Treg and suppressive cells (e.g., myeloid-derived suppressive cells) play crucial roles in establishing and maintaining peripheral tolerance and are known to be important for reducing GVHD (105–108). Waller and colleagues have demonstrated that transfer of donor BM pre-pDCs attenuated GVHD in mice (56). They identified that donor pDCs activated donor T cells to produce IFN-γ, which then enhanced pDC synthesis of indoleamine 2,3-dioxygenase (IDO). Increased production of IDO by pDCs altered the balance between donor Treg and alloreactive effector T cells, thereby limiting the severity of GVHD (56). Other studies showed that GVH reaction also impaired the antigen presentation function of de novo generated donor cDCs, leading to dramatically decreased Treg expansion and function, leading to severe chronic GVHD (109). These observations suggest that reconstitution of tolerogenic DCs from engrafted donor hematopoietic cells may be crucial for preventing the occurrence of severe GVHD.

#### RECONSTITUTION OF DONOR DCS AFTER ALLO-HSCT

Clinical studies indicated that impaired reconstitution of donor DCs correlates with the occurrence of severe GVHD (110–115). Wingard and colleagues examined the number of donor DCs in the circulating peripheral blood from a group of 50 allo-HSCT patients. They found that low number of circulating DCs was not only associated with significantly increased risk of relapse and acute GVHD, but also predicative of patient death after allogeneic HSCT (110). Notably, clinical studies from 39 children with allo-HSCT indicated that while normal cDC numbers were observed by 300–400 days after transplantation, pDC numbers were always lower than those of age-matched control patients during the entire follow-up period of up to 7 years (112). In contrast, patients with high pDC recovery profile often had improved overall survival (114). Data from preclinical studies also showed a marked deficit in all lineages of DCs (CD8+ DCs, CD11b+ DCs, and pDCs) in GVHD compared with non-GVHD mice (55, 109, 116, 117). Thus, the recovery of DCs from engrafted HSPCs in allo-HSCT patients may predict the occurrence of severe GVHD and non-relapse mortality.

#### Donor DCs Mediate Protective Immunity

DCs are critical for eliciting T cell immune responses, protecting the host against viral infection (18, 19, 118). Viral infection remains a major challenge for the success of allo-HSCT. Clinical studies have shown that after allo-HSCT, patients with lower numbers of circulating peripheral blood DCs often have increased risk of infections (1, 110). Cytomegalovirus (CMV) is a major cause of post-transplant mortality in patients subject to allo-HSCT, with ∼25% of CMV-seropositive recipients developing CMV-related disease within 3 months after transplantation (119–121). It is well-established that induction of adaptive T cell immunity is critical to control CMV replication and resolve viral reactivation-mediated disease (100, 122, 123). cDCs are essential to the generation of effector T cells reactive to CMV, especially during a primary response. However, GVHD induces a profound DC defect that leads to a failure in the generation of CMV-specific CD8+ T-cells and dramatically decreased antiviral immunity (116). Collectively, improving the reconstitution of DCs following allo-HSCT may represent an effective strategy to re-establish the protective immunity in the recipient. Since pDCs produce high levels of IL-12 and IFN-α upon activation (29, 124–126), improving pDC recovery after allo-HSCT may also provide efficient antiviral immunity.

### GVHD Impairs the Generation of DC Progenitors

GVHD-associated inflammatory responses may influence the reconstitution of donor DCs via a complex mechanism. DCs developed from HSPCs through successive steps of lineage commitment and differentiation: HSCs → multiple potent progenitors (MPP) → common DC progenitors (CDP) → cDCs and pDCs (22, 42, 125, 127–132). Inflammatory cytokines, such as TNF-α and IFN-γ, directly inhibit the proliferation of HSPCs and their generation of DCs (117). In addition, Matsushima and colleagues have shown that GVHD induced damage to the BM niche, leading to dramatically decreased hematopoiesis, including the reduction of CDP (133). However, the specific cellular component(s) within the niche that are responsible for the generation of DC progenitors have yet to be determined.

#### Transcriptional Regulation of DC Development

The generation of DCs is regulated by a group of functionally distinctive transcription factors (TFs). Analysis of gene-targeted mice has identified many critical TFs in DC development. Some of these TFs, such as Pu.1 and Stat3, influence the generation of all DC subsets. HSPCs lacking Pu.1 showed defective DC differentiation potential (134, 135). Targeted deletion of Stat3 impaired the generation of both cDCs and pDCs in vivo (128, 136). Thus, both Pu.1 and Stat3 are pioneer TFs in the regulation of DC commitment and differentiation from MPP (42, 128, 130).

DC subset-specifying TFs are required for committed CDP to become functionally distinct DC lineages (42, 127, 128, 130). For example, Batf3 has a non-redundant role in CD103+ cDC development and partial effect on inducing CD8α + DCs in lymph organs (52, 92, 127). Irf8-deficient animals lack spleenresident CD8α + cDCs and nonlymphoid tissue CD103+ cDCs (42, 127, 137). Other TFs, such as Irf4, Klf4, Notch2, and Relb, also play important roles in the regulation of other types of cDCs localized in different tissues (94, 138). Among them, Irf4 is required for cDCs to prime CD4+ T cells and promote Th17 differentiation in both the lung and intestine (60, 139). In addition, several TFs, such as Tcf4, Irf8, and Spib, are known to regulate pDC differentiation (140–142). The absence of Tcf4 leads to the loss of pDCs in mice (142).

There are limited number of studies investigating how GVH reactions influence the expression and function of these TFs required for DC development. Notably, a recent study revealed that inflammation cascades in GVH reaction favor the development of CD103+D11b− DCs in the GI tract (42), which require the presence of functional Irf4 (93). These data indicate that distinct TFs in DCs and their progenitors may have different susceptibility to the regulatory effect of inflammatory environments. This may result in a skewed expression and activation of transcriptional programs, promoting the generation of specific subset(s) of DCs and feed-forward action on alloreactive T cell responses during the GVHD progression. Delineating the mechanisms underlying this dysregulated donor DC reconstitution during GVH reaction will be important for understanding the pathophysiology of GVHD and the development of effective treatments for the disease.

#### DC MODULATION OF ALLOIMMUNITY

Manipulation of DC precursors in the HSPC graft may facilitate the establishment of a balance between GVHD and GVL effects (2, 9, 16, 34, 35, 56). Preclinical studies have shown that transfer of donor pre-pDCs derived from donor mice treated with Flt3L induced markedly augmented GVL activity of donor T cells without aggravating GVHD (56). These donor prepDCs persisted long in that they expanded in vivo for 2 weeks after transplantation (56). These findings perfectly supported the clinical value of donor DCs in modulating alloimmunity to improve the efficacy and safety of allo-HSCT.

#### Use of Tolerogenic DCs to Reduce GVHD

The capacity of DCs to induce tolerance has led to numerous therapeutic studies using these cells in an effort to control harmful immune responses in models of allograft rejection, GVHD and autoimmune disorders (18, 27, 34, 36, 37, 62, 87, 143). While transfer of immature CCR9+ pDCs reduced GVHD, transfer of mature donor pDCs did not as expected (144). Furthermore, transfer of highly purified immature pDCs derived from donors was technically challenging and typically required in vivo expansion step to generate the number enough for modulating GVH reaction in vivo (53, 56, 144).

With this technical bottle neck, many studies had to assess the therapeutic effect of ex-vivo-generated DCs. Tolerogenic DCs were tried to be generated through propagating human monocytes in vitro in the presence of various agents, such as IL-10, Vitamin D3, and immunosuppressive drugs (e.g., dexamethasone and rapamycin) (34, 36, 37, 87, 143). Yet, none of the approaches generated the best clinically applicable DCs with the expected tolerogenic capacity to modulate alloreactive T cells (34, 36, 37, 87, 143). Reddy and colleagues report that upon pre-treatment with the HDAC inhibitor SAHA, these moDCs produced high levels of IDO and promoted Treg expansion and function in vivo, leading to attenuated GVHD in mice (54). These findings indicate that targeting epigenetic regulators in DCs may prove to be an effective strategy to induce the generation of DCs with tolerogenic properties for reducing GVHD.

# DC Induction of GVL Effects After Allo-HSCT

Emerging evidence indicated that DCs were required for optimal GVL response without aggravating GVHD. Reddy and colleagues report that as compared to allogeneic wild-type (WT) hosts, allogeneic Batf3-deficient recipient mice developed severe GVHD but with significantly reduced GVL response (52). This indicates the importance of CD8α + DCs in GVL response. Indeed, co-transfer of WT host-type spleen DCs (which contain CD8α + DCs) and T cells into allogeneic B2m−/− recipients, which are functionally deficient in antigen presentation, induced a significant CD8+ T cell-mediated GVL response, leading to prolonged survival of recipients without tumor. In contrast, all of the B2m−/− mice receiving Batf3−/− spleen DCs, which lack CD8α + DCs (92), died from tumor despite the presence of other DC subsets (52). This confirms the crucial role of CD8α + DCs in eliciting anti-tumor immunity. However, these experiments did not examine the direct effect of CD8α + DCs on T cell-mediated GVL response.

In human recipients of unrelated donor BM grafts, but not granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood grafts, a higher number of donor pDCs is associated with increased survival and reduced GVHD (145). Data from experimental studies indicate that transfer of pDCenriched BM grafts preserved GVL effects without aggravating GVHD in mice (56, 146). pDCs produce high levels of IFN-α and IL-12 (147, 148), cytokines important to promote differentiation and expansion of antigen-specific effector cells (149). In these studies, Waller and colleagues have demonstrated that in vivo administration of Flt3L to donor mice induced 5-fold increase in pDC content without significant changes in the number of HSCs, T cells and B cells. Most importantly, transfer of pDCenriched BM graft from Flt3L-treated donors decreased GVHD while retaining GVL effects in allogeneic recipient mice (146).

We have recently established a novel platform to produce Dll4+ DCs from murine BM using Flt3L and TLR agonists (64). Upon allogeneic Dll4+ DC stimulation, CD4+ naïve T cells underwent effector differentiation and produced high levels of IFN-γ and IL-17 in vitro, depending on Dll4 activation of Notch signaling. Adoptive transfer of these Dll4+ DC-induced T cells eliminated leukemic cells without causing severe GVHD, leading to significantly improved survival of leukemic mice undergoing allo-HSCT. This strategy may potentially improve the antileukemic response after HSCT and overcome some barriers to the GVL response such as high disease burden and pharmacologic immunosuppression (64). Since DC activation of naïve T cells allows them to be primed with antigens, Dll4+ DCs loaded with leukemia-associated antigens may facilitate the selective expansion of leukemic cell-reactive T cells and specifically boost the anti-leukemia activity.

# CONCLUSION

While traditional therapies have been targeting T cells, extensive research in murine HSCT models convincingly showed the ability of DCs to preserve GVL response without aggravating GVHD. Targeting donor DCs in vivo or ex vivo may potentially subvert alloreactive T cell responses and reduce GVHD (53). Given the role of DCs in maintaining Treg after allo-HSCT (56, 109), cotransfer of tolerogenic DCs and Treg could be more effective on reducing GVH reactions in vivo. A randomized Phase I study has shown the safety of infusing the host tolerogenic DCs into diabetes patients (150). It will be interesting to test whether these ex vivo generated tolerogenic DCs given in the peritransplant period may prevent GVHD while preserving GVL effects.

One major challenge is to produce large number of donor-type tolerogenic DCs that can persist sufficient time to execute their function following adoptive transfer to modulate alloimmunity. We propose that donor-type DCs have several advantages compared to host-type DCs. For example, donors are healthy, and their hematopoietic system is not compromised by accompanied disease state and treatment conditions. Furthermore, available data from both clinical and pre-clinical studies suggest that donor-derived pDCs have potent capability to modulate GVH reactions (55, 56, 145, 146). These

#### REFERENCES

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Most recent studies have shown that the fate of pDCs is determined early at the stage of HSCs (127, 130, 136, 151– 153). This suggests that induction of tolerogenic DCs should start from the HSPC stage. Better understanding how the fate of tolerogenic DCs are determined and regulated may have significant implication in the production of DCs for efficiently modulating alloimmunity.

#### AUTHOR CONTRIBUTIONS

HY, YT, YW, SM, and YZ collected all materials for reviewing and wrote the review.

#### ACKNOWLEDGMENTS

This work was supported by Department of Defense (YZ) and NIH (CA172106-01, YZ, HL127351-01A1, YZ).

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**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 © 2019 Yu, Tian, Wang, Mineishi and Zhang. 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) and the copyright owner(s) 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.

# BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT

Sabrina N. Copsel 1,2 \*, Casey O. Lightbourn1 , Henry Barreras 1 , Ines Lohse2,3 , Dietlinde Wolf 2 , Cameron S. Bader 1 , John Manov 1 , Brandon J. Kale1 , Devangi Shah1 , Shaun P. Brothers 2,3, Victor L. Perez 4 , Krishna V. Komanduri 1,2,5, Claes Wahlestedt 2,3 and Robert B. Levy 1,2,4,5 \*

*1 Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, FL, United States, 2 Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL, United States, 3 Center for Therapeutic Innovation and Department of Psychiatry and Behavior Sciences, Miller School of Medicine, University of Miami, Miami, FL, United States, 4 Department of Ophthalmology, Miller School of Medicine, University of Miami, Miami, FL, United States, 5 Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL, United States*

#### Edited by:

*Xue-Zhong Yu, Medical University of South Carolina, United States*

#### Reviewed by:

*Dennis O. Adeegbe, Moffitt Cancer Center, United States Jessica Voss, Northwestern University, United States*

#### \*Correspondence:

*Sabrina N. Copsel<EMAIL_ADDRESS>Robert B. Levy<EMAIL_ADDRESS>
#### Specialty section:

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

Received: *28 September 2018* Accepted: *17 December 2018* Published: *24 January 2019*

#### Citation:

*Copsel SN, Lightbourn CO, Barreras H, Lohse I, Wolf D, Bader CS, Manov J, Kale BJ, Shah D, Brothers SP, Perez VL, Komanduri KV, Wahlestedt C and Levy RB (2019) BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT. Front. Immunol. 9:3104. doi: 10.3389/fimmu.2018.03104* A recent approach for limiting production of pro-inflammatory cytokines has been to target bromodomain and extra-terminal (BET) proteins. These epigenetic readers of histone acetylation regulate transcription of genes involved in inflammation, cardiovascular disease, and cancer. Development of BET inhibitors (BETi) has generated enormous interest for their therapeutic potential. Because inflammatory signals and donor T cells promote graft-versus-host disease (GVHD), regulating both pathways could be effective to abrogate this disorder. The objective of the present study was to identify a BETi which did not interfere *in vivo* with CD4+FoxP3+ regulatory T cell (Treg) expansion and function to utilize together with Tregs following allogeneic hematopoietic stem cell transplantation (aHSCT) to ameliorate GVHD. We have reported that Tregs can be markedly expanded and selectively activated with increased functional capacity by targeting TNFRSF25 and CD25 with TL1A-Ig and low dose IL-2, respectively. Here, mice were treated over 7 days (TL1A-Ig + IL-2) together with BETi. We found that the BETi EP11313 did not decrease frequency/numbers or phenotype of expanded Tregs as well as effector molecules, such as IL-10 and TGF-β. However, BETi JQ1 interfered with Treg expansion and altered subset distribution and phenotype. Notably, in Treg expanded mice, EP11313 diminished tnfa and ifng but not il-2 RNA levels. Remarkably, Treg pSTAT5 expression was not affected by EP11313 supporting the notion that Treg IL-2 signaling remained intact. MHC-mismatched aHSCT (B6→ BALB/c) was performed using *in vivo* expanded donor Tregs with or without EP11313 short-term treatment in the recipient. Early post-transplant, improvement in the splenic and LN CD4/CD8 ratio along with fewer effector cells and high Treg levels in aHSCT recipients treated with expanded Tregs + EP11313 was detected. Interestingly, this group exhibited a significant diminution of GVHD clinical score with less skin and ocular involvement. Finally, using low numbers of highly purified expanded Tregs, improved clinical GVHD scores were observed in EP11313 treated recipients. In total, we conclude that use of this novel combinatorial

**85**

strategy can suppress pre-clinical GVHD and posit, *in vivo* EP11313 treatment might be useful combined with Treg expansion therapy for treatment of diseases involving inflammatory responses.

Keywords: Tregs, bromodomain inhibitors, epigenetic regulation, GVHD, TNFRSF25, CD25

#### INTRODUCTION

Allogeneic hematopoietic stem cell transplantation (aHSCT) is utilized as a therapeutic modality for a variety of conditions including genetic disorders, immune deficiency syndromes, and hematologic diseases and malignancies. However, the limiting factor for successful aHSCT is the development of graft-versushost disease (GVHD). In fact, as many as half of the ∼8,000 aHSCTs performed in the U.S. each year will result in GVHD and therefore new strategies to ameliorate GVHD are needed. GVHD occurs when donor T cells are primed by recipient antigens subsequently eliciting an inflammatory response against the host (1). Acute GVHD is a multi-organ disorder resulting from inflammatory cytokines and donor T cells which primarily damage skin, liver, gastrointestinal tract, and the eye surface (2).

Because GVHD is promoted by donor T cells and inflammatory cytokines, we reason regulating both is the most rational strategy to abrogate this complication. Our lab and others have demonstrated that transfer of CD4+FoxP3+ regulatory T cells (Tregs) is a promising therapy to suppress donor T cells and inhibit GVHD (3–6). Our prior work identified a two-pathway in vivo strategy targeting TNFRSF25 and CD25 receptors which elicits a rapid and strong increase in Treg numbers and function (7). In fact, very low numbers of these in vivo expanded donor Treg cells demonstrated effective GVHD suppression in recipients following aHSCT (8). Recently, the targeting of bromodomain and extra-terminal (BET) proteins has provided a new strategy for reducing pro-inflammatory cytokine production (9). These readers of histone acetyled lysine residues are involved in transcriptional regulation of many genes involved in human diseases including inflammation, cancer and cardiovascular diseases (10, 11). Recent development of BET inhibitors (BETi) has generated enormous interest for their therapeutic potential (12–14). The BETi I-BET762 and JQ1 showed anti-inflammatory properties by disrupting the expression of pro-inflammatory cytokines (e.g., IL-1β, IL-6, and IL-12) in macrophages and suppressing genes involved in T cell-mediated pro-inflammatory functions (13, 15, 16). A prior study reported that BETi I-BET151 interfered with NF-κb function and diminished cytokine expression in dendritic cells and T cells, altered APC function and decreased experimental GVHD (17). Based on our previous work illustrating the effectiveness of expanded Tregs in ameliorating GVHD, we wanted to ask if BETi could be combined with this cell therapy to augment outcomes of aHSCT. Small biomolecule inhibition of CBP/EP300 bromodomains resulted in diminishment of Treg frequency and differentiation (18). It is notable that STAT5 activation is required for Treg proliferation and function (19, 20). Importantly, although JQ1 was shown to reduce STAT5 function in hematologic cancers and dendritic cells, there is no information regarding this or other BETi effects on (1) the IL-2 signaling pathway via STAT5 in Tregs as well as (2) IL-2 production which is required for Treg survival and their maintenance of suppressive function (21, 22).

The present studies examined if BETi could be combined with Treg cell therapy without interfering with Treg expansion, phenotype and function. We found that the BETi EP11313 did not decrease Treg numbers in treated mice and in Treg expanded mice, EP11313 diminished tnfa and ifng but not il-2 levels in non-Treg cells. Notably, Treg pSTAT5 expression was not affected by EP11313 supporting the notion that Treg IL-2 signaling remained intact. In the presence of this BETi, no alterations in Treg subsets or phenotype markers as well as effector molecules, such as IL-10 and TGF-β were observed. MHC-mismatched aHSCT (donor B6-BALB/c recipient) was performed using in vivo expanded donor Tregs with or without EP11313 treatment in the recipient. One week post-transplant we observed significant improvement in the splenic and LN CD4/CD8 ratio along with fewer effector cells and high Treg levels in HSCT recipients treated with expanded Tregs + EP11313. Remarkably, this group exhibited diminished acute GVHD. Finally, using low numbers of highly purified expanded Tregs, we found improved clinical GVHD scores in recipients treated with EP11313. We conclude in vivo treatment with selective BETi can be successfully combined with Treg expansion therapy for treatment of diseases involving inflammatory responses.

#### MATERIALS AND METHODS

#### Mice and Reagents

The FoxP3 reporter mice on a C57BL/6 background (B6- FoxP3RFP) (originally provided by R. Flavell, Yale University, New Haven, CT) (23) and B6-CD45.1 (H2b ) mice were bred in our facility. Wild-type BALB/c (H2d ) mice were purchased from Taconic (Rensselaer, NY). Mice were used at 6–12 weeks of age and were maintained in pathogen-free conditions at the University of Miami animal facilities. All animal use procedures were approved by the University of Miami Institutional Animal Care and Use Committee. EP11313 (provided by Neomed, Canada) and JQ1 (kindly provided by Dr. James Bradner) were reconstituted in DMSO and further diluted in Tween 80 and saline. The A20luc/YFP cell line (derived from BALB/c mice) was a generous gift of Dr. Robert Negrin (Stanford University).

#### Antibodies, Reagents, Flow Cytometry, and Cell Sorting

Commercial antibodies for use in flow cytometry were purchased from BD Biosciences (San Jose, CA), Biolegend (San Diego, CA), or eBioscience (Waltham, MA). Recombinant mouse IL-2 and α-IL-2 monoclonal antibody, clone JES6-5H4, were purchased from eBioscience. IL-2/αIL-2 complex was generated by incubating 1.5/7.5 µg recombinant mouse IL-2 with 8µg JES6-5H4 (∼8,000 IU/injection) for 15 min at room temperature and administered i.p. TL1A-Ig was generated in our laboratory as described previously (24) and administered i.p. at 50 µg/mouse/injection. Single-cell suspensions were prepared from different organs (spleen and lymph nodes). Peripheral blood was collected in heparinized tubes. Peripheral blood mononuclear cells were isolated by standard Ficoll density gradient centrifugation. Next, 106 cells were preblocked with anti-mouse CD16/CD32 and stained with different antibody combinations. Intracellular staining was performed according to standard procedures. The following mAbs to the indicated molecules, the fluorescent labels, and their sources were used in this study: CD4, CD8, CD19, CD25, CD44, CD62L, CD103, KLRG1, CD39, CD73, I-COS, Nrp-1, PD-1, CTLA-4, CCR8, Ly-6C, Ki-67, pSTAT5, and Annexin V (**Supplementary Materials and Methods, Table S1**). Flow cytometric analysis was performed on a BD LSR-Fortessa-HTS instrument (BD Biosciences, San Jose, CA) and the analysis was completed using FlowJo software (FlowJo, LLC, Ashland, OR). Splenic and pLN CD4−FoxP3−, CD4+FoxP3−, and CD4+FoxP3+ cells were sorted using a FACS Aria II cell sorter (BD Biosciences) after enrichment of T cells (surface immunoglobulin depletion of B cells).

#### RNA Isolation, RT-PCR, and Quantitative Real-Time PCR

Total RNA was isolated from unexpanded and expanded Tregs using RNAeasy mini kit following the manufacturer's instructions (Qiagen, Germantown, MD). cDNA was retrotranscribed from 1 µg of total RNA using qScript cDNA Mastermix (Quanta, Beverly, MA). Quantitative real-time PCR was (qPCR) was performed in triplicate using the ABI PRISM 7300 sequence detection system (Applied Biosystems, Whatman, MA) with the specific primers for tnfa, ifng, il-2, il-10, and gapdh (**Supplementary Materials and Methods**, **Table S2**). The PCR mixture contained 7.5 µl of 2X SYBR Green PCR master mix (Applied Biosystems) in a 15 µl final volume. The specificity of each primer set was monitored by analyzing the dissociation curve. The relative mRNA levels of each gene were calculated using the Livak method with GAPDH as the housekeeping gene.

#### Western Blot

Cells (0.5–1.0 × 106 ) were diluted with Laemmli sample buffer and boiled for 5 min. Proteins were separated by electrophoresis on 4–15% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The residual binding sites were blocked with 5% non-fat powdered milk in PBS containing 0.05% Tween 20, and membranes were incubated with anti-TGF-b mAb (0.5 mg/ml; ABCAM, Cambridge, United Kingdom) or anti-actin mAb (0.5 mg/ml; ABCAM) in PBS containing 0.05% Tween 20. All subsequent washes were performed with the same buffer. Reactivity was developed using HRP-coupled secondary polyclonal antibody (1:2,000, Jackson ImmunoResearch) and the SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).

#### In vitro Treg Functional Assays

For a standard suppressor assay, CD4+FoxP3− splenocytes (105 ) were cultured in 96-well plates and activated with 1 µg soluble anti-CD3 (clone 2C11) antibody in the presence of APCs (5 × 104 T cell depleted splenocytes) and titrating numbers of sorted CD4+FoxP3+ Tregs. Cultures were incubated for 72 h and pulsed with [3H]-Thymidine (0.5 µCi/well; Perkin Elmer) for the last 10 h. Incorporated isotope was measured by liquid scintillation counting (Micro Beta TriLux counter; Perkin Elmer). For a functional assay, spleen and LN suspensions were activated in vitro with 1µg/ml soluble anti-CD3. After 72 h, proliferating cells were counted by Trypan blue exclusion using the Vi-cell XR cell counter (Beckman Coulter, Brea, CA).

#### HSCT Experiments

For the HSCT in the major MHC-mismatch model (B6 → BALB/c), female BALB/c mice (H2d ) were ablatively conditioned with 8.5 Gy total body irradiation 1 day before transplantation. BM cells were obtained from femurs, tibias, and vertebrae from sex-matched B6-CD45.1 (H2b ; Thy1.2) donor animals. A singlecell suspension of marrow cells was prepared by flushing bones with a 21-gauge needle and the cells were filtered through a 100µm nylon mesh. Donor marrow cells were depleted of T cells via complement mediated lysis using anti–T-cell–specific antibody HO-13-4 (hybridoma supernatant, mouse anti-Thy1.2 IgM; ATCC, Manassas, VA) generously provided by Dr. Bruce Blazar (University of Minnesota), anti-CD4 mAb (clone 72.4), anti-CD8mAb (clone H02.2), and rabbit complement (Cedarlane Laboratories, Burlington, Ontario, Canada). The marrow cells were incubated at 37◦C for 45 min, washed twice in RPMI, and resuspended for HSCT. Marrow T cell depletion was routinely >99%. Donor T cells were prepared from spleens or LN obtained from C57BL/6-FoxP3RFP-expanded or unexpanded animals. Donor cells were stained for T cells (anti-CD4, clone RM4-5; anti CD8, clone 53-6-7) and adjusted to 1.0 × 106 T cells per mouse before mixing with BM. Recipient mice were injected twice a day with EP11313 10 mg/kg (from day −2 to 4) and underwent transplantation (day 0) with T cell–depleted (TCD) BM (5.5 × 106 ) and 1.0 × 106 T cells i.v. in a 0.2 ml volume via tail vein injection. GVHD was assessed by monitoring recipients for changes in total body weight, clinical signs, and overall survival. The clinical signs of GVHD were recorded for individual mice. Recipients were scored on a scale from 0 to 2 for 5 clinical parameters (25): weight loss, diarrhea, fur texture, posture, and alopecia and for ocular lid score on a scale from 0 to 4 (26).

#### Histologic Analysis

Briefly, tissues from animals 4–5 weeks after aHSCT were fixed in 10% formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin (H&E) and images were acquired using the Keyence BZ-X700 microscope (Itasca, IL). Slides were scored following a modified system described by Kaplan et al. (27). In brief, 3–4 parameters were used to compare pathology scores between groups in the skin and the colon.

#### Cell Survival Assay

Cells were seeded in 384-well micro-titer plates and incubated in a humidified environment at 37◦C and 5% CO2 and cultured for 24 h, followed by incubation with the EP11313, JQ1, iBET762, and iBET151. Individual drugs will be dissolved in 100% DMSO and tested in triplicates starting at a nominal test concentration of 10µM over a 20,000-fold concentration range to generate dose response curves allowing for calculation of half-maximal and maximal response concentrations. Cell viability was measured by bioluminescence after 72 h of drug exposure and dose response curves were generated for each compound.

# Cell Proliferation Assay

In order to monitor cell proliferation in response to treatment with EP11313, cells were seeded in 24-well plates (Corning, NY) at 104 cells per well. Cells were treated with 0, 0.05, 0.1, 0.5, 1, and 1.5µM EP11313 (n = 4 per treatment) and proliferation was evaluated using the Incucyte Zoom instrument (Essenbioscience, MI).

#### Determination of Serum TNF-α Levels

Serum was collected from animals treated in vivo with LPS 1 mg/kg and EP11313 10 or 30 mg/kg. TNF-α concentration was determined by Mouse TNF-α ELISA MAX (Biolegend, San Diego, CA), following manufacturer's protocol. Analysis was performed using Benchmark Plus microplate spectrophotometer at 405 OD.

#### Statistical Analysis

All graphing and statistical analysis were performed using GraphPad Prism (San Diego, CA). Values shown in graphs represent the mean of each group ± SEM. Survival data were analyzed with the Mantel-Cox log-rank test. Nonparametric unpaired two-tailed t-test was used for comparisons between two experimental groups, and multiple variable analysis was performed using ANOVA. A P-value <0.05 was considered significant. Brackets identifying the groups being compared are presented in each figure where appropriate accompanied by the level of significance or absence of significance (ns).

# RESULTS

#### A Bromodomain Inhibitor (BETi)—EP11313 Does Not Interfere With Treg Expansion, Subsets, and in vitro Function

To determine if Treg exposure to BETi in vivo impaired homeostatic proliferation of CD4+FoxP3+ Tregs, C57BL/6-FoxP3RFP (B6-FoxP3RFP) mice were administered EP11313 i.p. 2×/day (13 total injections) (**Figure S1A** in **Supplementary Material**). Spleen and lymph node (LN) cell analysis revealed no differences in the frequency of both the CD4+ compartment and Tregs (**Figures S1B**,**C** in **Supplementary Material**). Following Treg isolation via cell sorting (purity > 98%, data not shown) their in vitro functional activity was also not diminished as evidenced by a standard suppressor assay (**Figure S1D** in **Supplementary Material**). Since we employ expanded and proliferating Tregs for use to regulate GVHD (7, 8), we next addressed whether EP11313 altered expansion, subset distribution and function of Tregs undergoing two-pathway in vivo expansion following treatment with TL1A-Ig and low dose IL-2. Examination of peripheral (spleen, LN) lymphocytes indicated no change in the relative frequencies of CD4 and CD8 T cells in mice receiving two-pathway stimulation alone or together with EP11313 (**Figures 1A,B**, **S1E** in **Supplementary Material**). Importantly, the frequency and numbers of Treg cells within these two groups were also not altered (**Figures 1C,D**, **S1F** in **Supplementary Material**). Notably, following a 3× increase in EP11313 dose administration the percentage of Tregs within the CD4+ compartment was again not affected (**Figure 1E**). Utilizing Ly6C and CD62-L staining (28, 29), Treg subset evaluation demonstrated no change in the central Tregs (Ly6C−, CD62Lhi = cTregs) and effector Tregs (Ly6C−, CD62Llo = eTregs) frequency between animals which had Tregs expanded in the presence or absence of the BETi. We previously found a diminution in the Ly6C+ Treg fraction following two-pathway expansion (8). Here, we also observed the same diminution in this subset in the presence or absence of EP11313 (**Figure 1F**). Lastly, to directly assess the functional activity of the Tregs expanded in the presence and absence of EP11313, spleen and LN cells were removed and immediately stimulated in vitro with anti-CD3mAb. The decrease in proliferation by cells from animals following Treg expansion was not significantly different regardless of whether animals also received BETi treatment (equivalent Treg suppressive activity) (**Figure 1G**).

It is well established that BETi possess anti-tumor activity (14). Therefore, to demonstrate activity of the BETs, we assessed tumor cell viability and proliferation using a mouse lymphoma cell line. As anticipated, each BETi examined decreased tumor cell viability and numbers at varying concentrations (**Figure S2** in **Supplementary Material**). Since BETi are also known to inhibit transcription of inflammatory genes, we examined in vivo activity by the BETi EP11313. Following injection of two doses of this BETi, LPS was administered and TNF-α serum levels assessed after 1.5 h (**Figure S3A** in **Supplementary Material**). There was a clear decrease which was dose related in the serum levels of this inflammatory cytokine (**Figure S3B** in **Supplementary Material**).

#### In vivo Administration of the JQ1 Decreases Treg Proliferation and Alters Their Phenotype During Expansion of CD4+FoxP3+ T Cells

Next, we wanted to address if a prototypic BETi, JQ1 (12) exhibited the same pattern as EP11313 with regard to affecting Treg frequency, proliferation and subset distribution in Tregs undergoing expansion. Groups of B6-FoxP3RFP mice were treated 2×/day (13 total injections, **Figure S1E** in

**Supplementary Material**) with 5 mg/kg of JQ1 or 10 mg/kg of EP11313. In contrast to what we observed with EP11313 treatment, exposure of expanding Tregs to JQ1 resulted in a decrease in splenic and LN Treg frequencies (**Figures 2A,B**). A representative dot plot of Treg subsets showed that the cTregs (Ly6C−, CD62Lhi) fraction was decreased in animals receiving JQ1—but not EP11313—in animals undergoing twopathway Treg expansion (**Figure 2C**). Following Ki67 staining to assess cell proliferation, we observed that in contrast to EP11313, JQ1 treatment deceased the Ki67+ population within splenic and LN Tregs (**Figures 2D,E**). There was no effect of these BETi's on proliferation of conventional CD4+FoxP3− T cells (**Figure S4** in **Supplementary Material**). Lastly, IL-2 induced STAT5 phosphorylation of Tregs in vitro was not diminished in the presence of JQ1 or EP11313 (500 nM) (**Figure 2F**).

To more precisely analyze expanded Treg phenotype in the presence of BETi treatment, activation, function and differentiation markers were assessed using mAbs to defined Treg proteins (**Table S1** in **Supplementary Materials and Methods**). Notably, significantly decreased levels of activation and differentiation molecules, specifically ICOS, CD103, PD-1, CD44, and KLRG1 were identified in splenic Tregs undergoing expansion treated with 10 mg/kg of JQ1 (**Figure 3**). Additionally, the Treg functional suppressive mediators CD39, Nrp-1, and CTLA-4 were also diminished in this treated population. Similar results were obtained analyzing LN Tregs (data not shown). Notably, in contrast to the findings with JQ1, EP11313 treatment did not alter the expression of any of these phenotypic Treg markers (**Figure 3**).

#### EP11313 Regulates Inflammatory Cytokines but Spares the Il-2 Pathway and Effector Molecules in Treg Cells

The above findings demonstrate that EP11313 did not interfere with expansion and phenotype of expanding Treg cells. To examine whether treatment with this BETi altered molecules that mediate Treg function, highly purified CD4+FoxP3+ Tregs were isolated from animals undergoing expansion in the presence or absence of EP11313 treatment (**Figure 4**, **Figure S1E** in **Supplementary Material**). IL-10 RNA and protein levels were not altered in Tregs exposed to this BETi (**Figure 4A**). We also did not detect differences in TGF-β protein levels from sorted Treg populations obtained from the spleen and lymph nodes of treated mice (**Figure 4B**).

Since IL-2 is required for Treg function and survival, we addressed if this cytokine was present in recipients of EP11313 treated mice. Therefore, a Treg negative population (conventional CD4, CD8, NK, macrophage/monocyte and low numbers of contaminating B cells not depleted by sIg treatment) was examined for this cytokine. No differences in the RNA levels of il-2 were identified in these cells from animals undergoing expansion in the presence or absence of this BETi (**Figure 4C**). Since phosphorylation of STAT5 (pSTAT5) is required for IL-2R signaling, levels of this protein were examined within the Treg populations (**Figure 4D**). Importantly, no differences were observed in pSTAT5 expression in Tregs undergoing expansion from BETi treated vs. untreated animals (**Figure 4D**). To validate that EP11313 treatment affected gene transcription in treated animals, RNA from the sorted Treg negative populations was also examined for tnfa and ifng. These inflammatory cytokine RNA were significantly decreased in this population (**Figure 4E**).

#### Recipients Transplanted With Expanded Donor Tregs and Treated With EP11313 Demonstrate Diminished GVHD Early Post-HSCT

The above findings support the notion that selected BETi can be combined with Treg cells to prevent GVHD following allogeneic-HSCT. Accordingly, we performed a transplant using a fully MHC-mismatched aHSCT model (B6→ BALB/c) (**Figure 5A**). Groups of recipients received unfractionated spleen cells (adjusted to contain 1 × 106 T cells) from Treg expanded donors (TrED) or unexpanded donors (TrUD) in the presence or absence of short-term (Day −2 to 4) EP11313 treatment (**Figure 5A**). Using this protocol, treatment with EP11313 only did not diminish GVHD clinical scoring (**Figure 5A**). As we have reported, recipients of TrED did exhibit decreased GVHD clinical scores and increased survival compared to recipients receiving TrUD (8) (**Figures 5B,C**). Interestingly, the combinatorial strategy of TrED plus EP11313 treatment significantly lowered GVHD scores during the first 3 weeks post-HSCT and did not diminish overall (i.e., 100%) survival (**Figures 5B,C**). To obtain sufficient cell numbers, tissues were pooled from animals 1-week post-HSCT. TrED + EP11313 treatment resulted in increased CD4/CD8 ratios in the spleen and lymph nodes at this time (**Figure 5D**). More apparent in the lymph node T cells, there was a diminishment of the CD4 Teff/mem (CD44+CD62Llo) population and an increase in CD4 T naïve (CD44−CD62Lhi) cells from combination (TrED+BETi) treated mice (**Figure 5E**). As anticipated, recipients of TrED had higher levels of Treg cells compared with recipients of TrUD 1-week post-HSCT in the spleen (**Figure 5F**). Consistent with the EP11313 and Treg expansion findings above (**Figures 1**–**4**), mice receiving the combinatorial (TrED + EP11313) strategy contained similar splenic Treg levels compared with recipients of TrED alone (**Figure 5F**). In this context, our recent studies reported that <1.75 × 105 two-pathway expanded donor Tregs were not sufficient to ameliorate GVHD in fully mismatched aHSCT recipients (8). Therefore, transplants were performed using 1 × 105 highly purified donor expanded Tregs (**Figure S5** in **Supplementary Material**) alone or in combination with shortterm EP11313 treatment of recipients (**Figure 5G**). The results of two independent pooled transplants demonstrated a significant decrease (up to 3 weeks post-aHSCT) in the clinical GVHD scores between recipients of purified Tregs alone and those receiving the BETi from Days −2 to 4 TrED (**Figure 5H**). No differences in overall survival between these groups was detected (data not shown).

One and two months post-HSCT, clinical, histological and pathology assessments indicated lower ocular adnexa involvement with less clinical lid edema and closure (**Figure 6A**) and decreased skin involvement as assessed by overall thickening and fibrosis (**Figure 6B**). Thymic weight was superior in TrED + EP11313 vs. TrUD + EP11313 and colon length in the former was virtually identical to control BM alone transplanted recipients and significantly greater compared to recipients of TrED alone (**Figure S6** in **Supplementary Material** and **Figure 6C**, respectively). Moreover, histological assessment of the colon 5 weeks post-aHSCT showed mucosal thickening and severe inflammation with villi distortion in recipients of only 1 × 105 purified expanded Tregs. In contrast, colons from mice receiving 1 × 105 expanded Tregs with EP11313 exhibited a mild inflammation and no disruption of villi architecture (**Figure 6D**).

# DISCUSSION

The bromodomain and extra-terminal (BET) proteins have a central role in regulating transcription of inflammatory and oncogenic factors and have emerged as attractive druggable targets with therapeutic potential (14, 30). Increasing pre-clinical data, completed and ongoing clinical trials (14) (NCT01943851, NCT01587703, NCT01713582) have demonstrated that BET inhibitors (BETi) possess anti-cancer and anti-inflammatory activity (12, 13, 16, 31). The objective of the present study was to identify a BETi which did not interfere in vivo with CD4+FoxP3+

regulatory T cell (Treg) expansion and function so it could be utilized together with Tregs following aHSCT to ameliorate graft-versus-host disease (GVHD). Notably, in vitro analysis of a bromodomain inhibitor of CBP/EP300 reduced human Treg differentiation and suppressive function (18). Interestingly, our studies examining BETi in vivo demonstrated that JQ1 interfered with Treg expansion and altered subset distribution and phenotype. In contrast, we found that the BETi EP11313 did not impair the basal (un-manipulated) or the expanded (TL1A + low dose IL-2) Treg compartments. Remarkably, administration of low levels of EP11313 at the time of allogeneic HSCT together with adoptive transfer of expanded Tregs further diminished GVHD.

Regulatory T cells (Tregs) have a critical role in the immune system by maintaining immune homeostasis and preventing occurrence of autoimmune disease (32–34). IL-2 signaling via the high affinity IL-2R (CD25) results in phosphorylation of STAT5 and is necessary for the maintenance and expansion of CD4+FoxP3+ Tregs (19, 20). Adoptive transfer of regulatory T cells (Tregs) has emerged as a promising therapy for solid organ transplantation, autoimmune diseases and GVHD following aHSCT (3, 4, 6, 35, 36). Our group and others have shown the effectiveness of donor Tregs as a prophylactic strategy to prevent development of GVHD (3–5). We have previously reported that Tregs can be markedly expanded and selectively activated with increased functional capacity by targeting two receptors, i.e., TNFRSF25 and CD25 with TL1A-Ig and low dose IL-2, respectively (7, 8). Additionally, expanded Treg therapy was shown to be as effective as post-transplant cyclophosphamide for GVHD prophylaxis but the former promoted more rapid thymic reconstitution providing earlier recovery of recipient immune function (37). Acute GVHD occurs when donor T cells are primed by recipient antigens subsequently eliciting a rapid inflammatory response ("cytokine storm") in the host.

mice after phorbol 12-myristate 13-acetate (1 ng/mL) + ionomycin (1µM) stimulation for 6 h in the presence of monensin (right) (A). Western Blot analysis of TGF-β levels in sorted Tregs from LN and spleen of mice treated *in vivo* with TL1A-Ig + low dose IL-2 in the presence or absence of EP11313. β-actin was used as a loading control (B). qPCR analysis of il-2 mRNA levels (relative to gapdh) of splenic non-Treg population sorted from unexpanded, expanded or expanded + EP11313 treated mice (C). Representative pSTAT5 staining shown by flow cytometry in CD4+FoxP3+ Tregs from peripheral blood of expanded ± EP11313 treated B6-FoxP3RFP animals 1 h after final IL-2 injection (3 mice/group) (D). qPCR analysis of tnfa (left) and ifng (right) mRNA levels (relative to gapdh) of splenic non-Treg population sorted from expanded or expanded + EP11313 treated mice (E). Data representative of two independent experiments. ns, not significant. \*\**p* < 0.01; \*\*\*\**p* < 0.0001.

Because GVHD is promoted by inflammatory cytokines and donor T cells, we reasoned regulating both components is a rational strategy to abrogate onset of this disorder. Significant numbers of Tregs are required to inhibit alloreative T effector cells which induce GVHD. Accordingly, development of a successful combinatorial approach must include a BETi which does not interfere with Treg function or proliferation. During Treg expansion induced by TL1A-Ig + IL-2 stimulation, JQ1 treatment impaired their peripheral frequency and altered several key Treg differentiation and functional molecules including ICOS-1, Nrp-1, KLRG-1 as well as, PD-1 and CTLA-4 which were also reported to be reduced in Tregs by CBP/EP300 bromodomain inhibitors (18). While additional experiments are needed, based on JQ1's capacity to inhibit c-myc, is possible based on our data thus far, that this BETi is affecting more proliferative Treg subsets, i.e., cTregs rather than more differentiated eTregs.

means ± SEM. \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001.

Notably, JQ1 has also been found to inhibit frequency and function of tumor infiltrating Tregs in non-small cell lung cancer (38, 39). However, a non-structurally related BETi, EP11313 did not exhibit this pattern of alteration as no effect on frequency was observed in either these intentionally expanded—or homeostatic Treg compartments. It should be noted that although the ½ lives of both BETi are not different the results found that even in the presence of higher amounts of EP11313 (3X) vs. JQ1 there was no reduction in Treg proliferation by the former. These findings were consistent with the observations that IL-2 production by non-Treg cells was not diminished in EP11313 treated animals. Interestingly, Treg pSTAT5 levels were also not diminished after BETi treatment in vitro or in vivo. This finding contrasts reported observations that JQ1 inhibited STAT5 phosphorylation and transcriptional activity in monocyte/dendritic cells (22). However, similar to our results, it was previously reported that JQ1 or BRD2 downregulation diminishes STAT5 function through phosphorylation-independent mechanisms in lymphocytic leukemias (21). Although we anticipated that due to Treg proliferation differences in mice treated with either JQ1 or EP11313, STAT5 phosphorylation may have been differentially affected by these BETi's. In contrast to other cell populations, it appears BETi STAT5 regulation of lymphoid lineage populations involves a different mechanism. It was previously reported that STATs can be acetylated under certain conditions (40). Thus, it is possible that STAT5 or proteins involved in this signaling pathway in expanding Tregs or hematologic cancers does not contain acetylated lysine residues which are present following LPS stimulation of dendritic cells (21, 22).

Importantly, using a brief, i.e., 1 week protocol of EP11313 10 mg/kg (a low dose of BETi) as mentioned above, several differentiation and functional molecules (ex. ICOS-1, Nrp-1, PD-1) were not altered in Tregs. Moreover, this regimen of EP11313 did not reduce IL-10 and TGF-β–two key Treg suppressive molecules. As the objective of the present studies was to combine the use of BETi with donor Tregs to more effectively regulate GVHD onset, the findings above support the strategy of using both EP11313 together with Tregs. A BETi was reported to inhibit cytokine expression and APC function in dendritic cells and to decrease cytokine secretion and T cell expansion in vivo (17). Short-term administration of IBET151 early during BMT reduced GVHD severity supporting the notion that inhibiting BET proteins may serve as an approach for preventing GVHD (17). However, we observed that I-BET151 in vivo treatment for 1 week markedly reduced B cell levels in the spleen and therefore, would not be useful in the GVHD setting because it might affect recipients' immune reconstitution (data not shown). Importantly, EP11313 possesses distinct pharmacological properties, for example, this BETi is less extruded by P-glycoprotein (expressed on APC and activated T cells) across the cell membrane in comparison with I-BET762 and I-BET151 (MDR/PgP-MDCK efflux ratio BA/AB: 1.3, 27.9, 12.2, respectively, personal observation). EP11313 is therefore more highly retained intracellularly and this persistence may increase the regulation of inflammatory cytokines. Based on these properties together with the above mentioned findings showing that Tregs are not impaired by EP11313, short-term treatment (day −2 to 4) with expanded Tregs was utilized in an aHSCT. This approach was found to diminish early GVHD clinical scores including decreased ocular and skin involvement. Using highly purified donor TL1A-Ig + low dose IL-2 expanded Tregs, this second and more direct assessment of the combinatorial strategy further supported the notion that selective BETi can be used for treatment in combination with adoptive Treg therapy. Although short-term treatment with EP11313 did not enhance overall survival, BETi utilization in pre-clinical tumor models as well as clinical oncology trials have involved long-term (ex. months) administration of higher BETi doses (up to 50 mg/kg), therefore increasing the duration of BETi low dose treatment post-HSCT may further improve recipient outcomes.

It has been demonstrated that adoptive transfer of Tregs can effectively abrogate GVHD while maintaining graft-versus tumor or leukemia/lymphoma (GVT, GVL) (41, 42). In this context, we previously demonstrated using A20luc/YFP cells (murine B cell lymphoma) that transplanting donor TL1A-Ig + IL-2 spleen cells (containing ∼4 × 105 Tregs) GVHD was significantly reduced and GVL was preserved (7). In hematologic malignancies, BETi have demonstrated to possess effective antitumor activity by repressing aberrant oncogenic transcription (11, 43, 44). Importantly, here we showed besides GVHD amelioration, a direct effect of BETi EP11313 on A20 luc/YFP cell survival and proliferation. Examination of other mouse tumor cell lines i.e., EL4 (thymoma) and P815 (mastocytoma) indicated the latter was resistant to BETi effect on cell survival so not all tumors are equally sensitive to these compounds (SC, RBL unpublished observations). Our results indicate that JQ1

#### REFERENCES

1. Ferrara JL, Reddy P. Pathophysiology of graft-versus-host disease. Semin Hematol. (2006) 43:3–10. doi: 10.1053/j.seminhematol.2005. 09.001

and EP11313 have similar anti-cancer effects (IC50 = 0.19 and 0.28µM, respectively) in A20luc/YFP tumor cells. However, these BETi exhibit significantly different biological effects on Tregs, where EP11313 have no interference with Treg proliferation, phenotype and function. We hypothesize that a strategy using expanded Tregs and EP11313 may not impair GVL and could directly inhibit tumor growth. Thus, the overall mechanism proposed involves Treg mediated suppression of donor alloreactive T cells, BETi blockage of inflammatory cytokines; and direct BETi anti-tumor activity. In total, we posit that selected BETi treatment together with expanded Treg therapy represents a novel and potentially effective combinatorial strategy for ameliorating hematologic cancer and GVHD.

#### AUTHOR CONTRIBUTIONS

SC designed research studies, discussed data sets, conducted experiments, analyzed data and interpreted data, wrote the manuscript. CL, HB, JM, DS, and BK conducted experiments and analyzed data. IL, DW, and CB performed research, analyzed and interpreted data, and edited the manuscript. SB supported the research. KK discussed Treg studies and supported the research. VLP discussed Treg studies and BETi and supported the research. CW discussed data and manuscript and supported the research. RBL designed research studies, discussed data sets, wrote the paper, supervised and supported the research.

#### ACKNOWLEDGMENTS

We thank Dr. J. Bradner (Novartis Institutes for Biomedical Research, Cambridge, MA, United States) for kindly providing JQ1. We are grateful to Dr. Oliver Umland and the Sylvester Comprehensive Cancer Center Flow Cytometry Core for excellent assistance with flow cytometry and cell sorting. We are thankful to Dr. Robert Negrin (Stanford, CA) for generously providing the A20luc/YFP cell line. Research support: This work was supported by funds from the Sylvester Comprehensive Cancer Center and the NIH (RO1 EY024484-01), RBL and VLP; Sylvester Comprehensive Cancer Center, RBL and SC; NIH (DA035592, DA035055 and AA023781), Sylvester Cancer Center Molecular shared Resource, and Florida Department of Health (6AZ08 and 7AZ26), CW; NIH (R01NS092671 and R01MH110441), SB; Kalish Family Foundation, KK.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2018.03104/full#supplementary-material

transplantation. Blood (2005) 105:1828–36. doi: 10.1182/blood-2004-0 8-3213

bromodomain and extra-terminal (BET) bromodomain protein BRD2. Mol Cancer Ther. (2014) 13:1194–205. doi: 10.1158/1535-7163.MCT-1 3-0341

in diffuse large B cell lymphoma. Cancer Cell (2013) 24:777–90. doi: 10.1016/j.ccr.2013.11.003

**Conflict of Interest Statement:** RBL is a scientific advisory board member of Heat Biologics and a consultant for Heat Biologics and Pelican Therapeutics.

The remaining 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 © 2019 Copsel, Lightbourn, Barreras, Lohse, Wolf, Bader, Manov, Kale, Shah, Brothers, Perez, Komanduri, Wahlestedt and Levy. 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) and the copyright owner(s) 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.

# Blockade of TNFα to Improve Human CD34+ Cell Repopulating Activity in Allogeneic Stem Cell Transplantation

#### Vitalyi Senyuk, Pritesh Patel, Nadim Mahmud and Damiano Rondelli\*

*Division of Hematology/Oncology, Department of Medicine and UI Cancer Center, University of Illinois at Chicago, Chicago, IL, United States*

#### Edited by:

*Brian Christopher Betts, University of Minnesota Twin Cities, United States*

#### Reviewed by:

*Hung D. Nguyen, Medical University of South Carolina, United States Lohith Gowda, Yale University, United States*

> \*Correspondence: *Damiano Rondelli<EMAIL_ADDRESS>
#### Specialty section:

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

Received: *16 October 2018* Accepted: *31 December 2018* Published: *22 January 2019*

#### Citation:

*Senyuk V, Patel P, Mahmud N and Rondelli D (2019) Blockade of TNF*α *to Improve Human CD34*+ *Cell Repopulating Activity in Allogeneic Stem Cell Transplantation. Front. Immunol. 9:3186. doi: 10.3389/fimmu.2018.03186* Early release of TNFα after hematopoietic stem cell transplantation (HSCT) correlates with development of acute graft-vs.-host disease (GVHD). Here we tested the effect of TNFα and alloreactive T cells on early hematopoietic HSC genotype and function. Addition of TNFα (10 ng/ml) in liquid cultures with CD34+ cells for 6–72 h resulted in the downregulation of genes associated with stem cell activity, such as DNMT3A, DNMT3B, TET1, TET2, SOX2, NANOG, and OCT4, whereas no significant effect was observed on DNMT1 and GATA2 expression. These findings were reversed by using an anti-TNFα antibody. Similar gene downregulation was observed when CD34+ cells were co-cultured with alloreactive T cells CD34+ cells for 48–72 h, and this effect was partially prevented by rapamycin and an anti-TNFα antibody. CD34+ cells pre-incubated with TNFα for 48 h and transplanted in irradiated NOD-SCID È null (NSG) mice showed a reduced myeloid engraftment compared to control mice. By using a xenograft model recently developed in our lab, we co-transplanted CD34+ cells and allogeneic T lymphocytes at 1:0.1 ratio in one group that also received etanercept (TNFα inhibitor) at 100 µg intra-peritoneum (i.p.) on days −1,+1,+3,+5 post-HSCT, and in the control group. At 6 weeks post-transplant, mice that received etanercept had a significantly higher number of marrow huCD45+CD34+CD38- early stem cells (*p* = 0.03) and a reduced number of huCD45+CD3+ splenic T cells (*p* = 0.04) compared to controls. The repopulating activity of marrow cells from mice treated with etanercept vs. controls was tested in secondary transplants. Although the overall engraftment was similar in the two groups, CD34+ cells isolated from recipients of marrow from the etanercept group showed a significantly greater expression of stem cell-associated genes and a higher number of CD45+CD34+CD38- cells than in controls (*p* = 0.03). Our findings suggest that early TNFα increase post-transplant can affect long-term stem cell engraftment, and that blockade of TNFα early after transplant may limit a cytokine-mediated suppressive effect on repopulating stem cell function.

Keywords: CD34, TNFα, transplantation, GVHD, T cells, repopulating activity

# INTRODUCTION

The engraftment of donor hematopoietic stem cells (HSC) after transplantation requires a profound immunosuppression of the host to prevent the risk of rejection. However, the immunosuppression of the host also favors the expansion of donor immune cells that can target non-hematopoietic tissues and cause graft-vs.-host disease (GVHD) (1). The immunologic events determining GVHD start immediately after transplant and are largely based on an initial release of pro-inflammatory cytokines caused by the effect of chemotherapy/radiotherapy and antigen presenting cell: T cell interaction (1, 2). In fact, patients undergoing an allogeneic HSC transplant (HSCT) from HLA matched donors receive GVHD prophylaxis starting from the time of transplant and, in case of transplant from HLA haploidentical donors, with high doses of cyclophosphamide administered on days +3 and + 4 after transplant (3).

TNFα is a pro-inflammatory cytokine released particularly by donor T cells upon transplantation, and has previously been demonstrated to play a key role in the initial immune process leading to GVHD, both facilitating the activation of antigen presenting cells (APC) and the expansion of alloreactive T cells (4, 5). Moreover, detection of an increased serum level of TNFα receptor 1 in patients within the first week after transplantation is one of the biomarkers predicting future development of GVHD (6).

Previous findings from our lab demonstrated that TNFα mediates a direct effect of T cells on a subset of CD34+ cells hematopoietic progenitors, inducing their differentiation into the monocytic/dendritic lineage and increasing their direct as well as indirect antigen presenting function (7). Here we tested the question whether early hematopoietic stem cells with repopulating stem cell activity could also be a target of TNFα, thus hypothesizing that stem cell engraftment after transplantation could be affected by TNFα. We analyzed the in vitro effect of TNFα, as well as of allogeneic T cells, on CD34+ cell expression of genes regulating DNA methylation or pluripotency, such as DNMT1, DNMT3A, DNMT3B, NANOG, OCT4, SOX2 (8, 9). Then, we utilized a xenograft transplant (10) model to study the in-vivo effect of TNFα on HSC and the role of a TNFα inhibitor after co-transplantation of CD34+ and allogeneic T cells. The results shown here suggest that TNFα can affect early HSC and that blockade of TNFα may preserve a pool of stem cells with repopulating activity. Based on these findings, new therapeutic strategies may be tested to better protect stem cell engraftment after allogeneic transplantation.

#### MATERIALS AND METHODS

#### Cell Separation

Healthy donor G-CSF mobilized peripheral blood stem cells (PBSC) from AllCells (Alameda, CA) and PB cells from healthy volunteers were utilized in this study. Mononuclear cells (MNC), CD34+ cells and CD3+ T cells were purified as previously described (10). Isolated CD34+, or T cell samples were acquired on a FACS CaliburTM (Becton Dickinson) and analyzed using the Cell Quest TM software (Becton Dickinson), and showed, on average, >95% cell purity.

#### Flow Cytometry

Fluorescein isthiocyanate (FITC), or phycoerythrin (PE), or peridin chlorophyll protein (PerCP), conjugated mAbs (CD45, CD34, CD38, CD33, CD3) or isotype controls (Becton-Dickinson, San Jose', CA) were employed. Stained cells were washed twice in PBS and sample acquisition and analysis was performed within 2 h on a FACSCaliburTM (Becton Dickinson).

#### Co-cultures of CD34+ and T Cells

Purified human CD34+ cells (1–2 x 105 cells) were cocultured with human allogeneic T cells at 1:0.1, or 1:2 ratio in round-bottomed 96-well plates for 48–72 h at 37◦C in a 5% CO2 humidified atmosphere, as previously described. In selected experiments, CD34+ cells and T cells were cultured in the presence of the following molecules described: TNFα, Rapamycin, Cyclosporin A (Sigma-Aldrich (St. Louis, MO), Mycophenolate Motefil (Cayman Chemical Company, Ann Arbor, MI), Abatacept (Bristol Meyers Squibb, New York, NY), rabbit anti-thymocyte globulin (rATG, Thymoglobulin, Genzyme, Cambridge, MA), anti-TNFα antibody (AF-210-NA) from R&D Systems (Minneapolis, MN).

#### qRT-PCR

CD34+ cells re-isolated on human CD34+ MicroBead Kit UltraPure (Miltenyi Biotec, Bergisch Gladbach, Germany) after MLC or after transplantation were used for total RNA extraction with TRIzol reagent (Life Technologies Corporation, Grand Island, NY). RNA was transcribed into cDNA with SuperScript R III First-Strand Synthesis SuperMix (Life Technologies Corporation, Grand Island, NY) and analyzed with SYBR green (Applied Biosystems, Inc., Grand Island, NY) on the 7500 FAST Real Time PCR detection system (Applied Biosystems, Inc., Grand Island, NY). The human primers used are: ACTB, forward: 5-ggacttcgagcaagagatgg-3′ , reverse: 5′ -agc actcgtgttggcgtacag-3′ ; DNMT1, forward: 5′ -tgctgaagcctccga gat-3′ , reverse: 5′ -ttctgttaagctgtctctttcca-3′ ; DNMT3A, forward: 5 ′ -tacttccagagcttcagggc-3′ , reverse: 5′ -attccttctcacaacccgc-3; DNMT3B, forward: 5′ -gagattcgcgagcccag-3′ , reverse: 5′ -tctcca ttgagatgcctggt-3′ ; TET1, forward: 5′ -gagggaaaagaagcccaaag-3′ , reverse: 5′ -tcttccccatgaccacatct-3′ ; TET2, forward: 5′ -agaaaag ggaaaggagagcg-3′ , reverse: 5′ -gagagggtgtgctgctgaat-3; TET3, forward: 5′ -gccggtcaatggtgctagag-3′ , reverse: 5′ -cggttgaaggtt tcatagagcc-3′ ; NANOG, forward: 5′ -gatttgtgggcctgaagaaa-3′ , reverse: 5′ -cagggctgtcctgaataagc-3′ ; OCT4, forward: 5′ -gtggag gaagctgacaacaa-3′ , reverse: 5′ -ggttctcgatactggttcgc-3; SOX2, forward: 5′ -aaccccaagatgcaccaactc-3′ , reverse: 5′ -gcttagcctcgtcga tgaac-3,. GATA2, forward: 5′ - cacaagatgaatgggcagaa−3 ′ , reverse: 5 ′ - acaatttgcacaacaggtgc−3 ′ .

#### TNFα Blockade

In vitro TNFα blockade was tested in MLC assays with anti-TNFα antibody (AF-210-NA). In titration experiment, we tested 0.1µg/ml, 0.5µg/ml and 1µg/ml of anti-TNFα antibody, and in selected experiments at 5µg/ml. The tested anti-TNFα/ TNFα excess range (10x−100x) covers whole possible TNFα neutralization range, according to the manufacturer's guide. In vivo TNFα blockade was tested in NSG mice co-transplanted with CD34+ and allogeneic T cells at 1:0.1 ratio by injecting etanercept (Enbrel, Immunex Corporation, Thousand Oaks, CA) intra-peritoneum (i.p.).

#### Transplantation

Immunodeficient nonobese diabetic/ltsz-scid/scid (NOD/SCID) IL2 receptor gamma chain knockout mice (NSG) were purchased from the Jackson Laboratories (Bar Harbor, ME) and housed in a strict barrier environment. The study was approved by UIC Animal Care Committee and performed in accordance with national guidelines of laboratory animal care. Human CD34+ cells were initially incubated with or without TNFα (10 ng/ml) invitro for 48 h, re-isolated immune-magnetically using a human CD34+ MicroBead Kit UltraPure (Miltenyi Biotec, Bergisch Gladbach, Germany) and 2 × 105 cells were transplanted intravenously (i.v.) into sublethally irradiated (300 cGy) NSG mice as previously described (10). In a second set of experiments, purified CD34+ cells (2 × 105 /mouse) were mixed at 1:0.1 ratio with allogeneic T cells and then injected i.v. into sublethally irradiated (300 cGy) NSG mice. A group of mice was also injected intra-peritoneum (i.p.) with etanercept at 100 µg/mouse on days: −1, +1, +3 and +5 post-transplant (n = 10 mice/group). At day 42 post-transplant, marrow cells were collected to analyze engraftment, as well as to perform secondary transplants. To this purpose, marrow cells obtained after primary transplant were depleted of human T cells on a Miltenyi column after incubating the cells with MicroBeads conjugated to monoclonal anti-human CD3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). Then secondary NSG mice were transplanted with 5 × 106 T cell depleted marrow cells/mouse from the etanercept and control groups (n = 5 mice/group). Forty-two days following second transplant, mice were sacrificed and analyzed for stem cell engraftment in the marrow andspleen, as previously described (10). Human cell engraftment was assessed by measuring the expression of huCD45 marker. Analysis of all markers on marrow or spleen cells was performed on gated huCD45+ cells.

#### Statistical Analysis

Student t-test was performed to compare 2 series of data. Statistical tests were performed by using Graph Pad Prism version 7.0 (GraphPad Inc., San Diego, CA).

# RESULTS

#### TNFα-mediated Downregulation of Early Stem Cell Gene Expression in CD34+ Cells

It has been previously demonstrated that TNFα induces the differentiation of a subset of CD34+ cells committed to the monocytic-dendritic lineage within 3–5 days of liquid culture (7). To test whether also early hematopoietic progenitors can be targeted by the effect of TNFα, we incubated human CD34+ cells with or without TNFα at 10 ng/ml for 6–72 h and then extracted their mRNA to measure the expression of genes associated with self-renewal or pluripotent stem cell activity, such as: GATA2, DNMT1, DNMT3A, DNMT3B, TET1, TET2, TET3, NANOG, SOX2, and OCT4 (**Figure 1A**). In 5 separate experiments, GATA2 expression was not affected by TNFα at any time point (data not shown). On the contrary, all the other genes were significantly downregulated at either one or all three time points, compared to control experiments without TNFα. The low expression of DNMT3A, DNMT3B, TET2, NANOG, SOX2, and OCT4 at 24 and 72 h suggested that genes regulating stem cell proliferation can be rapidly downregulated by TNFα in CD34+ cells. We then incubated the CD34+ cells with TNFα and increasing doses of a blocking anti-TNFα antibody for 72 h. The expression of all the genes previously downregulated was restored to the level of control CD34+ in the presence of higher doses of the blocking antibody (**Figure 1B**). In control experiments, the anti-TNFa antibody alone did not affect gene expression on CD34+ cells (data unshown). These findings showed that TNFα directly modified the expression of multiple genes in CD34+ cells.

# T Cell-mediated Downregulation of Early Stem Cell Gene Expression in CD34+ Cells

We previously demonstrated that allogeneic T cells induce a rapid differentiation of a subset of CD34+ cells into monocytic/dentritic cells, mostly mediated by TNFα (7). Based on the observation above that TNFα downregulated the expression of genes associated with DNA methylation and pluripotent stem cell activity, we tested whether alloreactive T cells can induce a similar effect. We incubated CD34+ cells with allogeneic CD3+ T cells at 1:0 (control), 1:0.1 or 1:2 ratio as previously described (10). After 72 h, CD34+ cells were immunomagnetically re-purified and their mRNA was isolated to assess the gene expression. In the presence of low number of T cells (1:0.1 ratio), only DNMT3A, TET1, and NANOG expression was decreased, whereas at higher concentration of T cells (1:2 ratio) all the genes tested were downregulated with the exception of DNMT1 (**Figure 2**) and GATA2 (not shown). These findings suggested that alloreactive T cells may affect the expression of some of the genes associated with early stem cell activity in CD34+ cells.

#### Variable Effect of Standard Immunosuppressive Agents on T Cell-mediated Epigenetic Changes in CD34+ Cells

Since we observed that alloreactive T cells rapidly affect the expression of genes that could regulate stem cell long term engraftment, we investigated on whether an anti-TNFα antibody or immunosuppressive molecules commonly used as GVHD prophylaxis could prevent T cell effect on CD34+ cells. We performed 72 h liquid cultures with CD34+ and allogeneic T cells at 1:2 ratio or CD34+ alone, then we added standard immunosuppressive agents such as cyclosporine A (1µg/ml), rapamycin (1 µg /ml), mycophenolate mofetil (0.5µg/ml), abatacept (100 µg /ml), thymoglobulin (100 µg /ml) or an anti-TNFα antibody (5µg/ml) (**Figure 3**). After 72 h the CD34+ cells were immunomagnetically re-isolated in order to extract

the mRNA and test gene expression. Although none of the immunosuppressive drugs, or the anti-TNFα antibody, could completely prevent the T cell-mediated gene downregulation in CD34+ cells, a trend for a greater activity in preserving epigenetic and pluripotency gene expression was observed with rapamycin and anti-TNFα antibody, suggesting possible combination studies.

#### Short Exposure to TNFα Reduces CD34+ Cell Engraftment After Transplant

In order to test whether TNFα may affect the repopulating activity of CD34+ cells, initial experiments were designed to transplant CD34+ cells into NSG mice after short exposure to TNFα. Liquid cultures of CD34+ cells with or without TNFα at 10 ng/ml concentration were carried out for 48 h. Analysis of gene expression (not shown) confirmed downregulation of genes associated with early stem cell activity, as described above. CD34+ cells pretreated with TNFα for 48 h, or untreated CD34+ cells as control, were washed and then transplanted into sublethally irradiated NSG mice at 1 × 105 CD34+ cells/mice (n = 5 mice per group). Five to 6 weeks after transplant the mice were sacrificed and stem cell engraftment was measured in the marrow and spleen by flow cytometry. Mice transplanted with CD34+ cells pretreated with TNFα had a lower bone

DNMT1, DNMT3A, DBMT3B, NANOG, SOX2, OCT4 genes by qRT PCR. Gene expression in CD34+ cells cultured without T cells was arbitrary taken as 1 for each gene. ACTB expression was used as normalization control. The data is shown as mean values of 3 separate experiments and standard deviation bars are included. Differences in the expression of each gene in CD34+ cells co-cultured with T cells vs. control were calculated by *t*-test and significant *p*-values are shown.

*in-vitro*. CD34+ and allogeneic T cells were co-cultured at 1:2 ratio for 72 h with or without one of the following standard immunosuppressive molecules: cyclosporin A, rapamycin, mycophenolate mofetil, abatacept, anti-TNFα antibody, ATG. CD34+ cells were then re-isolated immunomagnetically and analyzed by qRT PCR. Gene expression in CD34+ cells cultured without T cells as control was arbitrary taken as 1 for each gene. ACTB expression was used as normalization control. The data is shown as mean values of 3 separate experiments and standard deviation bars are included.

marrow stem cell engraftment compared to controls, shown in a representative example in **Figure 4A**, and documented as lower percentage and absolute number of huCD45+ cells (**Figure 4B**). We also analyzed whether exposure of CD34+ cells to TNFα would affect the pool of marrow CD34+ cells after transplant, possibly responsible for long term engraftment. The absolute number of CD34+ cells was found significantly lower in the marrow of mice transplanted with TNFα-pretreated CD34+ cells (**Figure 4C**). These findings are consistent with the hypothesis that a brief exposure to TNFα may impair the engraftment ability of CD34+ cells.

# Etanercept Preserves the Engraftment of CD34+CD38- Cells After

Co-transplantation With Allogeneic T Cells

We recently observed that co-transplantation of CD34+ and allogeneic T cells at 1:0.1 ratio in a xenograft model results in low stem cell engraftment and in the expansion of T cells (10). Here we adopted this model to test whether in-vivo blockade of TNFα would improve the engraftment of CD34+ cells by reducing the effect of alloreactivity. Two groups of NSG mice were cotransplanted with CD34+ cells and T lymphocytes at 1:0.1 ratio

and one of these groups was injected i.p. with etanercept at 100 µg on days −1, +1, +3 and + 5 post transplant. Six weeks after transplant, the marrow of mice treated with etanercept and those in the control group showed similar percentages of huCD45+ cells and human myeloid CD33+ cells (**Figure 5A**). Instead, the percentage and absolute number of early hematopoietic

progenitors identified as CD45+CD34+CD38- cells were significantly higher in the etanercept group (p = 0.03), compared to control mice (**Figure 5B**). In addition, mice treated with

groups were analyzed by *t*-test and significant *p*-values are shown.

etanercept showed a significantly lower expansion of T cells in the spleen, as demonstrated by a lower percentage of CD45+ and CD45+CD3+ cells in the spleen (**Figure 5C**).

hematopoietic progenitors (CD45+CD34+CD38-) cells. The results are represented as mean values and standard deviation bars are shown. Differences between

These findings suggested that early blockade of TNFα after transplant may preserve the pool of early hematopoietic stem cells. To test whether these cells also had a greater repopulating stem cell activity, a secondary transplant with marrow cells obtained from the etanercept and control groups was performed. Secondary NSG mice were transplanted with equivalent number of marrow cells collected from primary transplants and 6 week later (total 12 weeks) the marrow was analyzed for myeloid engraftment and persistence of early progenitor cells. Recipients of marrow cells from the etanercept and control groups had comparable percentages of CD45+ and CD45+CD33+ myeloid cells after secondary transplant (not shown). However, a higher percentage and absolute number of CD34+CD38- cells were detected in recipients of marrow cells from the etanercept group (**Figure 6A**). Finally, CD34+ cells from both the etanercept and control groups were re-isolated after transplant. Because of the small absolute number of CD34+ cells, we could not analyze each single mouse separately and marrows from either group were pooled before CD34+ cell selection and RNA extraction. The results of this experiment (**Figure 6B**) showed a 4–8 fold increase of gene expression in the etanercept group compared to mice that had not received etanercept not during primary transplant. Persistence of high expression of pluripotent genes in CD34+ cells from the etanercept group after secondary transplant did not correlate with a skiewed differentiation capacity since myeloid engraftment was comparable in the two groups. These data are consistent with initial hypothesis that release of TNFα immediately after transplant may affect long-term reconstitution of hematopoietic progenitors.

#### DISCUSSION

This study demonstrates that TNFα and alloreactive T cells rapidly affect human early hematopoietic precursors by downregulating the expression of genes associated with selfrenewal and pluripotent stem cell activity, and affecting the engraftment of repopulating HSC in-vivo. A protective effect on repopulating HSC was elicited by blockade of TNFα in a xenograft model of co-transplantation of CD34+ and allogeneic T cells.

The role of TNFα in the development of GVHD has been extensively studied both in experimental models and in the clinical setting (1, 2, 4, 11–13). In this latter, it was also demonstrated that increased serum levels of TNFR1 on day 7 after transplant were shown to predict patients who then developed GVHD (6, 14–16). TNFα receptors (TNFR1 and TNFR2) were previously detected also on CD34+ hematopoietic progenitors (17, 18). Stimulation of TNF receptors with TNFα was then shown to upregulate the expression of interferon È (IFNÈR) and FasL receptors, thus increasing their susceptibility to inhibitory effects of TNFα or IFNÈ. However, although some experimental models suggested a negative effect of TNFα in regulating normal hematopoiesis in-vivo (19, 20), other studies observed an impaired long-term hematopoietic reconstitution in mice lacking TNFR1 (21). We previously showed that committed CD34+ progenitors can stimulate allogeneic T cell responses

in-vitro (22, 23) via B7:CD28 costimulation, and that ∼70% of PB CD34+ cells upregulated CD40 costimulatory molecule and increased their immunostimulatory activity following 24 h exposure to TNFα (24). Endogenous TNFα released in cocultures of CD34+ and allogeneic T cells was then demonstrated to mediate the rapid differentiation of a subset of CD34+ cells into monocytic/dendritic cells, whereas co-transplantation of CD34+ and T cells into NOD/SCID mice resulted in increased differentiation of mature dendritic cells (7). Our current study shows for the first time that within 6–72 h in liquid culture, TNFα downregulated the expression of some of the genes regulating stem cell methylation, such as DNMT3A/B, or TET1, TET2, TET3, and other transcription factor genes, such as NANOG, SOX2, or OCT4 that are associated with pluripotent status of stem cells (8, 9). Since it has previously reported that: de novo DNA methylation through DNMT3A/B activity is essential for stem cell long term reconstitution activity (25, 26); concomitant reduction of DNMT3A and TET-mediated DNA methylation can impair both self-renewal and differentiation of hematopoietic stem cells (27); and transcription factors such as OCT4, SOX2, and NANOG contribute to maintaining the pluripotency properties of early stem cells (9), it is conceivable that the effect of TNFα may target the epigenetic regulation of early CD34+ cells capable of post-transplant long-term hematopoietic reconstitution. Based on these observations on the downregulation of some of the genes affecting DNA methylation concomitantly with a reduced engraftment ability of HSC, new studies can be started to address the causality of each gene dysregulation and CD34+ cell post-transplant engraftment capacity. Because the same genes downregulated by TNFα were also rapidly downregulated in-vitro by allogeneic T cells, the cytokine storm caused by alloreactive T cells in the early phase post-transplant may affect CD34+ cell function either through TNFα alone, or through a combination of multiple soluble factors. Indeed, the T cell mediated epigenetic changes on CD34+ cells were only partially prevented by an anti-TNFα antibody. Interestingly, when we tested the effect of immunosuppressive molecules that are commonly used to prevent GVHD, such as cyclosporine A, rapamycin, mycophenolate mofetil, thymoglobulin, abatacept, or an anti-TNFα antibody in co-cultures of CD34+ and allogeneic T cells, none of them could completely prevent gene downregulation, and only rapamycin and anti-TNFα ab showed some partial protective effect. Future studies will test the effect of combination of these two agents. Consistent with in-vitro data, transplantation of CD34+ cells exposed to TNFα for 48 h resulted in a lower engraftment. In addition, by utilizing a xenograft model of co-transplantation of CD34+ and allogeneic T cells recently developed in our lab (10), we showed that injection of etanercept in the first week after transplant maintained a greater pool of CD34+CD38- hematopoietic progenitors after both primary and secondary transplant. These findings are consistent with a recent study where the authors transplanted high numbers of human umbilical cord (UC) cells into NSG mice and assessed the engraftment after only 4 weeks, because of xenogeneic GVHD limiting mice survival. In this study, they demonstrated that UC T cells produced high levels of multiple cytokines, including TNFα, which directly impaired stem cell engraftment (28). Although they did not test the repopulating stem cell activity in secondary transplants, they clearly showed a negative effect of T cell-derived TNFα on short and long-term hematopoietic stem cell subsets by inducing stem cell apoptosis, and proved that stem cell engraftment in mice transplanted with UC grafts plus etanercept was comparable to those receiving T cell-depleted

#### REFERENCES

grafts. Similarly, we demonstrated that in mice transplanted with CD34+ and allogeneic T cells TNFα blockade with etanercept in the first week of transplant allowed a greater engraftment of CD34+CD38- cells, not only in primary but also in secondary transplants. This could be due to limiting T cell expansion and preventing a TNFα-mediated epigenetic dysregulation of the physiologic balance of stem cell self-renewal and differentiation activity of CD34+ cells. This hypothesis was indirectly supported by our observation of higher expression of DNMTs, TETs and pluripotency transcription factor genes in CD34+ cells obtained from the etanercept group after secondary transplants.

All these findings expand the knowledge of an early effect of TNFα on the human hematopoietic system after allogeneic stem cell transplantation. In a clinical setting, it is conceivable that the pool of newly transplanted stem cells may be partially affected by the cytokines released by alloreactive T cells. Depending on the overall number of stem cells transplanted into a patient, a small loss of stem cell function may not be clinically relevant since the graft may have an abundant number of residual stem cells to guarantee the engraftment. However, patients fully engrafted with donor cells and yet experiencing late post-transplant cytopenias (29), or those with cytopenias in the context of GVHD, could be affected by a T cellmediated anti-stem cell reactivity. This could be identified as a cytokine-mediated graft-vs.-marrow disease. TNFα blockade was previously proven to ameliorate acute GVHD in combination with other immunosuppressive drugs (30–32). Our findings could prompt new studies testing etanercept in the context of GVHD prophylaxis, not only to better prevent GVHD, but also to protect donor HSC from the detrimental effect of TNFα and facilitate long-term engraftment.

#### AUTHOR CONTRIBUTIONS

VS, PP, and DR contributed to the study design. VS, NM, and PP performed all the experiments, VS and DR analyzed the data. VS and DR wrote of the manuscript. All the authors assisted in the critical review of the manuscript and approved the final version of the manuscript for submission.

#### ACKNOWLEDGMENTS

The study was partially supported by a grant from the Perry Family Foundation; a grant from the Michael Reese Research & Education Foundation; and a grant from the Thorek Memorial Foundation to DR.

posttransplantation cyclophosphamide. Biol Blood Marrow Transplant. (2008) 14:641–50. doi: 10.1016/j.bbmt.2008.03.005

based on biomarkers: a multicentre study. Lancet Haematol. (2015) 2:e21–9. doi: 10.1016/S2352-3026(14)00035-0

hematopoiesis at a stem cell level. J Exp Med. (1999) 190:1493–504. doi: 10.1084/jem.190.10.1493

**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 © 2019 Senyuk, Patel, Mahmud and Rondelli. 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) and the copyright owner(s) 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.

# Strategies for Enhancing and Preserving Anti-leukemia Effects Without Aggravating Graft-Versus-Host Disease

Ying-Jun Chang1 , Xiang-Yu Zhao1 and Xiao-Jun Huang1,2 \*

1 Peking University People's Hospital & Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing, China, 2 Peking-Tsinghua Center for Life Sciences, Beijing, China

Allogeneic stem cell transplantation (allo-SCT) is a curable method for the treatment of hematological malignancies. In the past two decades, the establishment of haploidentical transplant modalities make "everyone has a donor" become a reality. However, graft-versus-host disease (GVHD) and relapse remain the major two causes of death either in the human leukocyte antigen (HLA)-matched transplant or haploidentical transplant settings, both of which restrict the improvement of transplant outcomes. Preclinical mice model showed that both donor-derived T cells and natural killer (NK) cells play important role in the pathogenesis of GVHD and the effects of graft-versus-leukemia (GVL). Hence, understanding the immune mechanisms of GVHD and GVL would provide potential strategies for the control of leukemia relapse without aggravating GVHD. The purpose of the current review is to summarize the biology of GVHD and GVL responses in preclinical models and to discuss potential novel therapeutic strategies to reduce the relapse rate after allo-SCT. We will also review the approaches, including optimal donor selection and, conditioning regimens, donor lymphocyte infusion, BCR/ABL-specific CTL, and chimeric antigen receptor-modified T cells, which have been successfully used in the clinic to enhance and preserve anti-leukemia activity, especially GVL effects, without aggravating GVHD or alleviate GVHD.

Keywords: allogeneic stem cell transplantation, graft-versus-leukemia, graft-versus-host disease, relapse, G-CSF

# INTRODUCTION

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) remains a potentially curative therapeutic strategy for hematological malignancies (1–4). Currently, for patients who require transplantation, but have no related or unrelated donors with matching human-leukocyte antigen (HLA), haploidentical HSCT is an alternative modality, allowing everyone to have a donor (5, 6). Allo-HSCT benefits these malignancies due to a graft-versus-leukemia (GVL) effect that is mainly mediated by donor-derived alloreactive T cells and/or natural killer (NK) cells (7–12). However, T cells are also responsible for acute and/or chronic graft versus-host disease (GVHD), which leads to significant morbidity and mortality (13–15). Although the depletion of T cells from allografts alleviates GVHD either in human leukocyte antigen (HLA)-matched transplant settings or in HLAhaploidentical transplant modalities, removal of these cells results in increased graft failure and increased rates of leukemia relapse (16). Unfortunately, the immunosuppressive agents used for

#### Edited by:

Brian Christopher Betts, University of Minnesota Twin Cities, United States

#### Reviewed by:

Sergio Querol, Banc de Sang i Teixits, Spain Luisa Sisinni, Hospital de la Santa Creu i Sant Pau, Spain

\*Correspondence:

Xiao-Jun Huang<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 01 October 2018 Accepted: 10 December 2018 Published: 21 December 2018

#### Citation:

Chang Y-J, Zhao X-Y and Huang X-J (2018) Strategies for Enhancing and Preserving Anti-leukemia Effects Without Aggravating Graft-Versus-Host Disease. Front. Immunol. 9:3041. doi: 10.3389/fimmu.2018.03041 the prophylaxis and treatment of GVHD can also reduce the beneficial GVL effects. Therefore, the separation of GVL effects from GVHD remains the "holy grail" of allo-HSCT (17–20), which is urgently needed to allow a more effective therapy for hematological malignancies.

The challenge for the separation of GVL effects from GVHD is attributed to the underlying similarity of the alloreactive T responses between the two processes (4, 21, 22). In the past 20 years, great efforts have been made by researchers to elucidate specific distinguishing immune mechanisms of GVL vs. GVHD (23–25). In addition, a number of preclinical experiments have been performed to identify approaches that could be successfully used to separate GVL effects from GVHD (26–41). Clinically, a series of strategies, including donor selection, allograft engineering, adoptive immune cell infusion, and pharmacological agents have been established (42–44). More recently, the use of chimeric antigen receptor T (CAR-T) cells that target tumor cells with a limited capacity for GVHD induction, have been identified for enhancing GVL effects without aggravating GVHD (45–47). Previously, several reviews have been published related to the separation of GVL from GVHD (48–51) The present review briefly summarizes the underlying mechanisms related to GVHD and GVL effects, mainly focusing on recent advances in strategies for enhancing and preserving anti-leukemia activity without aggravating GVHD, especially approaches aimed at the separation of GVL effects from GVHD in preclinical mouse models and in the clinic.

#### MECHANISMS RELEVANT TO GVHD AND GVL EFFECTS

The pathophysiology of acute GVHD had been reviewed by several researchers (13–15), beginning with the activation of host antigen-presenting cells (APCs) by damage-associated molecular patterns and/or pathogen-associated molecular patterns expressed on damaged tissues. Activated host APCs then present host antigens to donor T cells, leading to alloactivation and inflammatory cytokine release, for example Interferon-γ (IFN-γ) and lipopolysaccharides (LPS). These inflammatory cytokines then recruit and induce the proliferation of additional immune effector cells, including Th1, Th2, Th17, neutrophils, and macrophages, which cause tissue injury and inflammation in a reaction that overwhelms any tolerance-promoting response from immune suppressor cells, such as regulatory T cells (Treg) (52), regulatory B cells (Breg) (22), mesenchymal stem cells (MSC) (25), and myeloid-derived suppressor cells (MDSC) (53).

The mechanisms underlying GVL are of interest (8, 54), as both T cells, natural killer (NK) cells, and cytokines, such as IFNγ and tumor necrosis factor-α, possess anti-leukemia activity. Two molecular path ways, including perforin and Fas, are mainly used by T cell to mediate cytotoxicity. CD3+CD4+ T cells utilize the Fas pathway and CD3+CD8+ T cells use both, while NK cells employ the perforin pathway. Interestingly, all of these cells also play an antileukemia role via cytokine release. Recently, more attention has been focused on the role of γδT cells (55), and iNKT cells in GVL effects. The target antigens for alloreactive T cells include major histocompatibility complex (MHC) and multiplex immunohistochemistry (miHC), or leukemia-associated antigens (56). The importance of MHC and miHC antigens in GVL is underlined by the close association between GVHD and GVL, although selective miHC antigens are considered to be attractive targets for anti-leukemia immunotherapy. More recently, a number of studies have demonstrated that the overall balance between regulatory cells, including Treg, MDSC, and effector cells might be related to the extent of organ damage in GVHD settings and the effects of GVL in anti-leukemia settings (**Figures 1**–**3**) (21, 22, 25, 52, 53, 57).

#### STRATEGIES FOR ENHANCING AND PRESERVING ANTI-LEUKEMIA EFFECTS WITHOUT AGGRAVATING GVHD IN PRECLINICAL MODELS

Several strategies, such as the use of cytokines (59), selectively depletion of alloreactive T cells, regulatory immune cells (60, 61), and pharmacological agents, such as bortezomib and azacitidine (AZA), have been investigated to enhance and preserve the antileukemia effects without aggravating GVHD after allo-HSCT (**Table 1**) (31, 72, 73).

#### Cytokines

Granulocyte colony-stimulating factor (G-CSF) is widely used during transplantation to mobilize hemopoietic stem cells, which is also a mediator of T cell tolerance (74, 75). Using a murine leukemia model, several researchers have demonstrated that G-CSF mobilization of peripheral blood stem cell transplantation could maintain GVL effects through T cells via a perforindependent pathway and/or NKTs and prevent GVHD by reducing systemic levels of LPS and TNF-α as well as inducing a type 2 cytokine profile, CD34+ monocyte, and tolerogenic APCs (19, 76). Subsequent studies have shown that allografts mobilized by G-CSF analogs, such as pegylated G-CSF and progenipoietins (engineered chimeric G-CSF and Flt-3L protein), have marked tolerogenic properties that reside in the T cell and APC compartments. Additionally, mobilization with G-CSF analogs allows the concurrent enhancement of NKT cell numbers and activities, promoting host DC activation and subsequent CD8-dependent GVL effects while promoting the generation of Tregs to prevent CD4-dependent GVHD.

Except for G-CSF and its anlogs, other cytokines (77), including KGF, IL-11, IL-18 (28), IL-35 (62), and interleukin-12/23p40 (78), can also be used to separate GVL effects from GVHD in animal models. Moreover, the roles in GVHD of IL-21 and IL-22, two proinflammatory cytokines produced by Th17 cells, have been assessed in several studies (79–81). Couturier et al. (59) and Hanash et al. (79), respectively, demonstrated that IL-22 deficiency in donor T cells and abrogation of donor T-cell IL-21 signaling, could alleviate murine acute GVHD mortality while sparing the GVL effects. Hartung et al. (82) indicated that allografts mobilized by G-CSF plus stem cell factor exerted significantly enhanced antileukemic activity compared with those harvested after treatment with G-CSF

alone, suggesting that a combination of different cytokines may be a better strategy for the separation of GVL effects from GVHD.

# Depletion of Alloreactive Cells

To investigate the subsets of T cells that were effector cells with anti-leukemia effects without causing GVHD, a murine transplant model of chronic phase chronic myelogenous leukemia was generated. Zheng et al. (63) found that CD4+CD62L−CD44+CD25− effector memory T cells (CD4+ TEMs), but not naïve T cells (TN), unprimed to recipient cells mediated GVL without causing GVHD, because they retained key cytolytic functions but lacked other features that are pivotal for initiating GVHD. In another study, Chen et al. (83) reported that sorted CD45RB+CD62L+CD44+ central memory T cells (TCM, a mix of CD4+ and CD8+ cells) did not cause GVHD in a fully MHC-mismatched transplant mouse model. However, using the same model as Chen et al. (83), Zheng et al. (64) demonstrated that highly purified CD8+ TCM induced GVHD, albeit less severe than that induced by TN. However, CD8+ TCM also contribute to GVL.

More recently, using multiple GVHD models (two murine allogeneic HCT models and a human → mouse xenogeneic HCT model), Ni et al. (58) showed that CD4+ T cell depletion increased serum IFN-γ levels, leading to an upregulation of PD-L1 in recipient tissues and donor CD8+ T cells. In GVHD target tissues, they also found that increased PD-L1/PD-1 interactions between recipient tissues and donor CD8+ T cells led to T cell exhaustion and apoptosis, thereby preventing GVHD. In lymphoid tissues, enhanced PD-L1/CD80 interactions between CD8+ T cells augmented T cell survival and expansion and preserved the GVL response. In summary, the data reported by Ni et al. (58) suggested that the separation of GVL effects from GVHD could be ascribed to the PD-L1–mediated effect on CD8+ T cells depending on whether CD4+ T cells were present, the nature of the interacting partner expressed by CD8+ T cells, and the tissue microenvironment (**Figure 1**) (19, 21).

In the clinic, depletion of TN from stem cell allografts has been successfully used to reduce the incidence of chronic GVHD, while preserving the transfer of functional T cell memory (84). Overall, these results suggest that depletion of alloreactive T cells may represent a promising method to preserve GVL effects with decreasing or without causing GVHD.

#### Adoptive Transfer of Effective Immune Cells

Adoptive transfer of effective cells represents another strategy for the separation of GVL effects from GVHD. Olson et al. (85)

suppressor cells; ILC2, group 2 innate lymphoid cells.

demonstrated that donor T cells exhibited reduced proliferation, CD25 expression, and IFN-γ production in the presence of NK cells. In addition, activated NK cells mediated direct lysis of reisolated GVHD-inducing T cells in vitro, both of which lead to the alleviation of GVHD. In addition, the GVL effects were maintained in the presence of NK cells. Using mismatched hematopoietic transplant models, Ruggeri et al. (86) demonstrated, for the first time, that donor-versus-recipient NK cell alloreactivity could eliminate leukemia relapse by killing host lymphohematopoietic cells, and protect patients against GVHD by eliminating recipient-type APCs. The effects of NK cells in enhancing anti-leukemia activity and mitigating GVHD has also confirmed by other researchers. Ghosh et al. (87) reported that adoptively transferred donor-type unsorted TRAIL+ T cells could potentially enhance the curative potential of allo-HSCT by increasing the GVT response via fratricide of alloactivated T cells and suppressing GVHD through limiting alloreactive T cell expansion.

Recently, CAR T-cells have been shown to possess a novel adoptive immune therapy (40, 88). Both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells. Jacoby et al. (88) found that, in a mouse model, CAR-mediated acute GVHD was only observed in the presence of leukemia, suggesting that CAR-target interactions induced GVHD. Additionally, Ghosh et al. (40) demonstrated that allogeneic donor CD19-specific CD28z CAR T cells could promote anti-lymphoma activity by non-alloreactive cells, which retained activity against CD19+ targets, with minimal GVHD by exhaustion and eventual deletion of the alloreactive CAR-T cells. They also reported that first-generation and 4-1BB-costimulated CARs increased GVHD. Overall, the data obtained from the mouse models suggest that CAR T cells could be used to enhance the anti-leukemia response, although its' effects on GVHD remain controversial.

# Regulatory Immune Cells

Regulatory cell subsets, including Tregs, Bregs, MDSCs, and MSCs, may not only control immune homeostasis, but they also reduce detrimental T cell responses to foreign antigens. In 2003, Edinger et al. (52) observed that, in a mouse model, CD4+CD25+ Tregs could suppress the early expansion of alloreactive donor T cells, their IL-2-receptor alpha-chain expression and their capacity to induce GVHD without compomising their GVT effects, mediated primarily by the perforin lysis pathway of T conv cells. Interestingly, recipient-type specific Tregs could also control GVHD while favoring immune reconstitution and maintaining GVL effects. (89) In addition, Zheng et al. (90) reported that ex vivo-induced CD8hi Tregs controlled GVHD in an allospecific manner by reducing alloreactive T cell proliferation as well as decreasing inflammatory cytokine and chemokine secretion within target organs through a CTLA-4 dependent mechanism in humanized mice. Currently reported data in the literatures suggest that Tregs might be the most important regulatory cells in preventing GVHD (4) through a series of approaches, including aurora A/JAK2 inhibition (91, 92), selective TNFR2 activation (93), DR3 signaling modulation (94), activated protein C signals (95), and IL-2 (96), which can be used to alleviate GVHD through a Tregs-dependent mechanism.

MDSCs are a heterogeneous group of immature immunosuppressive cells of the myeloid lineage, which can induce immunosuppressive cells such as Tregs and skew macrophages toward a proinflammatory type 2 phenotype via TABLE 1 | Representative approaches for the separation of GVHD and GVL in preclinical models.

(Continued)

TABLE 1 | Continued

GVL, graft-versus-leukemia; GVHD, graft-versus-host disease; Tregs, regulatory T cells; aGVHD, acute GVHD; G-CSF, granulocyte colony-stimulating factor; DCs, dendritic cells; TEM, effector memory T cell; TN, naïve T cells; IFN-γ , interferon-γ ; CAR-T, chimeric antigen receptor T; MSCs, mesenchymal stem cells; MDSCs, myeloid-derived suppressive cells; pDCs, plasmoid dendritic cells; TNF-α, tumor necrosis factor-α

IL-10 production. MDSCs can also suppress T-cells via arginase-1, NO, reactive oxygen species, heme oxygenase-1, TFG-β and IL-10, as well as promote Tregs. Highfill et al. (53) found that MDSCs generated in the presence of IL-13 could inhibit GVHD, migrate to sites of allopriming, and limit the activation and proliferation of donor T cells as well as induce a type 2 T cell response that was indispensable for GVHD prevention, but they did not diminish the GVL effect of donor T cells.

Another type of regulatory immune cells is the MSCs, which can inhibit the activation, proliferation, and function of T cells via arginase-1, NO, reactive oxygen species, chemokines, TGFβ, and IL-10. Interestingly, in vivo experiment have shown that MSCs are actively induced to undergo perforin-dependent apoptosis by recipient phagocytes that produced indoleamine 2,3-dioxygenase, which was essential to initiate MSC-induced immunosuppression (97). Directing the migration of MSCs by CCR7 from their broad battle field (inflammatory organs) to the modulatory center of the immune response could attenuate GVHD by exerting immunosuppressive effects on T cells, while preserving GVL effects by sparing the NK cell activity that contributes to GVL effects (25, 98).

Bregs can suppress immunopathology by prohibiting the expansion of pathogenic T cells and other pro-inflammatory lymphocytes through the production of IL-10, IL-35, and TGF-β (99). Our group showed that, in the acute GVHD mouse model, cotransplantation of Bregs prevented onset by inhibiting Th1 and Th17 differentiation and expanding regulatory T cells. In the GVL mouse model, Bregs contributed to the suppression of acute GVHD but had no adverse effects on GVL activity (22).

Excluding the abovementioned regulatory cells, group 2 innate lymphoid cells (ILC2) make up a large portion of the ILC population, which can polarize T cells to Th2 cells by secreting IL-4, and macrophages or DCs to an macrophage 2 or type 2 chemokine-secreting phenotype by secreting IL-13, respectively (100). ILC2 can alleviate GVHD by reducing donor Th1 and Th17 cells as well as accumulating MDSCs mediated by IL-13. Moreover, ILC2 do not inhibit the GVL response (101).

In summary, these preclinical studies suggest that cotransplantation or adoptive transfer of regulatory cells could be successfully used to alleviate GVHD without compromising the GVL effects. Therefore, pilot studies are warranted to evaluate the safety and feasibility of these regulatory cells in preventing and/or treating GVHD as well as preserving GVL effects in clinic.

#### Signaling Pathways

Several signaling pathways have been demonstrated to be correlated with T cell function. Janus kinases (JAKs) are intracellular signaling components of many type I/II cytokines (102, 103). There are 4 members of the JAK family that regulate the development and function of immune cells, including DCs, macrophages, T cells, B cells, and neutrophils, of which JAK1, JAK2, and JAK3 may be most relevant for the pathophysiology of GVHD (51). In murine models of GVHD and leukemia or lymphoma relapse, treatment with ruxolitinib reduced GVHD in the skin, liver, and gastrointestinal organs while preserving GVL activity, leading to improved survival (44, 104, 105). Betts et al. (91) found that the transfer of JAK2−−/−− donor T cells to allogeneic recipients led to attenuate GVHD by inhibiting Th1 differentiation, promoting Th2 polarization, and increasing and/or stabilizing CD8+ iTreg, yet it maintained GVL effects (106). In addition, pacritinib, a multikinase inhibitor with potent activity against JAK2, could significantly reduce GVHD and xenogeneic skin graft rejection in distinct rodent models and maintain donor anti-tumor immunity. Overall, these data suggest that JAK inhibition or other compounds, such as TG101348 (92), represents a new and potentially clinically relevant approach to separate GVL effects from GVHD.

Excluding JAKs, increasing data have demonstrated that targeting signaling pathways, such as the PKCα and PKCθ (66), MEK (68), NFAT (65), and IRE-1a/XBP-1 pathway (67), ikaros (107), toll-like receptor/myeloid differentiation factor 88 (108), DR3 signaling (94), and activated protein C signals (95), might provide strategies for alleviating GVHD, while enhancing or without compromising the GVL effects.

#### Pharmacological Agents

The roles played by biological agents in the separation of GVL effects from GVHD have been investigated in animal models (38, 71). Sun et al. (38) demonstrated that bortezomib might rapidly induce the preferential deletion of very highaffinity alloreactive T cells, thus allowing for expansion of the remaining T cells to maintain GVT responses yet with a reduced potential for promoting GVHD. Ehx et al. (71) found that AZA significantly decreased human T-cell proliferation as well as IFNγ and TNF-α serum levels, and it reduced the expression of GRANZYME B and PERFORIN 1 by cytotoxic T cells, leading to the prevention of GVHD. AZA could also induce the expression of tumor antigens by AML cells, leading to the generation of donor-derived tumor specific cytotoxic T cells, which have been demonstrated to prevent AML relapse (70). In addition, Stokes et al. (69) reported that bendamustine could alleviate GVHD by enhancing MDSC suppressive function without compromising GVL effects.

Caballero-Velázquez et al. (30) showed that the combination of sirolimus and bortezomib synergistically inhibited both the activation and proliferation of stimulated T cells. Additionally, the production of Th1 cytokines (IFN γ, IL-2, and TNF-α) was significantly inhibited. This effect was due, at least in part, to the inhibition of Erk and Akt phosphorylation. In vivo, the combination reduced the risk of GVHD without hampering GVL effects, as shown in mice that received GVHD prophylaxis with sirolimus plus bortezomib infused with tumor WEHI cells plus C57BL/6 donor BM and splenocytes. Overall, this study suggests a synergistic effect of the combination different pharmacological agents to prevent GVHD while maintaining the GVL effect.

In summary, experiment results from mouse models suggest that effective and regulatory immune cells play a key role in separation of GVL effects from GVHD. The approaches explored in preclinical settings have demonstrated, for example, that cytokines or inhibitors targeting signaling pathways of T cells might enhance and/or preserve antleukemia effects without compromising GVHD through regulating the functions of effective and regulatory immune cells (**Figures 2**, **3**).

#### STRATEGIES FOR ENHANCING AND PRESERVING ANTI-LEUKEMIA EFFECTS WITHOUT AGGRAVATING GVHD IN THE CLINIC

Several approaches, including donor selection, conditioning regimens, graft engineering and adoptive transfusion of immune cells, have been successfully used in the clinic to separate GVL effects from GVHD before and after allo-HSCT (**Figure 4**).

#### Donor Selection

In unrelated donor transplantation settings, Kawase et al. (109) suggested that donor selection made in consideration of these results might allow the separation of GVL from acute GVHD in patients with AML, ALL, or those with chronic myeloid leukemia (CML), especially in HLA-DPB1 mismatch combinations. Fleischhauer et al. (110) further demonstrated that avoidance of an unrelated donor with a non-permissive T-cell-epitope mismatch at HLA-DPB1 might contribute to a lower risk of mortality. In cord blood transplant modality, HLA-DPB1 mismatch was also associated with a significant reduction of leukemia relapse (HR 0.61, P = 0.001), and no significant effect of HLA-DPB1 mismatch was observed on the risk of acute GVHD, engraftment or mortality (111). Laghmouchi et al. (112) suggested that the allo-HLA-DP-specific T cell repertoire contained T cells with restricted recognition of hematopoietic cells, which might contribute to specific GVL effector reactivity without coincident GVHD (112).

In T cell depleted haplo-SCT settings, Ruggeri et al. (86) showed that increased NK cell alloreactivity in humans, based on the "missing self " model, was associated with a decreased CIR and improved survival in patients with AML but not in patients with ALL. In contrast, Huang et al. (113) following the Beijing Protocol, demonstrated that host MHC class I could determine NK cell responses. The functional recovery of donor-derived NK cells was higher in recipients that expressed ligands for donor inhibitory KIRs, and a high functional NK recovery correlated with better relapse control (114). In haplo-SCT with PT/Cy settings, Shimoni et al. (115) also demonstrated a trend toward higher relapse rates in patients with KIR ligand mismatching (HR 1.36, P = 0.09) in a total group of 444 acute leukemia patients. This trend was observed in patients with AML (HR 1.48, P = 0.07) but not in those with ALL (HR 0.95, P = 0.88).

In summary, these data suggest that donor selection according to HLA-DPB1 mismatch, NK cell alloreactivity, and other variables (116–119), could represent a strategy for the separation of GVL effects and GVHD, although further studies are still needed.

#### Conditioning Regimen

More recently retrospective registry studies and some, but not all, prospective randomized trials have demonstrated increased relapse rates in recipients of an RIC compared with an MAC regimen in patients with AML and MDS who underwent allo-HSCT (120, 121). However, these finding remain controversial (3). In a multicenter randomized controlled trial (122), 178 HR-AML patients received haplo-HSCT with conditioning regimens involving recombinant human G-CSF or non-rhG-CSF. The cumulative incidences of acute GVHD, chronic GVHD, transplantation-related toxicity, and infectious complications appeared to be equivalent. The 2-year probabilities of LFS and OS in the G-CSF-priming and non-rhG-CSF-priming groups were 55.1 vs. 32.6% (P < 0.01) and 59.6 vs. 34.8% (P < 0.01), respectively. This study suggests that the G-CSF-priming conditioning regimen is an acceptable choice for HR-AML patients, which may lead to partially separation of GVL effects from GVHD.

Overall, considering the central importance of regimen in determinng leukemia relapse risk based on the biological characteristics of disease and pretransplantation minimal residual disease (MRD), there remains an urgent need for randomized comparisons of different conditioning regimens to separate GVL from GVHD.

#### Allograft Engineering

In a single-arm clinical trial, 35 cases patients with high-risk leukemia received naïve T cell-depleted G-PBSCs following a myeloablative conditioning regimen. GVHD prevention includes tacrolimus immunosuppression alone. Bleakley et al. (42) reported that all the cases engrafted. GVHD in these patients was universally corticosteroid responsive, although the incidence of aGVHD was not reduced. Chronic GVHD was remarkably infrequent (9%) compared with historical rates of ∼50% with T cell-replete grafts. Memory T cells in the graft resulted in rapid T cell recovery and the transfer of protective virus-specific immunity. No excessive rates of infection or relapse occurred, and the OS was 78% at 2 years. These results suggest that the depletion of naïve T cells from allografts not only reduces the incidence of cGVHD but also preserves the transfer of functional T cell memory.

To decrease the incidence of GVHD in haploidentical allograft settings, the Perugia group established a protocol that includes TCD and a graft containing a mega-dose of highly purified CD34+ cells (average 10 × 106 /kg body weight), which is administered following a myeloablative conditioning regimen (86). This protocol ensures a high engraftment rate, despite the HLA barrier, without triggering GVHD. However, the benefit (the absence of GVHD) from this CD34 selected haplotype transplant approach is offset by a very slow immune recovery due to the small number of T cells infused and the ATG application, which result in high rates of opportunistic infections, such as viral and fungal infections, leading to a high TRM. To accelerate immune recovery, the Perugia group demonstrated, for the first time, that the adoptive transfer of Tregs promotes lymphoid reconstitution and improves immunity to opportunistic pathogens without weakening the GVL effects in the TCD haploidentical setting (16). This finding suggests that the adoptive transfer of gene modified T cells and/or pathogen-specific T cells may be needed to improve clinical outcomes. In a phase II study, researchers from Germany found that haplo-SCT with a negative CD3/CD19 depletion and reduced intensity conditioning allowed for a successful transplantation in an older, heavily pretreated patient population (16). The estimated 2-event free survival was 25%. The incidence of grade II-IV aGVHD was 46%, and the incidence of cGVHD was 18%. Therefore, new strategies are needed to further establish novel strategies for the separation of GVL effects and GVHD.

Luznik et al. (123) summarized that in vivo cyclophosphamide posttransplantation (PT/Cy) could induce the destruction of peripheral, alloantigen-reactive T cells, while a relative resistance of donor Teff/memory T cells to PT/Cy, as demonstrated in mice, might contribute to the overall reconstitution of peripheral T-cell pools and immune competence over the long term. These results suggest that in vivo allograft engineering with PT/Cy represents a novel method for GVL and GVHD separation, and it has been widely used in haploidentical and HLA-matched sibling donor transplant settings (6, 124).

#### Adoptive Transfusion of Immune Cells

Currently, adoptive transfusion of immune cells, such as donor lymphocyte infusion (DLI), cytotoxic T lymphocyte (CTLs), NK cells, and CAR-T, had been successfully used to separate GVL effects from GVHD.

#### DLI

In 1990, Kolb et al. (125) first reported sustained remission after DLI in patients with CML who relapsed after allo-HSCT. Since then, DLI had become the mainstay allogeneic cellular therapy. The NCI recommendations list DLI as the routinely considered method for patients who relapsed after allo-HSCT and do not have GVHD (126). Based on immune tolerance induced by G-CSF, such as the ability to polarize T cells from the Th1 to the Th2 phenotype and the hyporesponsiveness of T cells, Huang's group established a modified DLI protocol (127) that includes the following: (i) the use of G-CSF mobilized peripheral blood stem cell harvests (G-PBSCs) instead of a steady lymphocyte infusion; (ii) the introduction of short-term immune suppressive agents, including cyclosporine A (CSA) or methotrexate (MTX), to further decrease the incidence of GVHD. Impressively, the feasibility and efficacy of the modified DLI were confirmed either for treatment or prevention of relapse after haploidentical HSCT (127). Our group also demonstrated that MRD-directed DLI could significantly decreased the relapse rate without aggravating GVHD (128). The use of DLI was also demonstrated in patients who underwent haploidentical HSCT with PT/Cy (129).

Recently, Nikiforow et al. (130) undertook a phase I study of DLI depleted of CD25+ T cells in 21 patients with hematologic malignancies who had relapsed after allo-HSCT. Two dose levels were administered: 1 × 107 (n = 6) and 3 × 107 CD3+ cells/kg (n = 15). A median 2.3 log-depletion of Tregs was achieved. Seven subjects (33%) developed clinically significant GVHD by 1 year, including one patient who died. At dose level 1, five subjects had progressive disease and one had stable disease. At dose level 2, nine subjects (60%) achieved or maintained responses (8 CR, 1 PR), including seven with active disease at the time of infusion. A shorter period between relapse and infusion was associated with the response at dose level 2 (P = 0.016). The 1-year survival rate was 53% among patients treated with dose level 2. Four of eight subjects with AML remained in remission at 1 year. When compared to unmodified DLI in 14 contemporaneous patients meeting study eligibility, CD25/Treg depletion was associated with a better response rate and improved EFS.

Overall, the available data suggest that DLI represents a widely used approach in prophylactic, pre-emptive therapy and therapy for relapse either in HLA-matched HSCT or in haploidentical transplant settings. Furthermore, CD25/Tregdepleted DLI appears to be feasible and capable of inducing GVL effects without excessive GVHD (131), although multicenter, prospective study are warranted to confirm the results.

#### Leukemia Specific CTLs

Researchers from Italy have investigated the feasibility of expanding/priming p190BCR-ABL–specific T cells in vitro by stimulation with DCs pulsed with p190BCR-ABL peptides derived from the BCR-ABL junctional region and alternative splicing, and of adoptively administering them to patients with relapsed disease (132). Three patients were enrolled in this study. Patient 1 was a 61-year-old man experiencing a second molecular recurrence after matched unrelated donor (MUD) alloHSCT and unmanipulated DLIs. Patient 2 was a 30-yearold man diagnosed with Ph+ ALLwith hyperleukocytosis and central nervous system (CNS) involvement, experiencing his third hematologic relapse (BM blast 66%, F317L mutation) after MUD-HSCT, DLI, and rescue therapy with nilotinib. Patient 3 was a 62-year-old woman diagnosed with Ph+ ALL with CNS involvement, showing persistent molecular disease (last MRD before T-cell therapy 0.1% BCR-ABL/ABL) after induction, maintenance chemotherapy, and prolonged TKI treatment. She was not eligible for alloHSCT due to comorbidities. The results showed no postinfusion toxicity, except for a grade II skin GVHD in the patient who was treated for hematologic relapse. All patients achieved a molecular or hematologic CR after Tcell therapy, upon emergence of p190BCR-ABL-specific T cells in the BM. These results demonstrate that p190BCR-ABLspecific CTLs are capable of controlling treatment-refractory Ph+ ALL in vivo, and they support the development of adoptive immunotherapeutic approaches with BCR-ABL CTLs in Ph+ ALL. Therefore, further studies including large sample sizes are needed to confirm the abovementioned results.

Excuding BCR-ABL CTLs, the anti-leukemia effects of WT1 specific CTL were also observed in 11 relapsed or high-risk leukemia patients who underwent allo-HSCT (133). Chapuis et al. (133) found that CD8+ transferred T cells with a memory phenotype could be detected after long-term follow-up. An approach to generate multi-TAA-specific CTLs using peptide libraries of 15-mer peptides overlapping by 11 amino acids spanning the whole amino acid sequence of a target antigen was developed by Weber et al. (134) They also showed that TAAmixspecific CTLs could inhibit the colony formation of leukemia blasts. In summary, leukemia-specific CTLs might be a promising method for enhancing anti-leukemia activity.

#### NK Cells

The role played by NK cells in anti-leukemia activity had been fully investigated in allo-HSCT settings. In a dose-escalation study, Choi et al. (43) showed that, when given 2–3 weeks after haploidentical HSCT, donor-derived NK cells were welltolerated at a median total dose of 2.0 × 108 cells/kg. In a phase I study, the safety of haploidentical third-party NK cell infusion was further confirmed in 21 patients with high-risk myeloid malignancies who received a preparative regimen with busulfan and fludarabine followed by infusion of IL-2-activated NK cells with a dose ranging from 0.02 to 8.32 ×106 /kg. Lee et al. (135) demonstrated that five patients were alive, and 5 and 11 cases had died from transplant-related causes and relapse, respectively. Among the total patients, only 5 cases developed a maximum acute GVHD of grade 2, and 2 cases grade 3 GVHD. These results indicated that the infusion of third-party NK cells was well-tolerated and did not increase the rate of GVHD after allo-HSCT. Ciurea et al. (136) initiated a phase 1 dose-escalation study of membrane-bound interleukin 21-expanded donor NK cells infused before and after haploidentical HSCT for highrisk myeloid malignancies. NK cells were infused on days −2, +7, and +28 posttransplant. All NK expansions achieved the required cell number, and 11 of 13 patients enrolled received all 3 planned NK-cell doses (1 × 105 /kg to 1 × 108 /kg per dose). No infusional reactions or dose-limiting toxicities occurred. All patients were engrafted with donor cells. Seven patients (54%)

developed grade I-II acute GVHD (aGVHD), and no patients developed grade III-IV aGVHD or chronic GVHD. All other patients were alive and in remission at the last follow-up (median, 14.7 months). Overall, this trial demonstrated the production feasibility and safety of infusing high doses of ex vivo-expanded NK cells after haploidentical HSCT without adverse effects, increased GVHD, or higher mortality, which was associated with significantly improved NK-cell numbers and function, fewer viral infections, and a low relapse rate posttransplant. Further study deomonstrated that CD56+ donor cell infusion after PT/Cy and short-course cyclosporine were feasible with prompt engraftment, rapid reconstitution of CD4+ T, Tregs and NK cells and a reduced incidence of relapse and acute GVHD (137).

#### CAR-T Cells