays MICA diagonally in way that resembles αβTCR overlaying MHC I molecules. The central section of the MICA α2 domain is disordered when MICA is crystallized in isolated form, but it becomes ordered when MICA is bound to NKG2D and forms part of the interface between the two molecules (6).

MICA glycosylation was not essential, but it enhanced complex formation with NKG2D. Likewise, the glycosylation state of NKG2D had no substantial effect on complex formation (36).

MICA–NKG2D is considered a versatile ligand–receptor pair. As a matter of fact, NKG2D can act as a primary receptor or costimulatory molecule during infections, autoimmunity, or antitumor immune responses (6). For example, it has been shown that endothelial MICA triggeres an activating signal in allogeneic polyclonal NK cells through the immunoreceptor NKG2D, which may have account for a significant part in EC lysis by allogeneic NK cells. *In vitro* coculture assays show that interaction of endothelial MICA with NKG2D provides an immune suppressive pathway by downregulating NKG2D on the NK cell surface (14).

Boukouaci et al. (72) suggested that endocytosis of the NKG2D receptor, upon binding to sMICA, is considerably more rapid than the replenishment of cell surface NKG2D by *de novo* synthesis. The same authors also found that sMICA down regulates NKG2D receptor expression on CD8+ T cells. sMICA upregulates the IFNγ production only by cytokines-activated NK cells, while it has no effect on non-activated cells. The researchers demonstrated that sMICA upregulates IFNγ expression by IL-12/IL-18-activated CD3 CD56+ NK cells, demonstrating the pro-inflammatory effect of sMICA (72). A study with a mouse model found that Lewis rat hearts transplanted into BALB/c mice developed typical acute rejection (AR) in 6 days. The severity of xenograft rejection increased with time, from 2 to 6 days. Also increasing over time, the MICA protein and MICA mRNA reached their highest value after 6 h. The prevalence of anti-MICA was significantly higher among mice with severe AR. However, sMICA was significantly increased during AR at 2 h, then gradually decreased, and reaching its lowest value after 6 h (73).

# MICA–NKG2D AND KIDNEY TRANSPLANT

In the last few decades, the role of MICA and NKG2D in kidney transplants has emerged (**Table 4**). The involvement of NK cells was discovered in 1995 when some indirect evidence was reported during rejection of kidney transplants. Accumulation of CD56+ NK cells expressing granzyme in kidney biopsies of patients undergoing AR suggested a role of their cytolytic activity in kidney-allograft rejection (74). Over the years, the association between NK cells and the mechanisms of microcirculation injury during antibody-mediated rejection (AMR) in kidney transplants has become increasingly evident. The researchers proposed that donor-specific antibodies (DSA) were able to bind to the endothelium and to recruit NK cells that produce IFNγ and trigger antibody (Ab)-dependent cellular cytotoxicity (75).

# NK Cells and Kidney Damage in Mice and Cell Lines

Natural killer group 2 member D-ligand engagement delivers a strong dominant activating signal that overrides the inhibitory signal delivered by self-MHC class I, thus activating NKG2Dexpressing cells, resulting in innate and adaptive immunity activation (113).

Zhang et al. (116) reported a study on ischemia/reperfusion injury (IRI) on mice and discovered the capacity of NK cells to injure renal tubular epithelial cells *in vitro*. *In vivo* data supported the hypothesis that NK cells interact with tubular epithelia through NKG2D/Rae-1 interaction to mediate kidney damage following IRI.

Luo et al. (89) performed an *in vitro* study on human renal proximal tubular epithelial cell line (HK-2). They discovered that hypoxia-inducible factor-1-α (HIF-1α) plays a very important role in upregulating MICA expression and enhancing NK cell cytotoxicity toward target cells during hypoxia/reoxygenation in HK-2 cells. HIF is a heterodimer consisting of an α-subunit (HIF-1α) and a β-subunit (HIF-1β), the HIF-1β protein is constitutively

#### Table 4 | Relevant published work regarding NKG2D, MICA, and kidney transplants.

(*Continued*)

#### table 4 | Continued

(*Continued*)

#### table 4 | Continued

*"Yes" and "No" labels have been used if, in the studies analyzed, MICA has been valued as a possible biomarker ("Yes") or not ("No").*

present, while HIF-1α has a unique O2-dependent degradation domain, which leads to its degradation under normoxia conditions. The authors speculate that HIF-1α upregulates the surface expression of MICA on grafts during renal IRI, causing NK cells cytotoxicity against the organ (89).

#### Possible Causes of End-stage Renal Disease

A 2009 study of the possible causes of end-stage renal disease (ESRD) (70), while note directly related to kidney transplants, inevitably reported findings of consequences for kidney transplantation. Peraldi et al. evaluated seven patients with ESRD that were treated with peritoneal dialysis, and not with the hemodialysis procedure; NKG2D expression on NK cells was significantly decreased in these patients compared to healthy donors, indicating that reduction in NKG2D expression was independent of the dialysis procedure and linked with chronic renal failure. The authors also discovered that oxidative stress in presence of increased ROS production is one of the most significant consequences of chronic renal failure, alone or in concert with other mediators, and it seems to decrease the NKG2D levels on NK cells in ESRD and to favor the upregulation of MICA expression (70).

#### Anti-MICA Abs and Rejection

Some mechanisms have been proposed for MIC-mediated organ rejection. MICA antigens expressed in the allograft could induce the generation of anti-MICA Abs, which in turn might injure cells in the presence of complement.

This section contains no works that focus solely on NKG2D since most of the manuscripts are almost exclusively conserved with anti-MICA Abs: NKG2D is often just a side note; its presence and the link with MICA are given.

#### MICA-Sensitized Kidney Recipients and Higher Percentage of CD19**+**CD27**+**B Cells

CD19+CD27+ B cells are the subset of memory B cells that have the potential ability to secrete Abs. Li et al. (104) assessed the serum from 68 long-term survival kidney recipients and found 11 subjects who were MICA positive. They analyzed six MICAsensitized kidney transplant recipients and six healthy volunteers who did not receive a transplant (control group). Healthy controls had a higher percentage of CD19+CD27− in PBMCs than transplant patients, while the percentage of CD19+CD27+ in B cells was higher in transplant patients. The MICA-sensitized transplant patients had a significantly lower average percentage of CD19+ B cells in PBMC than healthy controls (3.58 ± 0.80 versus 8.53 ± 1.04%; *P* < 0.01). These results suggest that CD19+CD27+ B cells from sensitized patients have the potential ability to secrete Abs. In the same study, PBMC cells were isolated and cultured and stimulated with different molecules [toll-like receptor-9 ligand ODN-2006 CpG, PMA, B-cell activating factor (BAFF), CD40 ligand (CD40L), human recombinant IL-2 (rhuIL-2), rhuIL-10, rhuIL-4, rhuIL-21, CD40L, and BAFF] including MICA antigens. After stimulation, B cells from healthy controls and transplant patients had a lower percentage of apoptosis than non-stimulated cells. The average percentage of apoptosis cells from transplant patients was significantly higher than from healthy controls, and the IgM production (the first Ab produced by B cells after antigen stimulation) was higher in stimulated B cells from transplant patients than from healthy controls. The authors speculate that the B-cell population may be compromised by the transplant because patients are under immunosuppressive regimens, which may alter the apoptosis of B stimulated cells compared with healthy controls. The same study also performed an *in vitro* study with drugs and found that bortezomib and mycophenolic acid could inhibit B-cell Ab secretion (104).

#### MICA Abs

Hankey et al. (77) first reported that MICA and MICB expression on epithelial cells in transplanted kidneys and pancreases with histological evidence of rejection and cellular injury played a role in allograft rejection. The study showed that in a healthy kidney there was no immunochemical evidence of MIC expression. In contrast, the majority of biopsies with histologic proof of rejection or acute tubular necrosis (ATN) showed MICA positive staining of the tubular epithelium in the proximal and distal tubules. For this reason, it was concluded that alloantibodies against MICA might play a role in allograft rejection.

Zwirner et al. (76) found that several patients who had undergone a kidney transplant had specific Abs against MICA, and most of them were detected in serum samples collected at different times after organ rejection. However, these Abs were not directed against the alleles expressed by the patients, and it was speculated that if the presence of MICA Abs was probably caused by multiple blood transfusions received by the patients while awaiting a transplant, or resulting from a pregnancy or a previous transplant (76).

Lemy et al. (87) analyzed the MICA Abs from 494 controls and 597 patients with chronic kidney disease. They found a three times higher prevalence of MICA Abs in patients with chronic kidney disease when compared with controls (14.9 versus 4.7%). Nevertheless, they speculated that even if the increase in MICA Abs prevalence among patients affected by chronic kidney disease was probably related to previous renal transplantation and transfusions. Logistic regression analysis and analysis of chronic kidney disease patients who have not been subjected to transfusions and renal transplantations suggest that the increase of urea (and other nitrogenous waste) in the blood is connected to an increase of MICA immunization. The authors also reported that MICA Abs were more frequent in men than in women, despite pregnancy being an independent risk factor for the development of MICA Abs (87). This finding is in sharp contrast with other published work. The fact that nearly one-third of MICA chronic kidney disease stage V patients have never experienced any identifiable immunizing event indicates that there must be other causes for MICA sensitization. At the same time, one-fifth of the same patients showed the presence of autoreactive MICA IgG Abs, distinctly rare with respect to HLA Abs. The authors showed that patients with MICA Abs had a somewhat better overall graft

survival than MICA Abs− patients. Finally, Lemy et al. found in MICA Abs+ and MICA Abs− patients a similar incidence of AR episodes during the first year (10.2 versus 12.8%), as well as similar levels of proteinuria and creatinine (87).

Another study of MICA Abs screened 147 recipients with end-stage renal disease; 82 of these patients were Abs+ (55.8%). Forty patients had both anti-HLA and anti-MICA, 33 had only anti-HLA, and 9 only anti-MICA Abs in the posttransplant period. The authors found that patients who developed HLA alone, or both HLA and MICA Abs, rejected their grafts more frequently than Abs− recipients. The rates of HLA class I, class II, or both Abs+ were greater in the rejection patients than the nonrejection patients (*P* = 0.011, 0.037, and 0.0275, respectively). So the authors speculated that HLA class II and MICA Abs+ were the only important predictors of graft failure when both of them were present with HLA class I Abs+ (96).

In a retrospective study, Solgi et al. (98) analyzed sera samples of 40 living unrelated donor kidney recipients, looking at anti-HLA and anti-MICA Abs and the levels of soluble CD30 (sCD30) and sMICA. They found that patients with pre- and posttransplant HLA Abs had a higher incidence of AR episodes (*P* = 0.01 and *P* = 0.02), more graft loss (*P* = 0.001), and lower graft survival during a mean follow-up of 3 years. This group of patients also had higher levels of sCD30 and serum creatinine and decreased contents of sMICA early after transplantation, as compared to the patients without HLA Abs. Anti-MICA Abs were observed in 8/40 (20%) and 5/40 (12.5%) of all patients pre- and posttransplant, respectively. HLA and MICA Abs were both found in two out of four cases with graft loss. In a comparison of transplant rejecting to functioning graft groups, sCD30 levels increased at day 14 (*P* = 0.001), while sMICA levels were insignificantly lower in the first group (98).

Chaudhuri et al. (100) studied the evolution of humoral immunity in low-risk pediatric patients during the first 2 years after renal transplantation. They correlated the presence of serum anti-HLA DSA and serum MICA Abs with clinical outcomes and histology (the biopsies were performed at 0, 6, 12, and 24 months). They found anti-HLA Abs in 22% of patients, 6% of which were donor-specific, while 6% developed anti-MICA Abs. Three percent of patients developed *de novo* Abs to both HLA and MICA. The presence of *de novo* Abs was associated with significantly higher risks for AR (*P* = 0.02), chronic graft injury (*P*= 0.02), and decline in graft function (*P*= 0.02). Graft function was monitored by the difference between creatinine clearances. Anti-MICA and -HLA Abs were found in 25% of unsensitized pediatric patients. This was correlated with a greater risk of acute and chronic rejection (100).

Zhang et al. (107) associated the presence of *de novo* MICA Abs and proteinuria with graft failure, after renal transplantation. They investigated 275 patients without preexisting anti-HLA and -MICA Abs. Five years after renal transplantation, 25.8% showed *de novo* anti-HLA Abs, 12% showed *de novo* anti-MICA Abs, and 26.5% proteinuria. *De novo* anti-HLA Abs were associated with increased proteinuria after transplantation (relative risk, 3.12). Anti-HLA Abs and proteinuria were both associated with poor 5-year graft survival (*P* = 0.027 and *P* = 0.006, respectively). Patients with *de novo* anti-MICA Abs were also apt to have proteinuria. The authors concluded that *de novo* anti-HLA and -MICA Abs and proteinuria are all associated with poor graft survival (107).

#### Pretransplant Panel-Reactive Abs and Preexistent Circulating Abs

Opelz (78) studied the influence of pretransplant panel-reactive antibody (PRA) status on the long-term outcome of kidney grafts from HLA-A, -B, and -DR, identical sibling donors. In over 10 years of follow-up, he discovered that non-HLA-directed immunity and Abs against HLA had a similar influence for the long-term results for kidney recipients with PRA. Opelz suggested that the targets for Abs causing late rejections could be the so-called minor histocompatibility antigens (78).

Sanchez-Zapardiel et al. (105) studied 727 transplanted patients and showed that the effect of anti-MICA Abs occurs independently of the presence of anti-HLA Abs. Pacients were categorized into four groups according to the presence (+) or absence (−) of anti-HLA and anti-MICA Abs: HLA+MICA+ (*n* = 27); HLA−MICA− (*n* = 510); HLA+MICA− (*n* = 165), and HLA−MICA+ (*n* = 25). A notable difference was observed 3 months after transplantation, when HLA−MICA+ patients had a graft rejection rate of 8% compared with 2% in HLA− MICA− patients. The patients were also grouped according to the presence of preexisting anti-HLA Abs, as measured by % PRA (PRA+ or PRA−): PRA+MICA+ (*n* = 7), PRA−MICA− (*n* = 610), PRA+MICA− (*n* = 65), and PRA−MICA+ (*n* = 45). The incidence of rejection was found to be superior in PRA+MICA− cohort versus PRA−MICA− patients (24 months after transplantation), but allograft rejection rate was the highest when comparing PRA+MICA+ patients with PRA−MICA− patients 3 months after transplantation, a finding which was repeated at 6 months (105). This work is of interest because it performed a comparative study on the effects of anti-MICA and anti-HLA Abs on kidney transplants.

The Rodriguez Ferrero et al.'s (97) study included 22 recipients of kidney transplantations from deceased donors, and no differences between patients that showed preexistent circulating antibodies (CA) and those that did not were reported. In regards to the incidence of AR episodes, the only factor associated with CA was re-transplantation. So the authors concluded that CA monitoring is important for highly sensitized renal transplants, but they did not observe a difference in graft survival or renal function in the first 3-month follow-up (97).

#### Cd4 Deposition and C1q-Fixing Abs

A study of patients with acute antibody-mediated rejection (AAMR), who had MICA\*008 Ab, showed that the presence of anti-MICA Abs and the deposition of C4d in biopsies performed at the time of AAMR was associated with the detection of DSA or Abs against HLA (80). The observation that the control group of 30 patients with long-term functioning grafts did not have anti-MICA\*008 Abs provided indirect evidence of the importance of anti-MICA Abs in chronic rejection. Furthermore, all patients receiving an allograft fully matched at MICA had functioning grafts (80). It is also important to mention that MICA Abs are able to activate complement in *in vitro* experiments (80).

Alvarez-Marquez et al. (85) selected 58 patients that underwent a kidney biopsy because of primary non-function, delayed graft function or acute dysfunction of a previously functional graft, suspected by oliguria, increase of serum creatinine levels, or proteinuria. At the time of the transplant, all patients showed negative complement-dependent cytotoxicity crossmatches. Researchers demonstrated that 80% of a group of 19 patients with clinically evident graft dysfunction and with C4d deposition in kidney biopsies had Abs directed against donor-specific HLA class I, class II, MICA, or GSTT1 (glutathione-*S*-transferase T1) antigens (85).

In the Li et al.'s (88) study, a human ProtoArray platform was used to study 37 serum samples from 15 renal transplant patients (pediatric and young adult) with (*n* = 10) and without (*n* = 5) AR, and seven normal controls. To test serum Abs, they used a ProtoArray containing 5,056 non-redundant human proteins expressed in a baculovirus system, purified from insect cells and printed in duplicate onto a nitrocellulose-coated glass slide. Moreover, all patients were primary transplant recipients, and the biopsies were graded by the Banff classification. The authors found that the mean immune response signal in posttransplant patient serum showed an increase in anti-MICA Abs when compared with healthy normal controls (*n* = 7), but anti-MICA Abs signal intensity was unrelated to the sampling time interval posttransplantation. Mean MICA Abs signal intensity was higher in transplant patients with C4d+AR (121.4) versus C4d−AR (4.3), so a correlation between high MICA Abs levels and C4d+ graft rejection *r* = 0.54 (*P* = 0.039) was observed. On ProtoArray, each gene on the cDNA platform was compared between a specific kidney compartment versus all other compartments, by a two-unpaired class comparison and a multi-class comparison. The signal intensity of anti-MICA Abs ranked in the top 15 for glomerulus, so the MICA antigen was found to have a 2.7-fold higher expression in the glomerulus when compared to the other 6 normal kidney compartments. Cytoplasmic granular staining for MICA in normal and stable transplanted kidneys was observed solely in podocytes within glomeruli. In AR, in addition to the persisting glomerular staining, the infiltrating mononuclear lymphocytes also showed strong positive staining for MICA. So the authors demonstrated that Ab responses in patients are modulated by MICA after transplantation in patients, irrespective of graft rejection (88).

Another study correlates Cd4 deposition and creatinine levels. Ding et al. (101) evaluated serum anti-MICA Abs before and after kidney transplant, and they also examined PRA, serum creatinine, urine, graft ultrasound, lymphocyte subsets, and the pathology of graft biopsy. The study was split into two parts. In the first part, patients with AR were grouped into MICA+, MICA− (*P* < 0.05) and control groups. There were a significantly higher number of anti-MICA Abs positive patients with acute graft rejection compared with stable renal functions patients (control group).

Two to three days after the occurrence of AR, the anti-MICA Abs level increased gradually. Anti-rejection treatment had no effect on anti-MICA Abs but lowered serum creatinine to a normal level. In the second part, the authors analyzed chronic graft rejection patients. The number of anti-MICA Ab positive patients was significantly higher than those with stable renal function (*P* < 0.05), and the serum creatinine levels were significantly higher in MICA+ than in MICA− cases (*P* < 0.05). The authors also found that graft biopsy of all MICA+ cases showed C4d deposition (101).

Jin et al. (103) studied 53 cases of AR that showed C4d deposition in the peritubular capillaries, 50 cases of ARs without C4d deposition, 30 with peritubular capillaries alone, 28 with ATN, and 78 patients with surveillance biopsies (control group). The authors observed that the prevalence of PRA and anti-MICA Abs was increased among the peritubular capillaries alone group (30.0 and 43.3%, respectively), albeit not significantly different from the group with C4d+ AR (49.1 and 39.6%, respectively). They also observed that the immunophenotype of infiltrating T lymphocytes and serum Abs (85.9% of control biopsies presented) had a regulatory phenotype while in the peritubular capillaries cohort, 93.3% of biopsies showed the cytotoxic phenotype. These results showed that peritubular capillaries in biopsy specimens from patients with early renal-allograft dysfunction could be an indicator of AR, especially acute humoral rejection (103).

Cai et al. (111) collected samples from 975 kidney transplant recipients, and they tested for C1q-fixing Abs against denatured HLA class I, class II, and MICA antigens. Among 169 patients who lost renal grafts, 44% had c1q-fixing Abs against denatured HLA/MICA antigens, which was significantly higher in patients with functioning renal transplants (25%). They concluded that C1q-fixing Abs were significantly associated with graft failure caused by AMR (72.73%) and they affirmed that only c1q-fixing Abs were associated with graft failure and AMR (111).

#### MICA Allele Epitopes and Eplets

Regarding the anti-MICA Abs, Duquesnoy et al. (118) developed an eplet-based version of the HLA-Matchmaker algorithm as a tool to assess the epitope specificity of these Abs. A repertoire of 38 potentially immunogenic *MICA* eplets was selected (based on MICA structure molecular viewing and the amino acid sequence differences between *MICA* alleles). These eplets are based on a functional epitope structure (a configuration of amino acids within a 3 Å radius of an Ab accessible polymorphic residue on the molecular surface). In this study, the eplet frequencies were calculated from *MICA* allele frequencies in 1,245 European-Americans and 605 African-Americans. Many eplets are shared by very similar groups of *MICA* alleles. For instance, the combination of eplets called CMGWS "supereplet" is composed by 36C, 129M, 206GW, and 215S epitopes and shared by the same group of *MICA* alleles (*A\*001*, *A\*002*, *A\*007*, *A\*011*, *A\*012*, *A\*015*, *A\*017*, *A\*018*, *A\*021*, *A\*030*, *A\*041*, *A\*043*, *A\*045*, *A\*046*, *A\*047*, *A\*014*, *A\*020*, *A\*023*, *A\*026*, *A\*029*, *A\*036*, *A\*040*, *A\*050*, *A\*052*, and *A\*055*). The random chance that these eplets are a mismatch is 20.1% in African-Americans and 24.0% in European-Americans. Alternatively, the combination of eplets named AYVE "supereplet" is composed by 25AY, 129V, and 173E and was shared by another group of *MICA* alleles (*A\*004*, *A\*006*, *A\*008*, *A\*009*, *A\*010*, *A\*016*, *A\*019*, *A\*024*, and *A\*044*). The random chance of their being a mismatch is 28.2% in African-Americans and 20.1% in European-Americans (118).

Panigrahi et al. (82, 119) analyzed the presence of Abs against MICA\*001, MICA\*002, MICA\*004, MICA\*008, and MICA\*009 in serum samples of 185 patients transplanted with live related donor kidneys. Sixteen percent of all recipients developed anti-MICA Abs during the posttransplant period, 83% of the patients whose grafts eventually failed had both anti-HLA and anti-MICA Abs as compared to 29% patients who had only anti-MICA Abs, and 11% of those without any of the Abs (HLA or MICA) (82, 119).

Analysis of anti-MICA\*001, MICA\*002, MICA\*004, MICA\*008, and MICA\*009 Abs in serum samples from 1,910 kidney recipients showed that a correlation between the presence of anti-MICA Abs and the reduced in kidney-allograft survival was not influenced by the simultaneous presence of Abs against HLA (120). In this study, decreased renal-allograft survival is associated with anti-MICA Abs formed before transplantation. It was also found that patients with Abs against MICA before transplantation did not received more transfusions than patients without such Abs, in contrast with the Zwirner et al.'s study (76). So the authors speculate that cross-reactivity with substances from the environment may play a role in priming the immune system, facilitating anti-MICA Ab production (120).

Suarez-Alvarez et al. (84) screened 284 kidney transplant sera for anti-MICA Abs and mapped the epitopes of MICA by screening a library of synthetic overlapping peptides from the extracellular domains of the protein against the sera from kidney transplant patients with anti-MICA Abs. Anti-MICA Abs were detected in 50 of 284 patients (17.6%), and they correlated with the development of AR. The authors found that nine regions were reactive with anti-MICA Abs. Five epitopes were located in constant regions (II, III, IV, VI, and IX) and were present in all *MICA* alleles, while the other four regions (I, V, VII, and VIII) mapped to variable sites of polymorphic amino acids among the different alleles products of MICA. In particular, regions V, VII, and VIII were the regions with the highest amino acid variability. Three polymorphic residues, 173 (E/K), 175 (S/G), and 181 (R/T), had determined allele-specific epitopes. The aminoacid 208Y and 213T, instead, contributed in the cross-reactivity among alleles (84).

Cox et al. (90) identified MICA IgG Abs directed against MICA\*001, \*002, \*004, \*007, \*008, \*009, \*012, \*017, \*018, \*019, and \*027. Analysis of 116 healthy control subjects revealed only one subject with anti-MICA Abs (0.9%) and five subjects (4%) with anti-HLA class II Abs, while in a subgroup of 227 transplant recipients and their donors the coproduction of Abs to HLA and MICA significantly associated with acute cellular rejection (ACR). Analysis of patients with AAMR established strong associations with the presence of Abs against HLA class I and II, but not anti-MICA. By aligning *MICA* allele profiles present in the subgroup of 227 renal graft recipients and their respective donors, it was possible to establish the precise position of amino acid mismatches that correlate strongly with MICA Ab production. Mismatching at residues 36, 129, 173, 175, 213, and 251 showed the strongest association with anti-MICA Ab production in transplant recipients, while 91, 125, 156, and 221 residues were also mismatched between recipients and donors, but were not significantly associated with anti-MICA Ab production. There are two immunodominant motifs: MICA-G1 is characterized by residues 36 cysteine (C), 129 methionine (M), 173 lysine (K), 206 glycine (G), 210 tryptophan (W), and 215 serine (S). Alternatively MICA-G2 epitopes share residues 36 tyrosine (Y), 129 valine (V), 173 glutamic acid (E), 206 serine (S), 210 arginine (R), and 215 threonine (T). The majority of these recipients (10 out of 17 individuals, 59%) developed *de novo* donor-specific anri-MICA Abs posttransplantation, and there was a significant association of graft dysfunction with the presence of anti-MICA DSA alone after 2 years. In conclusion, it was discovered that mismatching *MICA* alleles lead to the development of anti-MICA Abs in some renal graft recipients, and the presence of anti-MICA DSA was independently associated with decreased glomerular filtration rate (eGFR) and poorer graft outcome (90).

Tonnerre et al. (106) went beyond the usual studies of anti-MICA Abs and focused on searching for a specific allele that could lead to a poorer outcome. The authors performed a study that showed that the MICA\*008 (A5.1) molecule is a major antigenic determinant and target for recipient sensitization of kidney transplant patients. *MICA A5.1* is associated with four alleles: *\*023*, *\*028*, *\*053*, and *\*008*. The authors divided primary EC cultures from transplant donors in *MICA A5.1* homozygous, heterozygous, and control. The MICA surface expression was significantly higher on ECs from *A5.1/A5.1* donors than from controls. The *MICA A5.1* allele also leads to a reduction of sMICA and an increase in the MICA level in exosomes in ECs. Anti-MICA (A5.1) Abs intensities in the sera of recipients with anti-MICA Abs were not higher than intensities observed for other anti-MICA (control) Abs. However, when tested on EC cultures expressing physiologic levels of membrane-bound MICA, the sera only bound to ECs from *MICA A5.1* donors. This seemed to show that anti-MICA Abs bind ECs' targets in an allele-specific manner.

In fact, the combination of the donor carrying *MICA A5.1* and the recipient having a non-*MICA A5.1* allele was overrepresented in the group of MICA-sensitized patients compared with the group of non-immunized recipients (106).

Sapak et al. (108) concluded that anti-MICA Abs could not be responsible for the rejection if they were not directly detected in the transplanted graft. In the sera of 124 renal recipients, the authors found only 22 patients positive for anti-MICA Abs. The most frequent anti-MICA Abs were directed against *MICA\*018* and *MICA\*001*. *MICA\*008* had the highest gene frequency (31%), followed by *MICA\*002* (14%). Comparing *MICA* allele profiles of donors and anti-MICA Ab epitopes of their respective recipients, Sapak et al. found a match in only in 9 donor–recipient pairs (41%) while the sera of the other 13 patients was negative for Abs against graft MICA molecules, but positive for Abs against other MICA antigens. The majority (59%) of anti-MICA Abs in patients were not donor-specific, so the authors suggested that anti-MICA Ab induction was not caused by renal graft allogeneic stimulation but was also probably stimulated by other still unknown immune mechanisms (108).

Sanchez-Zapardiel et al. (112) studied 727 kidney recipients. They found that PRA+MICA+ recipients exhibited a longer time to reach optimal serum creatinine level after transplantation (*P* = 0.005) had the lowest eGFR at 3 months and PRA+MICA+ status independently increased the risk for chronic kidney disease stage 5 at month 3. Pretransplant anti-MICA Abs were poly-specific; anti-AYVE supereplet reactivity was higher in HLA+MICA+ versus HLA−MICA+ patients and superior than anti-CMGWS supereplet within HLA+MICA+ patients. The authors also found that some preformed anti-MICA Abs might bind complement, using the C1q Luminex assay. Sanchez-Zapardiel et al. analyzed 13 anti-MICA+ pretransplant sera that were positive for the C1q binding assay and one of them (serum 3) exclusively recognized the AYVE supereplet with a strong reactivity against MICA\*027 antigen. The authors concluded that these preformed anti-MICA Abs are able to mediate cell death by fixing and activating the complement cascade. So they speculated that the anti-MICA Abs might contribute to worse early kidney graft function (112).

#### Correlation between Anti-MICA Abs and Creatinine Levels or Estimated Glomerular Filtration Rate (eGRF) or Death-Censored Graft Survival (DCGSs)

Yao et al. (92) included 29 sensitized recipient patients who had undergone living-related donor renal transplantation between 2007 and 2009. They found a statistical difference in postoperative serum creatinine levels within 1 week between anti-MICA Ab-positive (135.4 ± 21.4 mol/L) and anti-MICA Ab-negative groups (108.6 ± 31.6 mol/L), but no significant difference between the two groups at discharge. To decrease the preexisting Abs (mainly IgG, IgM, and IgE), all recipients were treated with protein A immunoadsorptions, and this therapy was effective in decreasing anti-MICA Abs (92).

Zhang et al. (93) studied patients receiving primary kidney transplants (all from deceased donors) between 2004 and 2007. No significant association was found between the presence of anti-MICA and -HLA Abs, nor between the presence of anti-MICA Abs and 1-year graft survival rate. However, during the follow-up period, eGFR decreased 24.0 ± 3.4% in the anti-MICA Abs positive group, while it decreased only 8.4 ± 3.0% in anti-MICA Abs negative patients. A strong correlation between the production of anti-MICA Abs and renal impairment was also found. For these reasons, the authors concluded that patients with anti-MICA Abs had a more rapid deterioration of graft function, compared to those without anti-MICA Abs (93).

In another study that did not recognize MICA as a biomarker, sera from 779 kidney transplant recipients was tested with two single-antigen flow bead assays 1 year after transplantation. Thirteen of the 779 patients were lost to follow-up, 50 had lost their graft, and 33 died with a functioning graft. The prevalence of anti-MICA Abs was 5.3% at 1-year posttransplantation, and that MICA+ patients were more frequently HLA sensitized and regrafted. However, 4-year DCGSs were not different between MICA+ and MICA− patients (97 versus 94%, *P*= 0.28), and 4- and 8-year survival rates were similar in MICA+ and MICA− patients. Thus, the hypothesis of an independent pathogenic role for MICA in long-term renal graft injury was not supported, and the authors questioned the utility of monitoring anti-MICA Abs posttransplant with single-antigen flow bead assays (94).

#### MICA Abs in Case Study

Narayan et al.'s (91) case study focused on a 14-year-old girl with branchiooto renal syndrome who underwent re-transplantation with an HLA crossmatch-negative deceased donor kidney. She lost her first kidney transplant to chronic rejection at the age of 10 and underwent allograft nephrectomy. She was highly sensitized, and to improve her chances for transplantation, she underwent desensitization with high-dose IVIG and rituximab. When she received a deceased donor renal transplant, the pretransplant anti-HLA Ab testing showed no anti-donor HLA Abs. The patient maintained good allograft function until postoperative day 10 when she presented with fever and anuric renal failure. The only Ab found was donor-specific anti-MICA Ab, specifically directed against MICA\*012 protein. Evaluation of the pretransplant serum revealed preformed anti-MICA\*012 Abs with levels that were elevated both before transplant and at the time of rejection. Anti-MICA Abs levels declined with the initiation of plasmapheresis and IVIG and correlated well with normalization of renal function and resolution of ACR and AMR. The authors speculated that the sensitization to the MICA\*012 protein was caused by prior sensitization from the first renal transplant or previous infections or transfusions. The conclusion of their research is that donor-specific anti-MICA Abs can be associated with both AMR and Banff type IIA ACR and may require treatment with plasmapheresis (91).

Ming et al. (109) studied a patient who suffered early aggressive AMR in the presence of DSA against MICA after her first renal transplant. The researchers found that anti-MICA–DSA in recipient serum could bind MICA-G1 antigens expressed in the cultured human umbilical cord vein endothelial cells (HUVECs). The recipient serum was cytotoxic to these HUVECs, but not against HUVECs that did not express MICA-G1 antigens in the presence of complement. The researchers discovered that the patient had been sensitized to MICA antigens and HLA, before transplantation, and the HLA alloantibodies were not specific to the first kidney donor, but the MICA alloantibodies were. In light of this discovery, the second renal transplant was with a negative MICA virtual crossmatch, and it was successful (109).

#### microRNA and mRNA's Analysis

Seiler et al. (62) showed that an elevated NKG2D mRNA expression in biopsy material was correlated with the severity of AR and detected NKG2D+ cells located in clusters around tubules in biopsies derived from patients diagnosed with acute and chronic rejection. The expression of *NKG2D* mRNA was also detected in urinary sediments obtained 2–3 days before the AR episode. However, significant levels of MICA mRNA were not detected in the patient groups analyzed (62). For the first time, the focus was on the importance of the role of the NKG2D molecule, which is responsible for MICA signal transduction.

Another controversial paper regarding the role of MICA is the Racca et al.'s study (86), in which the authors obtained peripheral blood samples from 29 renal-transplanted patients (19 men). They classified patients it into three groups: AR group (9 patients with acute grade I/II allograft rejection), chronic rejection group (10 patients with chronic allograft rejection), and stable evolution group (10 patients with clinically stable allograft evolution). The authors observed that MICA mRNA levels in peripheral blood mononuclear cells showed similar expression levels in all groups evaluated and in the control group. They also found similar levels of MICA expression in a comparison of biopsy specimens from AR and nephrotoxic ATN patients. They did not find a correlation between MICA expression and renal graft state (86). It is interesting to note that the MICA expression in biopsies did not have a healthy control group, while expression of MICA mRNA may be a posttranscriptional control that modules MICA expression on the cell surface. The Racca et al.'s (86) study still represents an interesting opportunity to discuss the role of MICA as a biomarker.

Xu et al. (110) studied miR-338-5p, a microRNA downregulated in AMR renal allografts, and negatively correlated with BAFF. This molecule plays an important role in the differentiation, development, and proliferation of B lymphocytes. BAFF could be released in a soluble form (sBAFF) after cleavage and would bind to BAFF receptor. The receptor-associated factor 3 is a sort of adaptor for the BAFF–BAFF-R connection, it is implicated in a signal transduction, and it appeared to be a candidate target for miR-338-5p. In the study, 49 follow-up renal-transplanted recipients and a healthy control group were examined, and it was found that anti-HLA II Ab, anti-MICA Ab, and anti-HLA + MICA mixed Abs were all statistically increased in recipients. Serum miR-338-5p was significantly downregulated in renal-transplanted recipients compared with healthy volunteers and was inversely correlated with sBAFF. The authors speculate that miR-338-5p may regulate the BAFF signal, and they suggested that sBAFF was significantly negatively correlated with anti-MICA Abs (110).

#### Cytomegalovirus (CMV) and Polyomavirus and Transplantation

Cytomegalovirus infection is the most common viral complication after renal transplantation and solid organ transplantation in general. One hundred ninety-six recipients who underwent kidney transplantation during the past 6 years were assessed with at follow-up of at least 12 months. In this study, it was shown that the activating receptor NKG2D was expressed in a significantly higher number of NK cells at day 0 and day 20 compared to day 180 (*P* = 0.01 and *P* = 0.003, respectively) and compared to the control group (*P* = 0.0003 and *P* = 0.0004, respectively) (121). This finding suggests a possible mechanism for the activation of NKG2D that goes beyond organ rejection, but it is closely related. In fact, in the Hadaya et al. (121) study, it was shown that an expansion of the NKG2D+ NK cell population occurred during acute CMV infection which decreased over time to a level very similar to that of the control group.

An interesting study that involved NKG2D, performed by Shabir et al. (117), demonstrated that CD4+CD28null T cell expansion is driven by latent CMV infection inflammation. The immune surveillance of CMV may have an unwanted consequence in the development of endothelial injury, which was proven to be mediated by CD4+CD27−CD28null cells in *in vitro* experiments. NKG2D was upregulated on CD4+CD27−CD28null cells isolated from patients in this study and might have an important component of the cytotoxic effects of these cells. In fact, CD4+CD28null cells were found predominantly in CMV-seropositive patients, and expanded in the posttransplantation period, and expressed markers of cytotoxicity (NKG2D and perforin) and endothelial homing (CX3CR1). Isolated CD4+CD27−CD28null cells previously exposed only to CMV-derived antigens showed signs of endothelial damage and apoptosis, and this effect was mitigated by NKG2D-blocking Ab. They concluded that the increase in CD4+CD28null cell frequencies was associated with delayed graft function and lower eGRF at end follow-up, and this could be mediated by NKG2D (117).

Another study by Tonnerre et al. (122) investigated the implication of MICA in BK polyomavirus (BKPyV) reactivation in a cohort of 144 transplant donor/recipient pairs including recipients with no reactivation (control). BKPyV is frequently reactivated in kidney transplant recipients receiving an immunosuppressive regimen and is associated with nephropathy (BKPyVAN) and graft rejection. They investigated the impact of the *MICA A5.1* mutation on recipient BKPyV reactivation, and they found that recipients carrying a *non*-*MICA A5.1 (nA5.1)* genotype transplanted with a kidney from a donor carrying the *A5.1 MICA* variant had a lower risk of BKPyV reactivation (*P* = 0.0148). So they speculated that *MICA A5.1* could be a protective allele toward BKPyV infection (122). Interestingly, these researchers also found that the donor (A5.1)–recipient (nA5.1) combination was overrepresented in the group of MICA-sensitized patients, but in the latter, MICA A5.1 seemed to be a protective factor for a virus related to graft rejection (106).

#### CONCLUSION

Since the *MICA* gene was first described, it has been the subject of many studies aiming to comprehend its immunobiology and the role it plays in fine-tuning the innate and adaptive immune response. MICA appears to be involved in transplant rejection, immune response against viruses and intracellular bacteria, inflammation, homeostasis of epithelia, immune response against tumors, and tumor immune escape mechanisms. However, there remain a number of open issues to be addressed surrounding MICA's functions and roles. Developing and implementing typing strategies for *MICA* alleles may increase the chance for positive outcomes in solid organ transplantation by allowing better matching. MICA's biological function is achieved through its interaction with the NKG2D receptor. This activating receptor and its ligands are deeply involved in the outcomes of transplanted grafts, in fact, the overexpression of NKG2DLs

#### REFERENCES

could be involved in rejection episodes and can contribute to graft loss (44).

Various studies have shown that anti-MICA Abs, binding to MICA molecules expressed at the endothelial allograft cell surface, may have relevance to kidney transplantation outcome (81, 106). However, it is important to note that some studies, such as that of Lemy et al. (87), where the presence of anti-MICA Abs do not show adverse effects in renal graft outcomes (87). Also, *MICA* mRNA level analysis in blood mononuclear cells did not show a correlation between *MICA* expression and renal graft state (86). Seiler et al. (62) did not find an enhancement of mRNA expression levels of MICA in kidney biopsies from patients undergoing AR or chronic allograft nephropathy, but they observed increased *NKG2D* expression. In an interesting study performed by Sapak et al. (108), 41% of the detected anti-MICA Abs were donor-specific, but an astonishing 36% were anti-MICA Abs against self-MICA antigens and several patients (27%) produced both (108).

Regarding NKG2D, there are studies that report that it is possible to prolong graft survival and to prevent CD28-independent rejection of cardiac allografts after blocking NKG2D (123).

We can conclude that the role of MICA and NKG2D in transplant outcome is not yet clear; MICA-mediated rejection probably is not just a reaction to the MICA non-self protein. The stress condition following a transplant causes a general inflammatory status in the recipient. This could increase MICA production, thus activating the response *via* the NKG2D receptor. The clinical impact of these interactions will remain unclear until further studies are performed.

#### AUTHOR CONTRIBUTIONS

MR: planning and organizing structure of the review; research and analysis of the papers; wrote the review; and planning and creation of figures and tables. MB: planning and organizing structure of the review and contributions to the sections writing/ critical review of the manuscript.

#### FUNDING

This work was funded by LIGH-FUNPAR ALLIANCE.

in glioma patients. *Neuro Oncol* (2010) 12(1):7–13. doi:10.1093/neuonc/ nop009

transplant patients. *Transplantation* (2009) 88(3 Suppl):S68–77. doi:10.1097/ TP.0b013e3181afeb7a

transplantation. *J Am Soc Nephrol* (2013) 24(4):655–64. doi:10.1681/ASN.<PHONE_NUMBER>

endothelial injury and kidney allograft dysfunction. *Am J Transplant* (2016) 16(4):1113–28. doi:10.1111/ajt.13614

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Risti and Bicalho. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Elusive Role of the CD94/NKG2C NK Cell Receptor in the Response to Cytomegalovirus: Novel Experimental Observations in a Reporter Cell System

#### *Aldi Pupuleku1 , Marcel Costa-García1 , Domènec Farré2 , Hartmut Hengel 3,4, Ana Angulo2,5, Aura Muntasell 6† and Miguel López-Botet 1,6\*†*

#### *Edited by:*

*Ulrike Koehl, Hannover Medical School, Germany*

#### *Reviewed by:*

*Christine Susanne Falk, Hannover Medical School, Germany Federico Simonetta, Stanford University, United States*

#### *\*Correspondence:*

*Miguel López-Botet<EMAIL_ADDRESS><EMAIL_ADDRESS>
*† These authors share credit for senior authorship.*

#### *Specialty section:*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology*

*Received: 18 July 2017 Accepted: 29 September 2017 Published: 24 October 2017*

#### *Citation:*

*Pupuleku A, Costa-García M, Farré D, Hengel H, Angulo A, Muntasell A and López-Botet M (2017) Elusive Role of the CD94/ NKG2C NK Cell Receptor in the Response to Cytomegalovirus: Novel Experimental Observations in a Reporter Cell System. Front. Immunol. 8:1317. doi: 10.3389/fimmu.2017.01317*

*1Department of Experimental and Health Sciences, University Pompeu Fabra, Barcelona, Spain, 2 Immunology Unit, Department of Biomedical Sciences, Medical School, University of Barcelona, Barcelona, Spain, 3 Institute of Virology, Albert Ludwigs University of Freiburg, Freiburg, Germany, 4Faculty of Medicine, Albert Ludwigs University of Freiburg, Freiburg, Germany, 5 Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain, 6Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain*

Human cytomegalovirus (HCMV) infection promotes the differentiation and persistent expansion of a mature NK cell subset, which displays high surface levels of the activating CD94/NKG2C NK cell receptor, together with additional distinctive phenotypic and functional features. The mechanisms underlying the development of adaptive NK cells remain uncertain but some observations support the involvement of a cognate interaction of CD94/NKG2C with ligand(s) displayed by HCMV-infected cells. To approach this issue, the heterodimer and its adaptor (DAP12) were expressed in the human Jurkat leukemia T cell line; signaling was detected by transfection of a reporter plasmid encoding for Luciferase (Luc) under NFAT/AP1-dependent control. Engagement of the receptor by solid-phase bound CD94- or NKG2C-specific monoclonal antibodies (mAbs) triggered Luc expression. Moreover, reporter activation was detectable upon interaction with HLA-E+ 721.221 (.221-AEH) cells, as well as with 721.221 cells incubated with synthetic peptides, which stabilized surface expression of endogenous HLA-E; the response was specifically antagonized by soluble NKG2C- and HLA-E-specific mAbs. By contrast, activation of Jurkat-NKG2C+ was undetectable upon interaction with Human Fetal Foreskin Fibroblasts (HFFF) infected with HCMV laboratory strains (i.e., AD169, Towne), regardless of their differential ability to preserve surface HLA-E expression. On the other hand, infection with two clinical isolates or with the endotheliotropic TB40/E strain triggered Jurkat-NKG2C+ activation; yet, this response was not inhibited by blocking mAbs and was independent of CD94/NKG2C expression. The results are discussed in the framework of previous observations supporting the hypothetical existence of specific ligand(s) for CD94/ NKG2C in HCMV-infected cells.

Keywords: human, natural killer cell, cytomegalovirus, CD94, NKG2C, HLA-E, UL40

# INTRODUCTION

Inhibitory receptors specific for MHC class I molecules with immunoreceptor tyrosine-based inhibitory motifs play a key role in preventing NK cell responses against normal autologous cells. This function is mainly fulfilled by members of the human killer-cell immunoglobulin-like receptor (KIR) family, which recognize sets of classical HLA class I (HLA-I) molecules, and by the CD94/NKG2A lectin-like heterodimer specific for HLA-E. Conversely, other KIRs and CD94/NKG2C, which display a lower affinity for HLA-I ligands trigger protein tyrosine kinase pathways through DAP12, an adaptor with immunoreceptor tyrosine-based activation motifs. Similar inhibitory and activating NK cell receptors (NKR) have been identified among the murine Ly49 and NKG2 lectin-like receptor families (1, 2). The hypothesis that MHC-specific activating NKR may contribute to the innate response against pathogens was supported by the evidence that Ly49H specifically interacts with the MHC class I-related murine cytomegalovirus glycoprotein m157, triggering NK cell effector functions and the development of a memorylike response that confers resistance against the viral infection in some mice strains (3–5).

With this remarkable exception, no formal proof has been thus far obtained supporting the involvement of other activating KIR, NKG2, or Ly49 receptors in direct recognition of pathogen molecules (6). In this regard, human cytomegalovirus (HCMV) infection has been shown to promote the differentiation and persistent expansion of a mature NK cell subset, which displays high surface levels of the activating CD94/NKG2C NKR (NKG2Cbright), together with additional distinctive phenotypic and functional features (7–12). The magnitude of such adaptive NK cell subset redistribution appears variable in healthy blood donors, being undetectable in some HCMV+ individuals. This NK cell response pattern has been as well observed following active HCMV infection in newborns and immunocompromised patients (13–17). Although expansions of NKG2C+ NK cells have been reported in the context of other infections (18–21), the effect appears restricted to individuals coinfected by HCMV, thus suggesting that it is specifically induced by this herpes virus, being potentially amplified along the immune response to other pathogens.

The mechanisms underlying differentiation and expansion of NKG2Cbright NK cells remain uncertain. Engagement of CD94/ NKG2C by specific monoclonal antibodies (mAbs) or HLA-E, expressed in the 721.221 (.221) HLA-I defective cell line, triggered NKG2C+ NK-cell effector functions and proliferation in response to IL-2 or IL-15, strongly suggesting that the receptor might play a direct role in the response to HCMV infection (22, 23). *In vitro* proliferation of NKG2C+ cells was observed coculturing PBMCs or purified NK cells from some HCMV+ donors with HCMV-infected fibroblasts. The response required the participation of cytokines (i.e., IL-12, IL-15) and was antagonized by anti-CD94 (22), -NKG2C, or -HLA-E mAbs (23). These observations supported the hypothesis of an instructive process driven by a cognate interaction of the CD94/NKG2C receptor with ligand(s) displayed by HCMV-infected cells (24). Paradoxically, no formal evidence has been obtained supporting an active role of the CD94/NKG2C receptor in triggering *in vitro* NK cell effector functions against HCMV-infected cells, suggesting that NKG2C-mediated NK cell activation might be hampered by viral immune evasion mechanism(s) (25). By contrast, antibody-dependent stimulation *via* CD16 (FcγR-IIIA) efficiently activates adaptive NKG2C+ NK cells to mediate specific cytotoxicity, cytokine production, and proliferation in response to HCMV- and other virus-infected cells (26–29). CD2 has been shown to play an important co-stimulatory role in antibody-dependent activation of NKG2C+ cells (30, 31). Recently, increased baseline proportions of adaptive NKG2C+ NK cells in kidney transplant recipients have been directly related with a reduced incidence of posttransplant HCMV infection (32), suggesting that they may play a role in antiviral defense, involving CD94/NKG2C and/or CD16-dependent activation (33).

Previous reports revealed that binding of HLA-E to a peptide from the HCMV UL40 leader sequence preserves its expression in infected cells, engaging the CD94/NKG2A inhibitory receptor (34, 35). On the other hand, viral MHC class I-modulating molecules (i.e., US2-US11) were shown to play a prevalent role in governing the response of NK cells against infected targets (36).

In the present study, we approached the identification of putative ligand(s) for CD94/NKG2C in HCMV-infected cells, reducing the complexity of NK cell-infected target interactions. To this end, both receptor subunits and DAP12 were stably expressed in the human Jurkat leukemia T cell line. Signaling was detected by transient transfection of a reporter plasmid encoding for Luciferase (Luc) under NFAT/AP1-dependent control. Our results are discussed in the hypothetical framework on the development of adaptive NKG2C+ cells in response to HCMV.

#### MATERIALS AND METHODS

#### mAbs and Flow Cytometry Analysis

Flow cytometry was performed using mAbs specific for the following surface molecules: anti-NKG2C-PE (clone 134591) R&D Systems (Minneapolis, MN, USA), anti-HLA-I-APC (clone HP-1F7) generated in our laboratory and conjugated by Immunostep (Salamanca, Spain). The following indirect antibodies were used as purified or culture supernatants: anti-HLA-E (clone 3D12) provided by Dr. D. E. Geraghty (Fred Hutchinson Cancer Research Centre, Seattle, WA, USA), anti-CD3 (clone SpvT3B); anti-NKG2A (clone Z199), anti-NKG2D (clone BAT221), anti-NKp46 (clone Bab281), anti-NKp30 (clone AZ20), anti-DNAM1 (clone F22), anti-CD16 (KD1) provided by Dr. A. Moretta (University of Genova), and Dr. D. Pende (National Institute for Cancer Research, Genova); anti-LFA1 (clone TS/18), anti-ICAM1 (clone HU5/3) provided by Dr. F. Sánchez-Madrid (Hospital Univ. de la Princesa, Madrid); anti-KIR3DL1 (clone DX9) provided by Dr. L. Lanier (University of California San Francisco, CA, USA); anti-KIR2DL2/S2/L3 (clone CH-L) provided by Dr. S. Ferrini (National Institute for Cancer Research, Genova, Italy); anti-KIR3DL1/3DL2/2DS4/2DS5/2DS2/3DS1 (clone 5.133), provided by Dr. M. Colonna (University of Saint Louis, MO, USA). Anti-CD94 (clone HP-3B1), anti-ILT2 (LILRB1, LIR1) (clone HP-F1), anti-CD2 (clone MAR206), anti-KIR2DL1 (clone HP-DM1), anti-KIR2DL1/2DS1/2DS3/2DS5 (clone HP-MA4), anti-KIR2DL5 (clone UP-R1), and anti-KIR2DL1/S1/ S4 (clone HP-3E4) were produced in our laboratory.

Briefly, cells were pretreated with human IgG (10 µg/ml) to block Fc receptors, incubated with individual NKR-specific mAbs for 30 min, washed, and further incubated with a secondary PE-tagged F(ab')2 rabbit anti-mouse Ig (The Jackson Immunoresearch, West Grove PA, USA); anti-myc mAb (9E10, IgG1) was used as negative control. Data were acquired on FACSCalibur flow cytometer (BD Biosciences) and processed using FlowJo software (TreeStar, OR, USA).

#### Cell Lines and Culture Conditions

The Jurkat leukemia T cell line and its transfectants were grown in RPMI-1640 medium (Gibco, Grand Island, New York, NY, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco), termed as complete medium. Jurkat-CD94+ cells were kindly provided by Dr. Lewis Lanier, obtained as previously described (37) and cultured in complete medium with G418 (1 mg/ml) (InvivoGen, San Diego, CA, USA).

The 721.221 (.221) HLA-I-deficient EBV-transformed B lymphoblastoid cell line and its transfectant .221-AEH (kindly provided by Dr. D. E. Geraghty, Fred Hutchinson Cancer Research Centre, Seattle, WA, USA) were cultured in RPMI-1640 complete medium .221-AEH cells were generated by stable transfection of .221 cells with a construct in which the leader sequence of the HLA-E\*0101 allele was replaced by that of HLA-A2 and were selected in the presence of 300 µg/ml hygromycin B (Invitrogen, Carlsbad, CA, USA) (38).

Synthetic leader sequence peptides from HLA-G (VMAPRTLFL) or the AD169 UL40 viral protein (VMAPRTLIL) were purchased from CRG-UPF proteomic core facility (Parc de Recerca Biomèdica de Barcelona, Spain). As described (39), to stabilize HLA-E surface expression, HLA-Ia-defective 721.221 cells were incubated overnight with peptides (10 mM) at 26°C; HLA-E surface expression was monitored before and after incubation with peptides by flow cytometry.

Human Fetal Foreskin Fibroblast (HFFF) cells provided by Prof. John Trowsdale (University of Cambridge, UK), and the human lung fibroblast cell line MRC-5 provided by Dr. A. Angulo, were maintained in Dulbecco modified essential medium (DMEM) (Gibco) supplemented with 10% FBS, penicillin, and streptomycin.

#### Generation of Jurkat-NKG2C**+** Reporter Cells

Jurkat-NKG2C+ cells were established using a retroviral expression system to stably express DAP12 and NKG2C proteins in Jurkat-CD94+ cells. DAP12 and NKG2C cDNA constructs were subcloned from pJFE14 expression vector to pBABE-puro retroviral vector using XbaI-EcoRI (DAP12) and BamHI-EcoRI (NKG2C) restriction sites. As described (40), the retroviral constructs were individually transfected using the non-modified polyethyleneimine reagent (PEI, Sigma-Aldrich, St. Louis, MO, USA) into the helper-virus free amphotropic producer cell line Phoenix-A, a derivative of the human embryonic kidney cell line 293T (provided by Dr. Ramon Gimeno, IMIM, Barcelona, Spain). At 48, 72, and 96 h post-transfection, supernatants containing retroviral particles of DAP12 and NKG2C were collected, filtered with 45 µm filter (Millipore, Billerica, MA, USA), and centrifuged with Beckman SW28 rotor at 25,000 rpm for 90 min at 4°C. Pelleted virus were resuspended in 1 ml of RPMI medium and used to transduce Jurkat-CD94+ cells. To this end, 500,000 cells were plated (48 well/plates) in 1 ml mixed concentrated retroviral medium (0.5 ml DAP12 and 0.5 ml NKG2C) in the presence of 8 µg/ml polybrene (Sigma-Aldrich) and spinned for 90 min at 930 *g.* Cells were supplemented with fresh medium at 6 h post-transduction and selected with 1.5 µg/ml puromycin (Sigma-Aldrich) for 48 h; subsequently, cells positive for NKG2C surface expression were sorted (Influx Cell sorter, BD Bioscience), cloned by limiting dilution and expanded.

# HCMV Preparations and Infection of Human Fetal Foreskin Fibroblasts (HFFF)

This work was carried out in an authorized UPF p2-level biohazard facility, in compliance with the official requirements for CMV manipulation. Stocks of concentrated HCMV strains AD169 and Towne, provided by Dr. A. Angulo, TB40/E, provided by Dr. C. Sinzger (Institute for Medical Virology, University of Tübingen), and two HCMV clinical isolates: UL1271 (41) and #119, provided by Dr. H. Hengel, were prepared as follows. Almost confluent MRC-5 fibroblasts were infected at low multiplicity of infection (MOI) and supernatants were recovered when maximum cytopathic effect was reached (7–10 days) followed by clearing of cellular debris by centrifugation at 1,750 *g* for 10 min (42). Thereafter, the virus was concentrated for 3 h by centrifugation at 29,000 *g* at 15°C. Pelleted virus was resuspended in serum-free Dulbecco medium, stored at −80°C, and titrated by standard plaque assays.

Human fetal foreskin fibroblasts were seeded in 48-well plates 2 days prior infection at 4 × 104 cells/well. Confluent cells were incubated alone (mock) or with different viral strains at MOI of 10. After 2 h of absorption at 37°C, cells were washed twice with PBS and then fresh DMEM medium was added. Depending on the experimental design, HFFF cells were washed again at 24, 48, or 72 h postinfection. The infection rate was assessed by monitoring expression of the IE1 protein by indirect immunofluorescence with mAb MAB810R (clone 8B1.2) (Millipore) and Alexa Fluor 488-Labeled F(ab')2 goat anti-mouse secondary (Invitrogen, Carlsbad, CA, USA). Alternatively, infected cells were indirectly identified assessing down-modulationof surface HLA-I expression at 72 h postinfection.

# Preparation and Activation of CD94-NKG2C**+** Reporter Cells

Human cytomegalovirus-infected HFFFs were cocultured with Jurkat-NKG2C+ cells previously transfected with a reporter plasmid encoding Luciferase (Luc) under the control of NFAT/ AP1 promoter (3X NFAT/AP1-Luc) generated as described (43) and provided by Dr. Jose Aramburu (Universitat Pompeu Fabra, Barcelona, Spain). Transfection of Jurkat cells was carried out using the Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA) following the protocol provided by the manufacturer. Luc-transfected Jurkat cells were cocultured with the different targets for 18–24 h. After coculture, cells were collected, lysed, and Luc activity was measured using Promega Luciferase Assay system (Promega, Madison, WI, USA). The data were normalized referring the specific luminescence counts to those of non-treated (NT) Jurkat-NKG2C+ cells and are represented as fold-change induction. Stimulation of Jurkat with plate-bound anti-CD3, -CD94, -NKG2C, or co-culture with 721.221 and .221-AEH cells were used as controls. To verify the involvement of NFAT/AP1 in the reporter activation, experiments were carried out in the presence of 1 µM FK506 calcineurin inhibitor (Sigma-Aldrich), pretreating Jurkat-NKG2C+ cells with the drug for 2 h. For antibody-blocking assays, Jurkat-NKG2C+ cells or infected HFFF cells were, respectively, preincubated for 2 h with anti-NKG2C (clone 134522, R&D Systems) or anti-HLA-E (clone 3D12) mAbs (5 µg/ml) prior to coculture; anti-CD94 F(ab')2 fragments obtained from the HP-3B1 clone (37) were employed in some experiments.

# Statistical Analysis

Jurkat-NKG2C+ cell activation, assessed by induction of Luc activity in response to control stimuli (i.e., anti-NKG2C or .221- AEH cells) in different experiments (*n*= 15), was verified to follow a normal distribution applying the conventional Shapiro–Wilk test. Statistical analysis of the results was carried out applying the Student's *t*-test.

# RESULTS

# Generation and Phenotypic Characterization of a Human CD94/NKG2C**+** Reporter T Cell Line

To study the role of the CD94/NKG2C NKR in recognition of HCMV-infected cells, a reporter cell system was developed expressing the receptor segregated from other NKR. For this purpose, NKG2C and DAP12 were stably transduced in an available CD94-transfected human Jurkat leukemia T cell line. Jurkat-NKG2C+ cells were sorted and cloned by limiting dilution assessing the expression of adhesion molecules and NKR. A clone (97), which expressed CD94/NKG2C was selected (**Figure 1A**). These cells had downregulated CD3, displayed adhesion/costimulatory molecules (i.e., LFA-1, CD2, ICAM-1, and DNAM-1), but lacked activating (i.e., NKG2D, NKp46, NKp44, NKp30, CD16, aKIR) and inhibitory (i.e., iKIR, NKG2A, TIGIT, ILT2) NKR (**Figure 1B**). The marginal expression of ILT2 was particularly important as this HLA-I specific inhibitory receptor, which interacts with the UL18 HCMV molecules, was detectable in the parental Jurkat-CD94+ cells (not shown).

# Specific Recognition of HLA-E by Jurkat-NKG2C**+** Reporter Cells

In order to detect signaling by the CD94/NKG2C-DAP12 complex, Jurkat-NKG2C+ cells were transiently transfected with

a plasmid encoding for Luciferase (Luc) under the control of NFAT/AP1-dependent promoter. Engagement of the receptor by solid-phase bound CD94- or NKG2C-specific mAbs triggered Luc expression (**Figure 2A**). Of note, a slightly higher background in Jurkat-NKG2C+ cells compared to the parental cell line suggested that the low constitutive HLA-E expression in Jurkat-NKG2C+ cells promoted a limited self-activation of the reporter (Figure S1 in Supplementary material). However, coculture of Jurkat-NKG2C+ cells with the .221-AEH cell line,

DX9 (KIR3DL1), UP-R1 (KIR2DL5). Gray histograms correspond to the

isotype control.

which displays HLA-E in the absence of classical HLA-I, induced Luc expression and the response was specifically antagonized by soluble NKG2C- and HLA-E-specific mAbs (**Figures 2A,B**). Similar results were obtained stimulating Jurkat-NKG2C+ cells with the .221 cell line preincubated with synthetic HLA-I leader sequence peptides, known to stabilize surface HLA-E expression (**Figures 3A,B**). These results validated the sensitivity and specificity of the reporter system to detect CD94/NKG2C receptor–ligand interaction.

## Response of Jurkat-NKG2C**+** Reporter Cells to Fibroblasts Infected by HCMV Laboratory Strains

On that ground, experiments were carried out incubating Jurkat-NKG2C+ cells with HFFF, infected at different time-points (24–72 h) with HCMV laboratory strains (AD169 and Towne). As compared to positive controls stimulated with anti-NKG2C mAb or .221-AEH cells, no differences were perceived comparing Luc expression in Jurkat-NKG2C+ cocultured either with mock- or HCMV-infected HFFF (**Figure 4A**); similar results were obtained incubating the reporter with MRC5 fibroblasts (not shown). Total HLA-I expression was downregulated in cells infected by both HCMV strains. By contrast, HLA-E was preserved by AD169, which displays a canonical UL40 leader peptide (VMAPRTLIL) binding to the HLA class Ib molecule, but was lost in Towne-infected cells (**Figure 4B**). As reported (44) and according to the annotated GenBank sequences, this HCMV strain contains a deletion spanning the UL40 leader sequence. These results point out that the undetectable response of Jurkat-NKG2C+ to fibroblasts infected by laboratory strains was unrelated to their ability to sustain surface HLA-E expression.

## Response of Jurkat-NKG2C**+** Reporter Cells to Fibroblasts Infected by HCMV Clinical Isolates

TB40/E HCMV strain retains the ability to replicate in endothelial and myelomonocytic cells, differing from AD169 and Towne (45). Moreover, it is well established that HCMV clinical isolates substantially diverge from laboratory strains, rapidly undergoing important genomic changes upon *in vitro* passage, as reviewed in Ref. (46). Thus, additional experiments were carried out coculturing Jurkat-NKG2C+ cells with HFFF infected with TB40/E, which encodes for a UL40 leader peptide variant unable to preserve HLA-E surface expression (VVAPRTLIL) (25), or with two HCMV clinical isolates

coculture with Jurkat-NKG2C+ cells; gray histograms represent the isotype control. Statistically significant differences are indicated (\*\**p* < 0.01).

(#UL1271 and #119), which share the canonical UL40-nonamer and preserved HLA-E expression (**Figures 5A,B**). Remarkably, a specific response of the reporter was detected upon infection with these viruses, which appeared greater for #119 and gradually increased along postinfection time. Yet, controls indicated that Luc expression in response to HFFF infected with HCMV clinical isolates was not inhibited by pretreatment with anti-NKG2C or -HLA-E mAbs (**Figures 6A,B**); similar results were obtained with anti-CD94 F(ab′)2 fragments (data not shown), ruling out the potential influence of an overlapping agonistic effect mediated by anti-NKG2C bound to virus-encoded FcR. Moreover, the effect was also induced in CD94+ and wild-type Jurkat cells, which lacked NKG2C, unequivocally revealing that reporter activation was independent of this NKR (**Figure 6C**); similar results were obtained with TB40/E-infected cells (data not shown). As this response was dominant, the possibility that it might eventually mask a subtle involvement of CD94/NKG2C cannot be entirely ruled out.

#### DISCUSSION

A number of observations indirectly support the hypothesis that a specific and direct interaction of CD94/NKG2C with infected cells contributes to drive the adaptive NK cell response; yet, molecular evidence remains thus far elusive (10). To explore the presence of putative ligand(s) for CD94/NKG2C in HCMVinfected cells, the receptor was stably expressed along with DAP12 in the human Jurkat leukemia cell line, transiently transfected with an NFAT/AP1-dependent Luc-encoding reporter. The rationale for using a human parental cell line was based on our previous experience with heterologous CD94/NKG2C+ rat basophilic leukemia cells (RBL), which failed to respond to receptor engagement by HLA-E+ .221-AEH cells (not shown). A Jurkat-NKG2C+ clone that shared with NK cells key adhesion receptors (i.e., LFA-1, CD2, and DNAM1) but lacked inhibitory NKR, was selected.

The possibility that constitutive HLA-E expression by Jurkat-NKG2C+ cells might promote self-activation of the reporter, potentially impairing its responsiveness to external stimuli, was beforehand considered a potential drawback. Yet, Luc expression in Jurkat-NKG2C+ cells was induced following stimulation with CD94 or NKG2C-specific mAbs. Importantly, reporter activation was detectable upon interaction with HLA-E+ .221-AEH cells, as well as with .221 cells incubated with synthetic peptides which stabilized surface expression of endogenous HLA-E; in these settings, inhibition by anti-NKG2C and -HLA-E mAbs supported specific receptor-ligand engagement. By contrast, reporter activation was undetectable upon interaction of

significant differences are indicated (\*\**p* < 0.01).

Jurkat-NKG2C+ cells with HFFF or MRC5 fibroblasts following infection with common HCMV laboratory strains (i.e., AD169, Towne), regardless of their ability to preserve surface HLA-E expression in infected cells.

Among variables that may potentially condition the expression of CD94/NKG2C ligand(s) by HCMV-infected cells, genomic differences between viral strains and changes associated to *in vitro* passage of the virus were considered potentially important. In fact, cells infected with two different clinical isolates and, to a lesser degree, with the endotheliotropic TB40/E strain, triggered Jurkat-NKG2C+ activation. Yet, this response was not inhibited by NKG2C- nor HLA-E-specific blocking mAbs and, furthermore, was independent of CD94/ NKG2C expression. Experiments were carried out to explore the molecular basis of this dominant effect, which might potentially mask CD94/NKG2C-specific signaling. Pretreatment of Jurkat-NKG2C+ with a calcineurin inhibitor (FK506) hampered Luc expression triggered by anti NKG2C mAb or .221-AEH cells, but did not prevent the response of Jurkat-NKG2C+ cells to HCMV clinical isolates (data not shown), pointing out that a nonconventional activation of the reporter plasmid was induced under these experimental conditions. Further studies are required

to assess the possibility of controlling this drawback, eventually re-designing the system.

NKG2C+ NK cells were reported to be activated upon interaction with primary aortic endothelial cells infected by the VHL/E HCMV strain (47); nevertheless, no data supporting an involvement of the receptor (e.g., blocking with anti-CD94 or -NKG2C mAbs) were provided. In our hands, Jurkat-NKG2C+ did not detectably respond to HCMV-infected MRC5 fibroblasts nor to human umbilical vein endothelial cells. Yet, the possibility that expression of putative CD94/NKG2C ligands might vary depending on the infected cell lineage is not ruled out.

Our observations should be discussed in the framework of previous indications supporting a CD94/NKG2C receptor interaction with infected cells. In this regard, NKG2C+ NK cells were shown to undergo *in vitro* expansion upon coculture with AD169 or Towne-infected fibroblasts (22, 23). The effect was dependent on cytokines (i.e., IL-12, IL-15) and was prevented by blocking anti-CD94, -NKG2C, or -HLA-E mAbs. The response was observed only in samples from some HCMV+ blood donors displaying baseline NKG2Cbright NK cell expansions, indicating that these experimental conditions promoted their proliferation, but did not strictly reproduce the differentiation process induced *in vivo* by primary HCMV infection. Of note, *in vitro* expansion was prevented upon infection by an HCMV BACmid mutant lacking the whole set of viral genes (i.e., US2-US11), which target HLA-I molecules, thus preserving their expression in infected cells (22). These experiments were interpreted as an indication of a relatively weak stimulation *via* CD94/NKG2C that was overridden by inhibitory KIR-HLA-I interactions which, by contrast, did not impair CD16-triggered NK cell effector functions against cells infected by the same virus (28).

On the other hand, a relation of NKG2C gene copy number with the magnitude of NKG2C+ NK cell expansion has been reported in HCMV+ healthy blood donors, in children with congenital infection and in renal transplant recipients (9, 14, 32, 48). Moreover, CD94/NKG2C surface expression levels and

activation upon engagement by HLA-E were also greater in NKG2Cbright NK cells from *NKG2C+/+* than *NKG2C+/del* individuals (9). These data supported that subtle differences in CD94/ NKG2C surface density may quantitatively influence adaptive NK cell differentiation/activation in response to HCMV, thus providing another indirect indication for a relatively low avidity of the hypothetical receptor–ligand interaction. The increased surface expression of CD94/NKG2C in differentiated adaptive NK cells (NKG2Cbright) from HCMV+ subjects as compared to the profile detected in non-infected individuals (NKG2Cdim) is also in line with this view. Of note, a limited affinity and/or reduced expression levels of the putative CD94/NKG2C ligand

in infected cells may presumably render this pathway particularly sensitive to viral immune evasion mechanisms, eventually accounting for the undetectable activation of NKG2C+ NK cell effector functions and of Jurkat-NKG2C+ cells, in that case, independently of HLA-specific inhibitory receptors.

Despite the negative results obtained with the reporter system, the existence of specific ligand(s) for CD94/NKG2C in HCMV-infected cells still remains a plausible hypothesis, compatible with the involvement of a viral glycoprotein or/and an HLA-E-peptide complex. The latter option is suggested by the blocking effect of an anti-HLA-E mAb in the expansion of NKG2C+ NK cells cocultured with infected fibroblasts (23). A first candidate to be considered is the UL40 leader sequencederived peptide reported to promote HLA-E expression in HCMV-infected cells by a mechanism refractory to the viral US6 TAP inhibitor (34) and favored by the intrinsic HLA-E resistance to other viral molecules targeting HLA-I (49). UL40-dependent HLA-E expression in infected cells was shown to inhibit NK cell activation by engagement of the CD94/NKG2A inhibitory receptor, presumably contributing to immune evasion (34). The canonical UL40-derived nonamer (VMAPRTLIL) binding to HLA-E is identical to that displayed by some HLA-I leader sequences, predictably promoting a low affinity interaction with CD94/NKG2C (50). Heatley et al. (51) compared the ability of a panel of UL40-nonamer variants to stabilize HLA-E in RMA-S cells, assessing the interaction of the corresponding HLA-E/peptide complexes with CD94/NKG2A or CD94/NKG2C by surface plasmon resonance assays, as well as their recognition by NK cell subsets, respectively, displaying these NKR. Of note, quantitative differences in the interaction of distinct HLA-E-peptide complexes were noticed, but none appeared preferentially recognized by CD94/NKG2C.

It is conceivable that HLA-E expression by HCMV-infected cells, in which HLA-I ligands for inhibitory KIRs are downregulated, might promote specific activation of NKG2C+ NKG2A-NK cells. Supporting this hypothesis, Jurkat-NKG2C+ cells were activated by .221 cells displaying HLA-E bound to the synthetic AD169 UL40-derived peptide (VMAPRTLIL), shared by some HLA-I alleles. Nevertheless, no reporter response was perceived following interaction with AD169-infected cells expressing HLA-E, pointing out to the influence of additional factors, and indicating that experimental approaches based on loading target cells with synthetic peptides provide valuable information but do not precisely reflect the complexity of HCMV infection. A putative role of UL40 in NKG2C+ NK cell expansion, detected following coculture with AD169-infected cells, was addressed employing a UL40 deletion mutant generated in an AD169 BACmid (HB5) (22, 23). In this setting, cells infected with ΔUL40 HB5 retained the ability to promote *in vitro* expansion of NKG2C+ NK cells; however, a caveat for interpreting these experiments is the gene deletion encompassing US2-US6 introduced for the generation of the HB5 BACmid. In fact, HCMV impairs surface expression of HLA-I molecules but not their biosynthesis and, therefore, endogenous HLA-I leader sequences in the absence of US6 may be presented by HLA-E in HB5-infected cells, eventually competing with the UL40-derived nonamer, or even replacing it in ΔUL40 HB5-infected HFFF. On the other hand, NKG2C+ NK cell expansions detected in cocultures with Towne-infected fibroblasts (22) may indirectly provide a case against the involvement of HLA-E expression, which was downregulated consistent with the existence of a genomic deletion spanning the UL40 leader sequence of this HCMV strain (44). Of note, no relation of the HLA-E dimorphism with the expansion of NKG2C+ cells was noticed in previous studies (7, 9).

In summary, molecular evidence supporting that a specific interaction of CD94/NKG2C with infected cells drives the adaptive NK cell response to HCMV remains elusive, leaving unanswered key interrelated questions, particularly, the cellular mechanisms underlying the stable expansion of NKG2Cbright

NK cells and the basis for the wide variability of this effect in HCMV+ individuals. Observations in immunocompromised patients suggested that this pattern of response may compensate an inefficient T-cell-mediated control of the primary infection, eventually determined in healthy individuals by viral/host genetic factors as well as by other circumstantial variables (e.g., age at infection, viral load, etc.).

We hypothesize that primary HCMV infection promotes the differentiation, proliferation, and survival of a pool of progenitors, possibly stemming from NKG2Cdim NKG2A-NK cells present in seronegative individuals. Increased surface expression of CD94/NKG2C presumably constitutes a key early event, facilitating activation of this NK cell subset upon interaction with low-avidity ligand(s) displayed by infected cells. Such NKG2C-driven selection is dependent on cytokines required for adaptive NK cell differentiation/expansion and may be tuned by specific KIR-HLA-I interactions, consistent with the oligoclonal KIR expression profile of adaptive NK cells (8, 52). Following a contraction phase after viral replication is controlled, a pool of long-lived NKG2Cbright NK cells with clonal expansion potential survive, and homeostatic proliferation contributes to their persistent increased numbers in the circulation. The process is reminiscent of the generation of memory cytotoxic T lymphocytes as proposed for the response of Ly49H in mice (53); in fact, responsiveness of differentiated NKG2Cbright NK cells to cytokines (e.g., IL-15, IL-2) becomes dependent on signaling by activating NKR (e.g., NKG2C or CD16). HCMV reactivation/reinfection as well as other infectious pathogens may boost antibody- and cytokine-dependent activation of adaptive NK cells, leading to their progressive acquisition of late differentiation features (e.g., FcRγ downregulation). Further efforts are warranted to understand how HCMV infection resets the NK-cell compartment homeostasis in some individuals, and the implications that such persistent reconfiguration of the NK cell compartment may have in the development of the immune response under different pathological conditions.

#### AUTHOR CONTRIBUTIONS

AP developed the reporter system, carried out the experimental work, and revised the manuscript. MC-G collaborated in the experimental development. DF and AA collaborated in the genetic analysis of HCMV as well as in critical discussion of the results. HH contributed with essential support for the analysis of HCMV clinical isolates as well as in critical discussion of the results. AM contributed to the design, follow-up, and interpretation of the results. ML-B proposed the experimental approach, contributed to the design, follow-up, and interpretation of results, and wrote the draft that was revised by all authors.

#### ACKNOWLEDGMENTS

The authors thank Gemma Heredia and Andrea Vera for technical help, and Ramón Gimeno for advice in retroviral vector expression.

#### FUNDING

The authors are supported by grants from: Plan Estatal I + D Retos (SAF2013-49063-C2-1-R; SAF2016-0363-C2-1-R), Spanish Ministry of Economy and Competitiveness (MINECO, FEDER); EU FP7-MINECO Infect-ERA program (PCIN-2015- 191-C02-01); AP is supported by EU-FP7 Marie Curie Training Network (NATURIMMUN FP7-PEOPLE-2012-ITN-317013); HH is supported by Infect-ERA grant BMBF 031L0090; AA is

#### REFERENCES

supported by Plan Estatal I+D Retos grant SAF2014-55683, MINECO, FEDER.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/fimmu.2017.01317/ full#supplementary-material.

induced by cytomegalovirus infection in preterm infants. *Hum Immunol* (2015) 76(2–3):118–23. doi:10.1016/j.humimm.2015.01.017

**Conflict of Interest Statement:** Authors individually declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

The reviewer CF and handling editor declared their shared affiliation.

*Copyright © 2017 Pupuleku, Costa-García, Farré, Hengel, Angulo, Muntasell and López-Botet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Regulation of the Functions of Natural Cytotoxicity Receptors by Interactions with Diverse Ligands and Alterations in Splice Variant Expression

#### *Tatiana Pazina1,2, Avishai Shemesh3 , Michael Brusilovsky4 , Angel Porgador3 and Kerry S. Campbell1 \**

*1Blood Cell Development and Function Program, Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA, 2 Federal State Budgetary Scientific Institution "Institute of Experimental Medicine", St. Petersburg, Russia, 3 Faculty of Health Sciences, The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Beer Sheva, Israel, 4Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA*

#### *Edited by:*

*Gianfranco Pittari, Hamad Medical Corporation, Qatar*

#### *Reviewed by:*

*Eric Vivier, Centre d'immunologie de Marseille-Luminy, France Alessandro Moretta, University of Genova, Italy*

> *\*Correspondence: Kerry S. Campbell<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology*

> *Received: 31 January 2017 Accepted: 14 March 2017 Published: 30 March 2017*

#### *Citation:*

*Pazina T, Shemesh A, Brusilovsky M, Porgador A and Campbell KS (2017) Regulation of the Functions of Natural Cytotoxicity Receptors by Interactions with Diverse Ligands and Alterations in Splice Variant Expression. Front. Immunol. 8:369. doi: 10.3389/fimmu.2017.00369*

The natural cytotoxicity receptor (NCR) family is constituted by NKp46, NKp44, and NKp30 in humans, which are expressed mainly on natural killer (NK) cells and are encoded by the *ncr1*, *ncr2*, and *ncr3* genes, respectively. NCRs have classically been defined as activating receptors that trigger cytotoxicity and cytokine responses by NK cells upon engaging with ligands on tumor cells. Several new findings, however, have challenged this model and identified alternative mechanisms regulating the function of NCRs. Recent reports indicate that ligand matters, since the interaction of NKp44 with distinct ligands on target cells can either activate or inhibit NK cells. Also, the NCRs have been found to interact with distinct specificities to various heparan sulfate glycosaminoglycans, which are complex polysaccharides found in extracellular matrix or on cell surface heparan sulfate proteoglycans (HSPGs). The NCRs can engage with HSPGs in trans as a co-ligand on the target cells or in cis on the NK cell surface to regulate receptor–ligand interactions and NK cell activation. A number of splice variants of *ncr2* and *ncr3* have also been identified, and a predominant expression of certain variants results in inhibitory signaling through NKp44 and NKp30. Several recent studies have found that the selective expression of some of these inhibitory splice variants can significantly influence outcome in the contexts of cancer, infection, and pregnancy. These findings establish that NCR functions are more diverse than originally thought, and better understanding of their splice variant expression profiles and ligand interactions are needed to establish their functional regulation in the context of human health.

Keywords: natural cytotoxicity receptors, natural killer cells, RNA splice variants, cytotoxicity, cancer immunology, virus immunity, human immunology, pregnancy

# INTRODUCTION

It has become increasingly clear that human natural killer (NK) cells use an array of germline-encoded cell surface receptors to spontaneously recognize and respond to the abnormal status of tumor cells, virus-infected cells, and stressed cells (1). Different NK cell surface receptors transduce either activating or inhibitory signals, directly or through associated adaptor proteins, to dynamically regulate the activation state of NK cells (2–4). Inhibitory receptors, such as killer cell Ig-like receptors (KIRs) and CD94/NKG2A, provide NK cells with a dominant, tolerizing immune checkpoint through their recognition of the MHC class I (MHC-I) molecules ubiquitously expressed on the surfaces of most normal cells. The loss of MHC-I by many transformed cells, however, overcomes this inhibitory checkpoint to favor activation. The loss of inhibitory restraint allows activating receptor signals to predominate and triggers exocytosis of perforin and granzymes to induce targeted apoptosis of the MHC-I-deficient cell and localized secretion of pro-inflammatory cytokines [especially interferon (IFN)-γ, TNF-α, and several chemokines].

The activation signals transduced in NK cells are derived from adhesion molecules (especially LFA-1), co-stimulatory receptors (such as NKG2D, DNAM-1, and SLAM family receptors), and several activating receptors physically linked to the immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane proteins, DAP12, TCR-ζ, and/or FcεRI-γ (2–4). The key ITAM-coupled activating receptors on human NK cells, include CD16 (FcγRIIIa), an activating subfamily of KIR (KIR2DS or KIR3DS receptors), CD94/NKG2C, and the natural cytotoxicity receptors (NCRs).

The human NCRs consist of three receptors, named NKp46 (NCR1, CD335), NKp44 (NCR2, CD336), and NKp30 (NCR3, CD337). These NCR were classically defined as germline-encoded receptors that play important roles in the activation of human NK cells toward transformed target cells (5, 6). Recent work, however, has established that the NCRs can also generate inhibitory responses under certain circumstances. Here, we will review our current understanding of the expression and function of NCRs on NK cells, particularly in humans, although it is important to note that the NCRs are also expressed on other innate lymphoid cells (ILCs) and a subset of T cells, which has been previously reviewed elsewhere (7, 8).

# NCRs AND THEIR STRUCTURES

The NCRs were initially discovered and characterized by the laboratories of Alessandro and Lorenzo Moretta in the late 1990s (9–13). They are type I transmembrane glycoproteins that were originally recognized as activating receptors and named in accordance with their molecular weight on SDS-PAGE (NKp30, NKp44, and NKp46). NKp46 is the only NCR also expressed in mice, although a receptor analogous to NKp30 has been shown to be expressed in 1 of 13 mouse strains examined (14) and in rats (15, 16). While the *ncr2* and *ncr3* genes encoding NKp44 and NKp30, respectively, are localized to human MHC class III locus on chromosome 6, the NKp46 encoding gene, *ncr1*, is found near the leukocyte regulatory complex on human chromosome 19 (10, 12, 13).

NKp46 has been shown to be a highly selective marker of all NK cells in mouse and man, although surface expression can be low on some NK cells, particularly in humans, and the receptor is also expressed on some ILCs and a small subset of T cells (9, 17, 18). Importantly, NKp46 is not expressed by CD1drestricted invariant NKT cells in mice and humans (17). NKp46 has been shown to provide NK cells with the capacity to recognize and kill a variety of tumor target cells (19–21). NKp46 ligands have been reported to be enriched in areas of high malignant potential and high proliferation within melanoma lesions, whereas surrounding normal melanocytes were found to lack NKp46 ligands (22). Evidence in mice also suggests that NKp46 also contributes to the development of type 1 diabetes by interacting with an uncharacterized ligand on pancreatic islet beta cells (21). As a tumor immunosuppressive mechanism, the surface expression of NKp46 on NK cells can be down-modulated by exposure to l-kynurenine, which is a tryptophan catabolism product generated by the indoleamine 2,3-dioxygenase (IDO) enzyme in tumor microenvironments (23).

NKp30, similar to NKp46, is expressed on nearly all human NK cells (13). This NCR has been shown to play important roles in crosstalk between NK cells and dendritic cells (DCs) through promoting both the maturation of and the cytotoxicity of immature DC (24, 25). Surface expression levels of NKp30 and NKp46 can be upregulated by IFN-α, IL-2, and prolactin and downregulated by cortisol and methylprednisolone (26–28). In addition, both receptors are also commonly downregulated in "adaptive" or "memory-like" NK cells that are found in some cytomegalovirus-infected individuals (29, 30). TGF-β has been shown to selectively down-modulate the expression of NKp30, but not NKp46 on NK cells (31).

NKp44 is distinct among NCRs, since it is unique to humans and only expressed constitutively on some CD56bright NK cells in a subset of individuals, but expression can be upregulated on essentially all NK cells after culture with IL-2, IL-15, or IL-1β (11, 32). Similarly, NKp44 can be upregulated on plasmacytoid DCs upon culture with IL-3 (33). Therefore, NKp44 may also be considered a marker of cytokine-activated NK cells in humans. IL-2 induced upregulation of NKp44 on NK cells can be inhibited by prostaglandin E2, which is readily produced by tumor-associated fibroblasts, especially when exposed to NK cells in culture (34). Similarly, prednisolone can suppress IL-2-mediated upregulation of NKp44 (26).

The extracellular domains of NCRs consist of one (NKp30 and NKp44) or two (NKp46) Ig-like domains that are responsible for ligand binding (10, 12, 13). Ligand binding and signaling function by NKp30 is highly dependent upon integrity of the membrane proximal stalk region (35). Crystal structures have revealed that NKp30 and NKp44 can form homodimeric structures with NKp30 dimerizing in a head-to-tail fashion to form an I-type Ig-like fold and two NKp44 V-type Ig-like domains form a saddle-shaped dimer with unique disulfide bridging (36, 37). On the other hand, the crystal structure of NKp46 demonstrates two C2-type Ig-like domains that are folded and oriented similar to the Ig-like domains of KIRs (38). Evidence for a homodimerization interface within the membrane proximal Ig-like domain of NKp46 has also been reported, and disruption of this dimerization interaction prevented ligand binding and activating function of the receptor (39). In conclusion, the three NCRs have unrelated structures, and grouping these receptors together is based more on their shared functional properties than related structure or genetic evolution (13).

The transmembrane domains of all three NCRs contain a positively charged lysine (NKp44) or arginine (NKp30 and NKp46) residue that interacts with acidic aspartic acid residues found in the transmembrane regions of the adaptor proteins DAP12 (NKp44) or TCR-ζ and/or FcεRI-γ (NKp30 and NKp46) (10). Physical association with DAP12 *via* these transmembrane charged residues is essential for surface expression of NKp44 (40). The reductions in surface expression levels of NKp30 and NKp46 on "adaptive" or "memory-like" NK cells is associated with the lack of FcεRI-γ expression in these cells (29, 30), exemplifying the importance of associating with this specific adaptor to transport a functional receptor to the cell surface. In addition to promoting surface expression, physical association with these associated transmembrane adaptors provides potent activation signaling function to the NCRs, since the tyrosine phosphorylation of their cytoplasmic ITAM domains results in the recruitment and activation of the Syk and ZAP-70 protein tyrosine kinases (2, 41). A unique activation signaling crosstalk has been reported between the NCRs, in which engagement of one NCR appears to initiate signaling through the others (41). Curiously, while several mRNA splice variants encoding NKp44 have been described, the major protein product or isoform was found to also contain a cytoplasmic ITIM-like domain. Although early work suggested that this domain was incapable of providing inhibitory signaling function in an NK-like cell line (40), more recent work has demonstrated ITIM-mediated inhibitory function by NKp44 upon recognition of a specific ligand, proliferating cell nuclear antigen (PCNA), as detailed below (42).

#### LIGANDS OF THE NCRs

Despite a great deal of work by numerous research groups, our understanding of the ligands for NCRs is still not clearly established. A diverse array of molecules have been report to interact with the extracellular domains of NCRs, including carbohydratebased contacts, cell surface proteins, and surprisingly, several intracellular-localized proteins that appear to reach the surface of infected or transformed cells. While engagement with most of these reported ligands stimulates activation of NK cells, some have been found to inhibit their functions. Our current understanding of putative ligands for NCRs and their functions are described below and summarized in **Figure 1** and **Table 1**.

#### Viral Ligands

Several viral-derived NCR ligands have been reported. Early work showed that the viral hemagglutinin (HA) of influenza virus on the surface of infected cells can readily engage with branched α-2,3- and α-2,6-sialylated *O*-glycan sequences conjugated on NKp46, and influenza-infected target cells can be killed by human NK cells in a NKp46-dependent manner (65–68). In fact, exposure of NK cells to influenza virions or free HA has been found to decrease NCR-mediated cytotoxicity, which was associated with loss of TCR-ζ protein expression (71). In addition, influenza virus-infected DCs stimulate IFN-γ production by NK cells in an NKp46- and HA-dependent manner (69). Furthermore, it has been demonstrated that NKp46-deficient mice are more susceptible to death after infection with influenza virus (72). These interactions are consistent with the known sialic acid-binding properties of viral HAs.  Similarly, both NKp46 and NKp44 were found to interact with the HA from avian Newcastle disease virus and this interaction potentiates cytotoxicity of target cells infected with this paramyxovirus (56).

Hemagglutinin from the orthopox family viruses, human vaccinia virus, and murine ectromelia virus has been shown to interact with NKp46 and NKp30 (43). Late-stage vaccinia virusinfected target cells were further shown to be less susceptible to NK cell cytotoxicity compared to uninfected targets, and this reduced killing was dependent upon viral HA in the target cells and NKp30 in the NK cells (43). The results from this study suggest that the HA on the surface of vaccinia virus-infected cells interacts with NKp30 to either block its activating function or to mediate inhibitory signaling in NK cells, whereas NKp46 engagement with vaccinia virus-derived HA on target cell surfaces stimulates cytotoxicity responses.

NKp44 has been shown to recognize the envelope glycoproteins from West Nile and Dengue flaviviruses (57). In particular, NKp44 was found to directly bind domain III of WNV envelope protein, but does not appear to involve viral HA, since independent of sialylation of oligosaccharides on NKp44. Consistent with this finding, West Nile virus-infected cells more readily bind a soluble recombinant form of NKp44 and stimulate NK cells to degranulate and produce IFN-γ in an NKp44-dependent manner (57). It should be noted that expression of Dengue viral nonstructural proteins in target cells reduces susceptibility to NK cell cytotoxicity through upregulating MHC-I expression (73).

In addition, NKp30 has been shown to directly interact with pp65, which is the main tegument protein of human cytomegalovirus (HCMV) (44). HCMV infected target cells were found to be less susceptible to NK cell-mediated killing, and this inhibition was lost if the target cells were infected with pp65-deficient HCMV or if anti-NKp30 blocking antibodies were added (44). The authors of this report further provided evidence that treating NK cells with a recombinant soluble form of pp65 resulted in the dissociation of the TCR-ζ signaling adaptor protein from NKp30 (44). In this way, pp65 appears to provide HCMV with a mechanism to avoid NK cell-mediated immunity by disrupting activation signaling through NKp30.

#### Other Ligands Expressed by Pathogens or Pathological Conditions

Natural cytotoxicity receptors have also been shown to directly recognize bacterial and parasite pathogens. It has been demonstrated that NKp30 (and to a lesser extent, NKp46) can interact with the Duffy binding-like (DBL)-1α domain of *Plasmodium*

*falciparum* erythrocyte membrane protein-1 to mediate cytolysis of malaria-infected erythrocytes (45). The interaction appears to be direct, since the effect can be inhibited by the addition of recombinant soluble forms of these NCRs or peptides matching the sequence of DBL-1α (45).

It has been reported that *Mycobacterium bovis bacillus* Calmette-Guérin (BCG) can directly interact with NKp44, and exposure to BCG can increase NKp44 expression on CD56bright NK cells (58). This study also found that additional *Mycobacterium* family members can bind NKp44, such as *Nocardia farcinica* and *Pseudomonas aeruginosa*. Interactions of these bacteria with NKp44 did not activate NK cell functions, however, so the relevance of these interactions is currently unclear.

NKp44 has been reported to recognize an uncharacterized ligand on cartilage-derived chondrocytes, and cytotoxicity of primary chondrocytes by long-term IL-2 activated-NK cells was inhibited by an NKp44-blocking antibody (59). These results suggest that NKp44 activation signaling may promote NK cell-mediated autoimmunity in chronic inflammatory cartilaginous disease.

#### Heparan Sulfates

Heparan sulfate (HS) glycosaminoglycans (GAGs) have also been shown to interact with all of the NCRs, with different affinities for the three receptors (46–48, 60). While these carbohydratedirected interactions likely do not represent primary ligands for the NCRs, they appear to have the capacity to regulate NCR function or may play a supporting role as co-ligands (61). HS GAGs consist of long, unbranched, anionic polysaccharides that are found on cell surfaces and the extracellular matrix (74). The HS GAG polysaccharides are composed of repeating disaccharide units of uronic acid (iduronic or glucuronic acid) and glucosamine that are differentially sulfated at *N*, 2-*O*, 3-*O*, and 6-*O* positions to generate highly diverse structures with unique protein binding properties (75). HS GAG can be conjugated to a small subset of proteins to form HS proteoglycans, including syndecans and glypicans, and their negative-charged configurations can provide docking sites for basic domains on chemokines, FGF, and wnt ligand family members, thereby "presenting" them to cell surface receptors (76–79).

Interestingly, HS GAG are highly diverse structures, and the three NCRs preferentially recognize highly sulfated HS structures *via* basic amino acid patches on the receptor surfaces, and each NCR demonstrates distinct HS binding specificity (47, 48, 60). Therefore, it is conceivable that each NCR has the capacity to distinguish particular configurations of HS GAG primary and tertiary structures that might be uniquely expressed in the

contexts of tumor microenvironments or sites of infection or inflammation. We have also shown the HS GAGs can interact with another NK cell receptor, KIR2DL4, and the binding can modulate receptor function (80). In addition, an interaction of NKp44 with the heparan sulfate proteoglycans (HSPGs), syndecan-4, in cis on the NK cell surface can modulate the surface distribution and function of the receptor (61). Based on this and other reports (47), we have proposed that interactions with HSPGs in cis (on the surface of NK cells) may be impacting KIR2DL4 and NCR functions through masking interactions with HS GAG or other ligands on adjacent target cells (trans interactions) and/or may be affecting the trafficking of NCR to intracellular degradation and recycling pathways upon endocytosis (61, 81). In this way, cis interactions between NCR and HSPGs may provide an allosteric regulation mechanism. It is also intriguing to speculate that treatment of patients with structurally related heparin as a therapeutic agent could impact NK cell functions through binding to NCRs and other NK cell surface receptors, including KIR2DL4.

# Intracellular Proteins As Cell Surface Ligands

The expression of a ligand for NKp44, named NKp44L, was first shown to be induced by the HIV-1 envelope protein gp41 on infected CD4+ T cells, and the expression increased in patients with increasing viral load (62). NKp44L is an activating ligand, since NK cell-mediated lysis of HIV-infected CD4+ T cells was inhibited by antibodies to NKp44 or NKp44L (62). The NKp44L was subsequently identified as a unique splice variant isoform of mixed-lineage leukemia-5 (MLL5) protein (63). While full-length MLL5 is a nuclear protein, the NKp44L splice variant is localized near the plasma membrane in the cytoplasm and expressed in several tumor tissues and transformed cell lines, but not in normal tissues (63).

It was reported that NKp46 is involved in NK cell-mediated cytolytic attack of monocytes infected with *Mycobacterium tuberculosis* (20). Subsequent work established that this is due to an interaction with vimentin, which is expressed at high levels in infected monocytes and appears on the cell surface (70). Since vimentin is a type III intermediate filament of the cytoskeleton, however, it is unexpected to find on the cell surface, but this follows an emerging theme of several traditionally intracellular proteins serving as putative cell surface ligands for NCRs. NK cells were more efficient at lysing target cells transfected to overexpress vimentin, and this cytotoxicity was inhibited by antibodies targeting NKp46 or vimentin (70).

In addition, NKp30 has been shown to interact with the HLA-B-associated transcript 3 (BAT3)/Bcl2-associated anthogene 6 (BAG6) protein to stimulate NK cytolytic responses (49). BAT3/ BAG6 is predominantly expressed in the nucleus, but can move to the plasma membrane in cells exposed to heat shock and can be secreted in exosomes by tumors and stressed cells (49, 82). BAT3/BAG6-expressing exosomes can stimulate cytokine release from NK cells upon interaction with NKp30, and BAT3/BAG6 expression by DC is responsible for activation NK cells to mediate the crosstalk with DC (49, 50). Similarly, RIG-I stimulation of melanoma cell lines was shown to trigger the extracellular release of BAT3/BAG6-containing vesicles that can stimulate NK cell cytolytic responses (51). In contrast, a soluble form of BAT3/ BAG6 has been found at high levels in the plasma of CLL patients and can suppress NK cytolytic responses, apparently by blocking recognition of this and other ligands on tumor cells (82, 83).

It was also reported that NKp44 can interact with PCNA, at target cell surfaces (42). PCNA is highly expressed in proliferating cancer cells, where it is usually tightly associated with DNA and involved in DNA replication and repair mechanisms (84). Surprisingly, PCNA was found to migrate to the plasma membrane of target cells within the immunological synapse with NKp44-expressing NK cells, and this interaction inhibited cytolytic function and IFN-γ production by the NK cells (42). A second report has also described the interaction of PCNA and NKp44 and the association of PCNA with MHC-I molecules at the plasma membrane of tumor cells as a potential surface transport mechanism (64). The PCNA-induced inhibition was found to be mediated through the ITIM-like sequence in the cytoplasmic domain of NKp44 (42), despite earlier work in which the ITIM-containing NKp44 cytoplasmic domain was shown to lack inhibitory function in the context of a chimeric receptor construct (40). It appears that PCNA interaction with the full NKp44 receptor establishes a unique conformation that transduces an inhibitory signal. Inhibitory function was also previously reported for NKp44 expressed on ILCs and plasmacytoid DCs (33, 85).

#### B7-H6

NKp30 has been found to bind to a cell surface protein member of the B7 family, named B7-H6 (52). B7-H6 is not normally expressed on healthy cells, but can be upregulated on human tumor cells through a Myc-mediated mechanism (86) or upon stimulation of monocytes and neutrophils with TLR ligands or pro-inflammatory cytokines (87). Upon recognition by NKp30, B7-H6 triggers cytotoxicity and cytokine production by NK cells (52). The interaction of B7-H6 with NKp30 is the most rigorously characterized of NCR ligands, since it is the only NCR–ligand interaction so far confirmed in an X-ray crystallography structure (53). It has also been shown that some tumors can escape NKp30 recognition by shedding B7-H6 from their surfaces with the metalloproteases, ADAM-10, and ADAM-17 (88). Soluble and tumor-associated expression of B7-H6 in the peritoneum of ovarian cancer patients has also been shown to correlate with reduced surface expression of NKp30 on peritoneal NK cells, presumably due to chronic interaction with ligand (89).

## SPLICE VARIANTS OF NCRs RESULTING IN DISTINCT RECEPTOR ISOFORMS

A variety of mRNA splice variants encoding different isoforms of NCRs have been recognized for many years, but only recent work has established that some of these variant NCR isoforms can facilitate inhibitory functions. Distinct splice variant expression patterns have also been shown to correlate with outcomes in cancer and infectious disease, suggesting potential prognostic value in patients. A summary diagram of current reported functions of distinct isoforms of NKp30 and NKp44 is presented in **Figure 2**.

# Splice Variants of *ncr3*

The expression of distinct isoforms of NKp30 is of great interest, because this NCR is involved in DC-to-NK cell crosstalk (25), can facilitate tumor cell recognition (13, 49, 90), and can influence the prognosis of different infectious diseases (91). Six splice variant transcripts have been identified from the *ncr3* gene, which was originally called 1C7 (92, 93). The most highly expressed *ncr3* variants are designated a, b, and c that encode NKp30 proteins with an extracellular V-type Ig domain, while d, e, and f isoforms encode NKp30 receptors possessing a C2-type Ig domain that lacks 25 amino acids (93). The three members of each subgroup share three distinct cytoplasmic domains encoded by splice variations within exon 4. Although the d, e, and f protein isoforms of NKp30 have not been studied to date, several groups have examined the functions and interesting clinical outcomes associated with differential expression of the a, b, and c isoforms, as discussed below.

Alternative splicing of the *ncr3* gene impacts functions of NKp30 isoforms that can be immunosuppressive or immunostimulatory. Delahaye and colleagues expressed isoforms NKp30a, NKp30b, and NKp30c in the human NK cell line NKL to characterize their functions. It was shown that antibody- or B7-H6-mediated engagement of NKp30 on NKL transfected with either NKp30a or NKp30b isoforms stimulated production of large amounts of IFN-γ, degranulation, and cytotoxicity responses. In contrast, engagement of NKp30c on NKL cell transfectants did not result in degranulation or elicit cytotoxicity, but instead produced the inhibitory cytokine IL-10 and very little IFN-γ (94). Similar immunostimulatory functions for NKp30a and NKp30b isoforms were observed when NKp30 transfectants were cocultured with DCs, whereas NKL-NKp30c transfectants demonstrated minimal responsiveness in these assays. In all of these experiments, NKp30a stimulated the most potent activating responses, whereas NKp30c was inhibitory or non-responsive, and NKp30b induced intermediate activation (94).

It was also shown that NKp30a associated more tightly with TCR-ζ upon crosslinking, as compared to NKp30c (94). Surprisingly, p38 MAP kinase activation was more pronounced when NKp30 was engaged in NKL-NKp30c cells then for NKL cells transfected with NKp30a or NKp30b. Furthermore, treatment of NKL-NKp30c cells with a p38 inhibitor produced IFN-γ upon exposure to immature DCs (94). Therefore, NKp30b and especially NKp30a are stimulatory isoforms that can induce cytotoxicity and cytokine production, whereas NKp30c induces an immunosuppressive response that appears to involve activation of p38 and the production of IL-10.

#### NKp30 Isoforms and Cancer

In addition to characterizing different functions for distinct NKp30 isoforms, Delahaye performed a retrospective analysis of NKp30 expression profiles in 80 patients with gastrointestinal stromal tumors (GIST), which is a malignancy that expresses NKp30 ligands. In that analysis, predominant expression of the immunosuppressive NKp30c isoform over the immunostimulatory NKp30a/b isoforms was found to be associated with reduced overall survival in imatinib-treated patients (94). Moreover, a subset of GIST patients with predominant expression of the NKp30c isoform and a distinct haplotype involving two SNPs in the *ncr3* gene were found to be associated with particularly poor survival (94). For GI carcinomas and variety of other cancers, we observed that both cancerous and matched normal tissues manifested balanced NKp30c inhibitory and NKp30a/b activation profiles; yet, we found skewed NKp30 splice variant profiles in about 50% of a variety of tumor tissues compared to their matched normal tissues (95).

Neuroblastoma is another malignancy where NCRs are involved in the tumor cell recognition (96–98). Semeraro et al., found that neuroblastoma tumor samples express the NKp30 ligand, B7-H6, and some patients with metastatic neuroblastoma had high levels of soluble B7-H6 in their serum, which was associated with reduced expression of NKp30 on the surface of NK cells and higher degree of metastases (99). Furthermore, serum from patients with high soluble B7-H6 suppressed NK cell IFN-γ responses (99). Analysis of NKp30 isoform expression

signaling through DAP12 (pathway "B" for NKp44-1) that triggers cytotoxicity and IFN-γ production.

suggested lower expression of NKp30a/b isoforms in patients with metastatic neuroblastoma compared to patients with localized disease or healthy donors. Comparison of the ratios of expression of mRNA for individual NKp30 isoforms revealed that high expression of the suppressive NKp30c isoform over the activating NKp30b isoform was associated with shorter progression-free survival of patients with metastatic disease (99). Surprisingly, IFN-γ was found to suppress the expression of *B7-H6* and *BAT3* mRNA, whereas IL-10 increased *B7-H6* mRNA expression on neuroblastoma tumors. The results of this study suggest that high expression of IL-10 by NKp30c-expressing NK cells induces B7-H6 expression on tumor, thereby potentiating further expression of this immunosuppressive cytokine.

The potential prognostic value of NKp30 isoforms was also recently investigated in melanoma patients (100). The overall mRNA expression levels of each of the NKp30 isoforms were found to be reduced compared to expression in healthy donors, but expression ratios between the isoforms did not differ. Levels of mRNA expression of the immunosuppressive NKp30c isoform were found to be higher on NK cells from stage IV melanoma patients, but relative expression levels of each NKp30 isoform did not predict overall survival of patients. Interestingly, long-term surviving melanoma patients were found to express higher levels of stimulatory NKp30a transcript. In accordance with this finding, the long-term survivors were more likely to have SNPs associated with reduced expression of suppressive NKp30c and increased expression of NKp30a. Furthermore, NK cells from long-term surviving patients exhibited increased degranulation potential in response to NKp30 stimulation (100). Therefore, higher expression of NKp30a in melanoma patients appears to be beneficial.

#### NKp30 Isoforms and Infectious Diseases

Surface expression NKp30 on NK cells was found to be downregulated in HIV-1 patients, but expression patterns of NKp30 isoforms do not affect disease progression or survival (101). NK cells from hepatitis C virus (HCV)-infected patients also showed reduced expression of NKp30 on the surface of NK cells, and expression of the immunosuppressive NKp30c transcript was found to be significantly decreased in infected patients compare to healthy controls (102). Also NKp30a/NKp30c ratio was significantly higher compared to healthy individuals suggesting an immunostimulatory profile in infected patients. If stratified according to mRNA expression levels, patients with low expression of all three isoforms had lower surface expression of NKp30 on NK cells, but higher ratio of NKp30a/NKp30c isoform transcripts. Accordingly, NK cells from these patients exhibited more potent degranulation and cytokine production responses upon engagement of the receptor, as compared to healthy controls (102). Interestingly, positive correlations were observed between NKp30a isoform mRNA levels and liver stiffness and between NKp30a/NKp30c ratio and a measure of liver fibrosis, suggesting that reduction of immunosuppressive NKp30c isoform and increase of immunostimulatory NKp30a isoform is associated with advanced liver disease in HCV-infected patients.

#### NKp30 in the Decidua of Pregnant Women

Natural killer cells from decidual tissue in pregnant uterus (dNK) have unique phenotypical and functional properties compared to NK cells found in peripheral blood (pNK) (103). Mouse studies originally demonstrated that dNK cells function to support embryo implantation by secretion of numerous factors, including IFN-γ, to promote angiogenesis and trophoblast invasion (104). Comparison of freshly isolated pNK and dNK cells from the same pregnant donors showed that pNK cells express high levels of NKp30a and NKp30b isoforms and low levels of NKp30c isoform, while dNK cells had high levels of NKp30c but significantly lower amounts of NKp30a/b (105). While crosslinking NKp30 on IL-15-stimulated pNK cells induced degranulation, freshly isolated dNK cells did not degranulate, and co-crosslinking NKp30 in dNK cells did not inhibit NKp46-mediated degranulation, consistent with the lack of activation by NKp30c in previously described studies by Delahaye et al. (94, 105). Coculturing pNK cells in presence of IL-15 and IL-18 induced increased expression of NKp30a/b isoforms, whereas further addition of TGF-β suppressed the induction of expression of all three isoforms, although expression of NKp30b and NKp30c isoforms was greater than NKp30a upon addition of TGF-β (105). Overall, the combination of IL-15, IL-18, and TGF-β, which are found together in the decidual stromal microenvironment, shifted pNK cells toward higher expression of inhibitory NKp30c isoform and expression of other markers characteristic of dNK cells (105).

Shemesh et al. found significantly increased mRNA encoding the activating NKp30a/b isoforms in the placenta of women who had experienced sporadic or recurrent miscarriage within the first trimester, whereas this shift toward activating isoforms was not evident in the peripheral blood (106). The increase in activating isoforms did not correlate with higher expression of TNF-α, IFN-γ, IL-10, and placental growth factor mRNAs in the placental tissue of those women who experienced sporadic miscarriage, as compared to those that had undergone elective abortions. These results suggest that increased expression of these activating isoforms of NKp30 on dNK cells may be in some way contributing to failed pregnancies through promoting dysregulated cytokine production in the placenta.

#### Splice Variants of *ncr2*

Three major mRNA splice variants of *ncr2* have been recognized, and one of these (NKp44-1) encodes the classic receptor that possesses a cytoplasmic ITIM, while the others (NKp44-2 and NKp44-3) have alternative sequences in the cytoplasmic region that lack ITIMs. It has long been known that IL-2-cultured NK cells upregulate expression of an activating form of NKp44-1 associated with the transmembrane signaling protein, DAP12, which becomes tyrosine phosphorylated upon receptor engagement with an antibody (11, 40, 41). Recent analysis of the major NKp44 isoforms, however, has demonstrated that isolated human NK cells cultured in IL-2- or IL-15 express predominantly NKp44-1 mRNA and have reduced capacity to kill PCNA-transfected target cells in an NKp44-dependent manner (12, 107). Furthermore, NK-92 cells transduced to overexpress NKp44-1 show suppressed cytotoxicity and diminished immune synapse formation toward PCNA-transfected target cells, as compared to NK-92 transduced to overexpress the other isotypes. These results indicate that the NKp44-1 isoform is an inhibitory receptor when engaged with the PCNA ligand expressed by target cells, whereas NKp44-2 and NKp44-3 do not transduce inhibitory signals (107).

#### NKp44 Isoforms in Cancer

Shemesh et al. studied the impact of NKp44 isoforms on overall survival of patients with acute myeloid leukemia (AML) using TCGA RNA-Seq data (107). Whereas no survival advantage was found in newly diagnosed patients who expressed mRNA encoding NKp44, as compared to those lacking expression, survival was significantly diminished in patients who exclusively expressed NKp44-1, as compared to patients who also expressed at least some detectable level of NKp44-2 and/or NKp44-3 or lacked NKp44 expression altogether. These results imply that NKp44 plays a key role in NK cell responsiveness toward AML tumors, but exclusive expression of the ITIM-containing NKp44-1 isoform can stifle these beneficial responses, presumably through inhibitory signaling, thereby resulting in poor patient outcome.

The same group found higher incidence of NKp44 mRNA in various solid tumor tissues, as compared to surrounding normal tissues (95). Those tumor samples that expressed NKp44 mRNA were found to consist of predominantly NKp44-1 isoform. Thus, NK cells in the tumor microenvironment predominantly express the inhibitory NKp44 isoform.

# NKp44 in the Decidua of Pregnant Women

Siewiera et al. found that while NKp44-2 mRNA is expressed more by freshly isolated pNK cells than NKp44-1 and NKp44- 3, dNK cells from women undergoing elective first trimester abortions (healthy pregnancies) express all three isoforms of NKp44 at similar levels (105). While crosslinking NKp44 on IL-15-stimulated pNK cells induces degranulation, crosslinking on freshly isolated dNK cells did not result in degranulation and co-crosslinking suppressed degranulation in response to crosslinking NKp46, demonstrating predominant inhibitory function for NKp44 in decidual/placental NK cells (105).

Shemesh et al. also found that NKp44-2 and NKp44-3 isoforms predominated in decidual tissue obtained from the majority of first trimester spontaneous abortions, while a NKp44- 1-dominant (inhibitory) profile was found in dNK cells from most elective abortions or term deliveries (healthy pregnancies), which is consistent with the inhibitory function of dNK cells in the study by Siewiera et al. (95).

#### Splice Variants of *ncr1*

Recent work by Shemer-Avni et al. has also provided new insights on differential splicing of *ncr1* (108). Five major splice variants of *ncr1* have been described, and three of these encode NKp46 protein isoforms containing both extracellular domains, while two lack the first Ig-like domain (D1, encoded by exon 3). Surprisingly, NK-92 cells transduced with a D1-negative *ncr1* cDNA degranulated significantly more efficiently toward HEK293T target cells, as compared to NK-92 cells transduced to express conventional NKp46 protein containing both Ig-like domains (108). Using NKp46-reactive antibodies, D1-negative NKp46 was not observed in fresh peripheral blood from healthy donors, but a subset expressing the NKp46 D1-negative receptor was found after long-time culturing of NK cells in IL-2 (108). This subset of NK cells containing D1-negative NKp46 degranulated more robustly in response to a combination of plate-bound anti-NKp46 and anti-NKp30 antibodies (108).

This group also studied NKp46 isoform expression in upper airway lavage samples from pediatric patients with respiratory tract viral infections. While most of these samples expressed both NKp30 and NKp46 mRNA, none contained mRNA encoding NKp44. When *ncr1* splice variants were analyzed, most of the lavage samples from virus-infected patients were found to contain D1-negative NKp46 isoform transcripts (108). Taken together, these results suggest that NK cells by IL-2 or viral infection can express isoforms of NKp46 lacking the D1 Ig-like domain, and NK cells expressing these domain-deficient receptors exhibit increased functional capacity.

# CONCLUSION

Significant progress has been made in recent years to improve our understanding of the functions and ligand recognition capacities of NCRs. Clearly these receptors play important roles in NK cell recognition of tissue changes in cancer, viral infections, decidual tissues in pregnancy, and immature DC. This new knowledge is crucial for establishing the basis of molecular mechanisms controlling NK cell responses under these diverse conditions. In

#### REFERENCES

contrast to the original dogma that NCRs are exclusively activating receptors, several new findings have revealed inhibitory functions for these receptors.

Numerous ligands or co-ligands for NCRs have now been described. Several of these can surprisingly trigger inhibitory signals or induce production of inhibitory cytokines when engaged with NCRs. Furthermore, some of these putative ligands are classically nuclear or cytosolic proteins that appear to relocate to the cell surface in cancer cells, where they can engage with NCRs. Also, heparan sulfates seem to have capacity to interact with all three of the NCRs and may regulate their functions in trans and cis.