Alzheimer's disease (AD) is a devastating age-related neurodegenerative disorder, affecting over 34 million people worldwide (1). AD is characterized by progressive loss of memory and cognitive functions (1–5). The cognitive decline in AD is associated with hallmark protein aggregates, amyloid-beta (Aβ) plaques and neurofibrillary tangles, which are accompanied by neuroinflammation, and synaptic and neuronal loss (1–5). Aβ plaques play a central role in the pathogenesis of AD and are generated from proteolytic cleavage of amyloid precursor protein (APP) (6, 7). Aβ can accelerate neuronal cell death and neuronal tangle formation, affect synaptic function adversely and eventually cause neuron loss (2–4, 8). The accumulated Aβ plaques and neuroinflammation have led to numerous attempts over the years to treat AD, either by removing the Aβ plaques, or by systemic anti-inflammatory drug administration to arrest brain inflammation. However, the drugs tested thus far for AD have largely failed in the clinic (4, 9–12). These failures suggest that, although removal of Aβ plaques may be important, this approach alone is not enough to arrest or reverse cognitive loss. Furthermore, recent data also suggest that mitigating neuroinflammation in AD necessitates stimulation, rather than suppression of the immune system, to drive an immunedependent cascade needed for the Aβ clearance and brain repair (4, 13, 14). It has been shown that anti-inflammation is an active mechanism mediated by recruitment of circulating immune cells to sites of brain pathology (15–17). In addition, systemic immune deficiency is associated with cognitive dysfunction (18) and accelerated AD pathology (19, 20).

T cells are the major component of the immune system. Multiple lines of evidence have suggested that T cells play an important role in the CNS maintenance and repair. For example, systemic T cell deficiency is associated with increased neuronal loss in animal models of CNS injury or AD (14, 19, 21). Systemic T cells not only participate in CNS repair, but are also needed for life-long brain plasticity (18, 22, 23). Both T cells and monocytederived macrophages recognizing brain antigens are required for coping with and helping heal brain damage during central nervous system (CNS) injuries (24–29). T cells present in the periphery play an important role in adaptive–innate immunity cross-talk and help in CNS repair (16, 19, 20). Furthermore, it has been suggested that autoreactive T cells that recognize CNSspecific antigens augment the recruitment of monocyte-derived macrophages to the brain (14, 26).

The thymus is the primary organ for T cell generation. It, however, undergoes age-dependent thymic involution, resulting in decreased numbers of T cells in the elderly. This reduction has direct etiological linkages with many diseases (30–34), including acceleration of the development and progression of AD (19). T cell development in the thymus depends on the thymic microenvironment, in which thymic epithelial cells (TECs) are the major component (35–41). However, TECs undergo both qualitative and quantitative loss over time, which is believed to be the major factor responsible for age-dependent thymic involution (30–34). It is well-known that embryonic stem cells (ESCs) have the dual ability to propagate indefinitely in vitro in an undifferentiated state and to differentiate into many types of cells (42). We have reported that ESCs can be selectively induced to generate TEPs in vitro (43–46). When transplanted into young or old mice, the ESC-TEPs further develop into TECs, reconstitute the normal thymic architecture, and promote T cell generation, resulting in increased number of functional T cell in the periphery (43–46).

We hypothesized that AD aged mice and patients have a very severe defect in the thymic microenvironment and that transplantation of ESC-TEPs into AD mice would rejuvenate the aged thymic microenvironment, leading to an increased number of functional T cell in the periphery, resulting in attenuated AD pathology. It is well-known that TECs, especially medullary TECs (mTECs), are involved in the deletion of autoreactive T cells. We have demonstrated that transplantation of ESC-TEPs expressing disease-causative self-antigen results in the deletion of the antigen-specific autoreactive T cells (47, 48). Our hypothesis further proposes that transplantation of APP gene-deleted ESC-TEPs would lead to the generation of Aβ-specific autoreactive T cells that could help the production of other Aβ-specific immune cells to clear the Aβ plaques in the CNS.

We show here that transplantation of APP gene deleted (APP−/−) or their wild-type (APP+/+) mouse ESC (mESCs) derived-TEPs results in enhanced thymopoiesis, increased T cell number, especially IFN-γ-producing cells, in the periphery, enhanced choroid plexus (CP) gateway activity, and enhanced recruitment of macrophages into the brain. Consequently, these mice have reduced Aβ deposits in the brain and improved cognitive performance. Furthermore, transplantation of APP−/− mESC-TEPs has a greater effect than that of APP+/+ mESC-TEPs in clearance of Aβ deposits in the CNS and reversal of cognitive decline. This is related to the generation of Aβ-specific T cells, increased numbers of anti-Aβ antibody (Ab)-producing B cells in the spleen, increased levels of anti-Aβ Ab in the serum, and enhanced function of macrophages to phagocytose Aβ in the brain. Our results suggest that human ESC (hESC) or induced pluripotent stem cell (iPSC)-derived TEPs, especially APP−/− hESC or iPSC-TEPs, may serve as a novel tool to modify AD pathology.

#### MATERIALS AND METHODS

#### Mice

3xTg-AD, APP/PS1, C57BL/6 (B6) mice were purchased from Jackson Laboratory. The mice were used in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Connecticut.

 For TEP differentiation, mESCs were first induced to differentiate into definitive endoderm, and then TEPs in the presence of BMP-4, FGF 7, FGF10, and EGF, as well as rFOXN1 and rHOXA3 protein as we previously described (43).

#### Genome Editing

The APP gene in mESCs was knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) genome editing. B6 mESCs were transfected with APP-specific double nickase plasmids or control double nickase plasmids (from Santa Cruz Biotechnology). The cells were screened to obtain APP−/− and APP+/+ mESCs. The information of the plasmids and gRNA sequences are shown in **Supplemental Figure 1**.

#### Intrathymic Injection

Mice were anesthetized and injected with 5 × 104 cells in 10–20 µl PBS into the thymus posterior to the upper sternum using a 26–28 gauge needle as described (49).

### Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Real-Time Qualitative RT-PCR (qRT- PCR)

Total RNA was extracted from tissues or cells using a Nucleo Spin RNA II kit (Macherey-Nagel, Düren, Germany). The RNA was converted into complementary DNA using High Capacity cDNA Reverse Transcription Kit (Invitrogen, USA). RT-PCR was performed with GoTaq R Green Master Mix (Promega, USA). qRT- PCR was performed with the Power SYBR green master mix (Applied Biosystems, UK) using the 7500 real-time PCR system (Applied Biosystems, UK). The primer sequences are shown in **Supplemental Table 1**.

# Western Blot Analysis

GFP+ mESC-TECs were purified from the thymocytes using a magnetic-activated cell sorter immunomagnetic separation system (Mitenyi Biotec). The cells were collected and lysed. Equal amounts of denatured proteins were loaded onto a 4– 12% Bis-Tris gel (Invitrogen, Carlsbad, CA), electrophoresed and transferred onto a PVDF membrane (Invitrogen). The membranes were blocked with 5% nonfat milk in TBST (mixture of Tris-Buffered Saline and Tween 20), and incubated with anti-mouse APP monoclonal antibody (Invitrogen) at 4 degree overnight. The membranes were then incubated with goat antimouse IgG HRP-conjugated secondary antibody and developed with a SuperSignal West Pico chemiluminescence substrate (Thermo Scientific, Rockford, IL).

#### Immunohistochemistry

The brain tissues were incubated in a fixative solution, embedded in OCT medium, snap frozen, and subsequently cut into 6µm sections. The cultured cells were incubated with primary antibodies. The following primary antibodies were used: mouse anti-Aβ (clone 6E10,) and rabbit anti-GFAP (Biolegend, USA). After washing, the sections were incubated with fluorochromeconjugated secondary antibody, counterstained with 4′ , 6′ diamidino-2-phenylindole (DAPI) and observed under a Nikon A1R Spectral Confocal microscope (Nikon, Kanagawa, Japan). To quantify the staining intensity, total cells and background fluorescence intensity were measured using ImageJ software (NIH, USA), and the intensity of specific staining was calculated as described (4).

# Flow Cytometry Analysis

To analyze TECs, the thymi were incubated at 37◦C in 0.01 (w/v) liberase (Roche, Nutley NJ) and 0.02% (w/v) DNAse I (Roche) with regular and gentle agitation as described (50). A single-cell suspension of tissues was stained with fluorochromeconjugated antibodies directly or indirectly as described (51). For intracellular staining, the cells were first permeabilized with a BD Cytofix/Cytoperm solution for 20 min at 4◦C. The following antibodies were used: CD4, CD8, EpCAM1, CD45, CD11b, F4/80, IFNγ, Ly6c, and SRA1 (BioLegend, San Diego, CA, or ThermoFisher Scientific), Keratin (K) 5 (Covance, Dallas, TX), and K8 (US Biological, Salem, MA). The samples were analyzed on an LSRFortessa X-20 Cell Analyzer (BD Biosciences). Data analysis was performed using FlowJo software (Ashland, OR).

# T Cell Proliferation Assay

Splenocytes were stained with 5µM CFSE (ThermoFisher Scientific) for 15 min. at 37◦C. The cells were then cultured in a 96-well flat-bottom plate in the presence of plate-bound Aβ40 or Aβ42 (Anaspec, USA) and anti-CD3 antibody for 3 days. The cells were then stained with anti-PE labeled-CD4 and APC labeled-CD8 antibodies and analyzed for CFSE levels by T cells using flow cytometry.

# ELISA Assay for Anti-Aβ40 or Anti-Aβ42 Antibody

Aβ40 or Aβ42 (Anaspec, USA) was coated on 96-well microplates overnight at 4◦C, then blocked with blocking buffer (2% BSA+5% goat serum in PBS) for 2 h at room temperature. The serum samples diluted into 1:1000 were added to the plates and incubated 2 h at room temperature. After washing, HRPconjugated goat anti-mouse IgG (Biolegend) was added to the plates and incubated for 1 h. The reaction was developed by TMB substrate (Thermo Scientific, USA) and stopped with 0.1 N HCl. The microplate was read at 450 nm under a microplate reader (Bio-Tek, ELX800, USA). The antibody concentrations were calculated using a standard curve generated with known concentrations of anti-Aβ antibody.

# Soluble Aβ Protein (sAβ) Isolation and Quantification

Brain parenchyma was dissected, snap-frozen and kept at −75◦C until homogenization. The samples were homogenized, and the supernatants were collected and detected for the concentrations of Aβ1−40 and Aβ1−42 by ELISA as described (13).

#### B Cell ELISpot Assay

MultiScreen-IP plates (Millipore, Billerica, MA) were washed with 70% ethanol, rinsed three times with PBS, coated with Aβ40 (4µg/ml) or Aβ42 (4µg/ml) at 4◦C overnight. The plates were blocked with blocking buffer (2% BSA in RPMI medium). 1 × 104 splenocytes were added into the plates and incubated for 48 h. The plates were washed 6 times with 0.25% Tween 20 (Sigma, USA) in PBS, incubated with HRP-conjugated goat anti-mouse IgG (H+L) (Biolegend) for 1 h, developed with a DAB Peroxidase Substrate Kit (Vector, USA), and counted for ELISpots (52).

#### Amyloid Phagocytosis Assay

HiLyte Fluor 647 Beta-Amyloid (1–42) (Anaspec) was resuspended in Tris/EDTA (pH 8.2) at 20 mM and then incubated in the dark for 3 days at 37◦C to promote aggregation. Macrophages in suspension were pretreated in low serum medium as described (53). The HiLyte Fluor 647 Beta-Amyloid was added and incubated for 5 h. Cells were stained with macrophage markers; amyloid phagocytosis by the macrophages was determined by flow cytometry (53).

#### Barnes Maze

Barnes Maze was conducted as previously described (54, 55). Briefly, each mouse was placed in the center of the maze and subjected to aversive stimuli. Mice were trained 4 training trials per day for 5 days, and a probe test was performed 24 h after the last training trial. The latency and number of errors were recorded for the training tail and probe test.

#### Novel Object Recognition (NOR) Test

A NOR test was conducted as previously described (54–56). Briefly, mice were trained by allowing them to explore two identical objects placed at opposite ends of the arena for 10 min. 24 h later, mice were tested with one copy of the familiar object and one novel object of similar dimensions for 3 min. The time spent on exploring and sniffing of each object was recorded. The NOR index represents the percentage of time mice spent exploring the novel object.

#### Statistical Analysis

P-values were based on the two-sided Student's T-test. A confidence level above 95% (p < 0.05) was determined to be significant.

### RESULTS

1. Deletion of the APP gene in mESCs to generate APP−/− mESCs and generation of TEPs from APP−/− and APP+/+ mESCs in vitro.

Since Aβ is produced from proteolytic cleavage of APP, we deleted the APP gene in mESCs using CRISPR and Cas9 genome editing. B6 mESCs were transfected with APP-specific double nickase plasmids that contain the APP-specific-guide RNAs, and the Cas9 nuclease and GFP genes. The cells were screened in puromycin to obtain APP−/− mESCs. The gene deletion was confirmed by RT-PCR with one of primers spanning the gRNA region (**Figure 1A**). The cells that were transfected with control double nickase plasmids containing non-targeting scrambled gRNA, Cas9, and GFP genes were used as a control (APP+/+ mESCs).

Both APP−/− and APP +/+ mESCs were positive for alkaline phosphatase (AP) activity, indicating that the mESCs were in an undifferentiated state (**Figure 1B**). We then induced the APP −/− and APP+/+ mESCs to differentiate into TEPs in vitro following our protocol (43). After the differentiation, both APP−/− and APP+/+ mESC-derived cells contained comparable numbers of EpCAM1 positive cells that co-expressed K5 and K8, a phenotype of TEPs (**Figure 1C**). We purified EpCAM1+ TEPs from APP−/− and APP+/+ mESC-derived cells and injected an equal number of the TEPs into the thymus of syngeneic mice. Two months later, the mESC-TEPs generated comparable numbers of GFP+ mESC-derived TECs that accounted for 51– 58% of total TECs.

Western blot analysis showed that purified GFP+ APP+/+ mESC-TECs expressed APP protein, whereas GFP+ APP−/− mESC-TECs did not (**Figure 1D** and **Supplemental Figure 2**). Together, these results indicate that the APP gene has been deleted in the APP−/− mESCs, and that the deletion does not affect the differentiation ability of mESCs into TEPs and TECs.

2. Both APP−/− and APP+/+ mESC-TEP-transplanted AD mice have an improved cognitive performance, and APP−/− mESC-TEP-transplanted mice perform better than APP+/+ mESC-TEP-transplanted mice.

To determine whether transplantation of APP−/− and APP+/+ mESC-TEPs affects cognitive performance in AD mice, 3XTg-AD mice aged 12 months, an age of advanced cerebral pathology, were injected intrathymically (i.t.) with APP−/− or APP+/+ mESC-TEPs. APP−/− or APP+/+ mESC-derived EpCAM1− non-TEPs (control cells) were used as controls. Two months later, the mice were evaluated for spatial learning and memory. It has been reported that the Barnes maze, a hippocampal-dependent spatial task (57, 58), is the most sensitive test for detecting cognitive deficits in 3XTg-AD mice (59). We found that both APP−/− and APP+/+ mESC-TEP-treated mice had significantly greater Barnes maze learning curves than control cell-treated mice (**Figure 2A**). Furthermore, APP−/− mESC-TEP-treated mice performed better than APP+/+ mESC-TEP-treated mice, almost reaching the performance level observed in wild-type (WT) non-AD mice (**Figure 2A**). Since there were no significant differences between APP−/− and APP+/+ mESC-EpCAM1− control cell-transplanted mice in all of the results in this paper (data not shown), we pooled the data from the two groups and named this group as control cell (Ctrl)-transplanted mice.

Both APP−/− and APP+/+ mESC-TEP-treated mice also had decreased latency to find the target zone during the probe trial conducted 24 h after the final training session (**Figure 2B**), indicating an improved memory performance. In addition, the number of errors committed in the APP−/− or APP+/+ mESC-TEP-treated mice was also significantly reduced, as compared to control cell-treated mice (**Figure 2C**).

The NOR test is to study learning and memory in rodents based on their spontaneous tendency to have more interactions with a novel than with a familiar object (60). NOR is a more cortically-dependent novel object recognition preference task (57, 58). In agreement with the results in the Barnes maze task, APP−/− or APP+/+ mESC-TEP-treated mice performed significantly better than control cell-treated mice (**Figure 2D**). In all of these studies (**Figures 2A–D**), APP−/− mESC-TEP-treated mice performed significantly better than APP+/+ mESC-TEPtreated mice. Together, our data suggest that transplantation of APP−/− or APP+/+ mESC-TEPs results in improved spatial learning and memory in 3xTg-AD mice and that transplantation of APP−/− mESC-TEPs demonstrates greater effectiveness than APP+/+ mESC-TEPs.

3. Both APP−/− and APP+/+ mESC-TEP-transplanted AD mice have reduced AD pathology with greater reduction in APP−/− mESC-TEP-transplanted mice.

We then determined whether transplantation of APP−/− or APP+/+ mESC-TEPs leads to improved AD pathology. After the Barnes maze and NOR tests (**Figure 2**), the brains were harvested and immunohistochemical analysis performed. We found that both APP−/− and APP+/+ mESC-TEPtransplanted 3XTg-AD mice had a reduced cerebral Aβ plaque

load in the hippocampus, specifically in the dentate gyrus (DG) and in the cerebral cortex (layer V) (**Figures 3A–C**), areas showing robust Aβ-plaque pathology in AD mice. Comparatively, APP−/− mESC-TEP-transplanted mice had the greater reduction in Aβ-plaque load (**Figures 3A–C**). Astrogliosis, as assessed by glial fibrillary acid protein (GFAP) immunoreactivity, was also reduced in APP−/− and APP+/+ mESC-TEP-treated mice, as compared to control cell-treated mice (**Figures 3A,D**). Again, APP−/− mESC-TEP-transplanted mice had, by comparison, a greater reduction in GFAP immunoreactivity (**Figures 3A,D**).

Since impaired synaptic plasticity and memory deficits in AD are associated with elevated cerebral soluble Aβ1- 40/Aβ1-42 (sAβ) levels (61), we then measured sAβ levels in the AD mice by ELISA. Consistent with the immunohistochemical results, both APP−/− and APP+/+

mESC-TEP-treated mice had reduced cerebral sAβ, as compared to control cell-treated mice (**Figure 3E**). Transplantation of APP−/− mESC-TEPs demonstrated the greater reduction (**Figure 3E**).

We likewise examined the effect of mESC-TEPs in another AD model, APP/PS1 mice, which develop Aβ-plaque pathology at a more advanced age than do 3XTg-AD mice. Transplantation of APP−/− or APP+/+ mESC-TEPs reduced hippocampal Aβ plaque load, as compared to control cell-treated mice, and APP−/− mESC-TEP-treated mice had more Aβ plaque load reduction than APP+/+ mESC-TEP-treated mice (**Figures 3F,G**). Taken together, our results suggest that transplantation of APP−/− or APP+/+ mESC-TEPs into AD mice results in clearance of Ab plaques and reversal of cognitive decline, and APP−/− mESC-TEP-treated mice perform better than APP+/+ mESC-TEP-treated mice.

4. Both APP−/− and APP+/+ mESC-TEP-transplanted AD mice have increased T cell numbers, and APP−/− mESC-TEP-transplanted mice have enhanced T cell proliferation in response to Aβ stimulation.

cell group, \*\**p* < 0.05 vs. APP+/+ mESC-TEP group.

We have previously demonstrated that transplantation of mESC-TEPs results in enhanced thymopoiesis and increased T cell numbers in the spleen (43–45). Consistent with the previous reports, APP+/+ or APP−/− mESC-TEP-transplanted mice had increased numbers of thymocytes in the thymus and T cells in the spleen compared to control cell-treated mice. The number of T cells in the thymus and the spleen between APP+/+ and APP−/− mESC-TEP-transplanted mice were not significantly different (**Figures 4A,B**).

Thymocytes can be divided into four major subsets: CD4 and CD8 double negative (DN), double positive (DP), CD4 single positive (SP), and CD8 SP thymocytes. DN thymoctyes

can be further divided into DN1 to DN4 subsets based on the expression of CD44 and CD25. APP+/+ or APP−/− mESC-TEP-transplanted mice had decreased percentages of DN subsets (**Supplemental Figure 3**), suggesting improved thymocyte development. Because APP+/+ or APP−/− mESC-TEP-transplanted mice had increased numbers of total thymocytes, and numbers of each thymocyte subsets in these mice were higher than those in control cell-treated mice (**Figure 4A** and **Supplemental Figure 3**). We also analyzed the percentage and number of regulatory T cells (Tregs) in the thymus and the spleen. Although the percentages of Tregs were not significant different among the groups, the number of Tregs in APP+/+ or APP−/− mESC-TEP-transplanted AD mice was higher than WT or control cell-transplanted AD mice (**Supplemental Figures 3**, **4**). Similarly, the percentages of CD11c+ dendritic cells (DCs) and CD19+ B cells in the spleen were not significant different among the groups (**Supplemental Figure 4**).

We then determined the proliferation of the splenic T cells in response to Aβ40 and Aβ42 protein stimulation in the presence of anti-CD3 antibody in vitro. The proliferation of CD4 and CD8 T cells from APP+/+ mESC-TEP-transplanted mice was slightly higher than that from control cell-transplanted mice (**Figures 4C,D**), which may be due to enhanced T cell function after transplantation of mESC-TEPs. Furthermore, the proliferation of both CD4 and CD8 T cells from APP−/− mESC-TEP-transplanted mice was significantly higher than that from APP+/+ mESC-TEP-transplanted mice (**Figures 4C,D**). The latter results suggest that Aβ-specific autoreactive T cells might not be deleted in the thymus of APP−/− mESC-TEP-transplanted mice, leading to presence of Aβ-specific autoreactive T cells in the periphery, resulting in greater proliferation response. Of note, although the proliferation of both CD4 and CD8 T cells in response to anti-CD3 antibody alone (without Aβ40 or Aβ42 peptide) from APP+/+ or APP−/− mESC-TEP-transplanted mice was greater than that from control cell-transplanted mice, there was no significant difference between APP+/+ and APP−/− mESC-TEP-transplanted groups (data not shown).

It has been reported that PD-1 blockage reduced AD pathology involves an IFNγ-dependent immunological response (4). We then analyzed IFNγ-producing T cells in the spleen and found that the percentage of IFNγ-producing CD4 T cells in APP+/+ or APP−/− mESC-TEP-transplanted AD mice was significantly higher than that in control cell-treated mice (**Figures 4E,F**). This is consistent with our previous reports that transplantation of mESC-TEPs leads to the generation

of functional T cells including enhanced production of IFNγ (44, 45). Compared with APP+/+ mESC-TEP-transplanted mice, the percentage of IFNγ-producing CD4 T cells in APP−/− mESC-TEP-transplanted mice showed a larger increase, which is probably due to enhanced anti-Aβ autoimmunity in APP−/− mESC-TEP-transplanted AD mice. Furthermore, more IFNγ was detected in the supernatant of cultured splenocyts from APP−/− mESC-TEP-transplanted AD mice in response to Aβ40 or Aβ42 protein stimulation (**Supplemental Figure 5**).

5. APP−/− mESC-TEP-transplanted mice have an increased number of anti-Aβ Ab-producing B cells in the spleen and increased level of anti-Aβ Ab in the serum.

It is well-known that T cells can help B cell functions. We then determined whether the enhanced T cell proliferation to Aβ stimulation in mESC-TEP-transplanted AD mice led to increased production of anti-Aβ Ab-producing B cells. We used Aβ40 and Aβ42 as antigens for an ELISpot assay to measure anti-Aβ Ab-producing B cells in the spleen. The number of anti-Aβ Ab-producing B cells in APP+/+ mESC-TEP-transplanted mice was higher than that in control cell-transplanted mice, while the number of anti-Aβ Ab-producing B cells in APP−/− mESC-TEP- transplanted mice was higher than that in APP+/+ mESC-TEP-transplanted mice (**Figures 5A,B**).

We also analyzed the levels of anti-Aβ Abs in the serum. Consistent with the ELISpot results, the levels of both anti-Aβ40 and anti-Aβ42 antibodies in the serum of APP+/+ mESC-TEP-treated mice were higher than those in control cell-treated mice, and the levels of these antibodies in APP−/− mESC-TEPtreated mice were significantly higher than those in APP+/+ mESC-TEP-treated mice (**Figure 5C**). The results suggest that increased T cell number in APP+/+ or APP−/− mESC-TEPtreated mice, especially Aβ-specific T cells in APP−/− mESC-TEP-treated mice, help to generate anti-Aβ Ab-producing B cells that secret anti-Aβ Abs into the serum.

6. Transplantation of both APP−/− and APP+/+ mESC-TEPs enhances the brain's choroid plexus (CP) activity.

The CP, the epithelial layer that forms the blood–CSF barrier, is a selective gateway for leukocyte entry to the CNS (13). AD mice have a defect in the CP gateway, as indicated by significantly lower levels of leukocyte homing and trafficking molecule expression in the CP (13). In contrast, IFNγ signaling enhances the expression of leukocyte trafficking molecules (15). Since we have demonstrated that IFNγ-producing T cells were increased in the spleen of APP−/− or APP+/+ mESC-TEPtreated AD mice (**Figure 4E**), we analyzed IFNγ availability at the CP in these mice. qRT-PCR analysis revealed a higher IFNγ mRNA expression level in the CP of APP−/− or APP+/+ mESC-TEP-transplanted AD mice, compared with control cell-treated mice (**Figure 6A**). Flow cytometric examination confirmed a significantly higher percentage of IFNγ-producing CD4+ immune cells in this compartment in APP−/− or APP+/+ mESC-TEP-transplanted AD mice (**Figure 6B**). Again, the IFNγ mRNA expression levels and the percentage of IFNγ-producing CD4+ immune cells in the CP APP−/− mESC-TEP-transplanted AD

mice were higher than those in APP+/+ mESC-TEP-transplanted AD mice (**Figures 6A,B**), consistent with the results for the percentage of IFNγ-producing CD4+ splenic T cells among the mice (**Figure 4E**).

Since increased IFNγ availability can enhance CP activity in this compartment (4, 13), we analyzed the expression of leukocyte homing and trafficking molecules, including intercellular adhesion molecule 1 (icam1), chemokine C-C motif ligand 2 (ccl2), vascular cell adhesion molecule 1 (vcam1), and C-X-C motif chemokine 10 (cxcl10) in the CP. As shown in **Figure 6C**, the mRNA expression levels of these leukocyte trafficking molecules in the CP of APP+/+ mESC-TEP-treated AD mice were higher than those in control cell-treated mice. The mRNA expression levels of these molecules (except vcam1) in the CP of APP−/− mESC-TEP-treated AD mice were higher than those in APP+/+ mESC-TEP-treated mice (**Figure 6C**). These results suggest that administration of APP−/− or APP+/+ mESC-TEPs results in an increased CP activity, which is likely due to the increased IFNγ availability in this compartment.

7. APP−/− mESC-TEP-transplanted mice have an increased number of Aβ phagocytosing macrophages in the brain and the spleen.

Increased CP activity can result in recruitment of monocytederived macrophages to the brain to attenuate AD pathology (4, 13). Since transplantation of APP−/− or APP+/+ mESC-TEPs led to an increased CP activity, we analyzed whether there was an increased number of monocyte-derived macrophages in the brain. It has been shown that CD45hi/CD11b+ cells represent a myeloid population enriched with CNS-infiltrating monocyte-derived macrophages in the brain (4, 13). We found both APP−/− and APP+/+ mESC-TEP-transplanted AD mice had an elevated proportion of CD45hi/CD11b+ cells in the brain, as compared to control cell-treated mice (**Figures 7A,B**). The proportion of CD45hi/CD11b+ cells in the brain of APP−/− mESC-TEP-transplanted AD mice was higher than that in APP+/+ mESC-TEP-transplanted mice. Furthermore, the CD45hi/CD11b+ cells in APP−/− mESC-TEP-transplanted mice had a higher percentage of lymphocyte antigen 6c (Ly6C) positive cells than those in APP+/+ mESC-TEP-transplanted mice (**Figure 7C**). The CD45hi/CD11b+ cells in APP−/− mESC-TEP-transplanted mice also expressed higher levels of chemokine receptor ccr2 and scavenger receptor A (SRA1) (**Figures 7D,E**). It has been reported that Ly6C and ccr2 are related to myeloid cell neuroprotection in AD (62), whereas SRA1 is an Aβ-binding scavenger receptor associated with Aβ-plaque clearance (63). We also analyzed the phagocytosis ability of CD45hi/CD11b+ cells and found that the cells in APP−/− mESC-TEPtransplanted mice had a higher ability to phagocytose

FIGURE 7 | Transplantation of APP−/− mESC-TEPs results in an increased number of Aβ phagocytosing macrophages in the brain and the spleen. 3XTg-AD mice were injected i.t. with APP−/− mESC-TEPs, APP+/+ mESC-TEPs or control cells as in Figure 2. Two and half months later, (A–F) the brain and (G,H) the spleen were harvested. (A–C) The brain was analyzed for the percentage of CD45hi/CD11b+ cells and the expression of Ly6C by CD45hi or CD45lo cells. (A) Flow cytometry gating strategy is shown. (B,C) Statistical analysis of the percentages of (B) CD45hi/CD11b + cells in CD11b + cells and (C) Ly6C+ in CD45hi/CD11b + cells. (D,E) The brain was analyzed for (D) the expression of ccr2 and SRA1 mRNA by qRT-PCR (the expression level of the genes in control cell-treated mice is defined as 1), and (E) the expression of SRA1 protein by CD45hiCD11b + cells using flow cytometry. (F) CD45hi/CD11b + cells were isolated from brains and analyzed for phagocytosis using HF647 Aβ42. (G,H) The splenocytes were analyzed for (G) the percentage of F4/80+ macrophages and (H) the ability of F4/80+ macrophages to phagocytose Aβ42. The data are expressed as mean ± SD from one of three independent experiments with similar results (4–8 mice per group in each experiment). \**p* < 0.05 vs. control cell group, \*\**p* < 0.05 vs. APP+/+ mESC-TEP group.

Aβ42 than those in APP+/+ mESC-TEP-transplanted mice (**Figure 7F**).

We then analyzed macrophages in the spleen and found the percentages of F4/80+ macrophages in APP−/− or APP+/+ mESC-TEP-transplanted AD mice were higher than that those in control cell-treated mice (**Figure 7G**). Although the percentage of F4/80+ macrophages in APP−/− mESC-TEP-transplanted mice was slightly higher than that in APP+/+ mESC-TEP-transplanted mice, the difference did not reach statistical significance (**Figure 7G**). Furthermore, F4/80+ macrophages in APP+/+ mESC-TEP-transplanted mice were more able to phagocytose Aβ42 than those in control cell-transplanted mice, and the macrophages in APP−/− mESC-TEP-transplanted mice had a greater ability than those in APP+/+ mESC-TEP-transplanted mice (**Figure 7H**), in agreement with the data for the macrophage function in the brain (**Figure 7F**).

#### DISCUSSION

AD is hallmarked by the accumulation of Aβ plaques in the brain, which can adversely affect synaptic function and eventually cause neuron loss (2–4, 7, 8). The brain has been traditionally considered a site of immune privilege and exempt from systemic immune surveillance (16, 24). It is now accepted that neuroimmunological cross-talk, in which circulating immune cells enter the CNS, play an important role in brain tissue maintenance and repair, especially in pathological conditions (18, 24–26, 64– 66). Like the situation in cancer immunology, onset of AD may reflect systemic immune suppression and the loss of immune surveillance (13, 67), impairing the ability to mount an immune response to fight brain pathology (13, 14, 68). For example, it has been shown that AD severity is greater in immunocompromised mice (19). In contrast, replacement of the missing adaptive immune populations, such as T cells and B cells, can dramatically reduce AD pathology (19). Boosting recruitment of monocytederived macrophages to sites of brain pathology also facilitates Aβ plaque clearance and relieves AD pathology (4, 13, 69–72). Therefore, systemic immunity in AD should be driven, rather than suppressed, to initiate an immune-dependent cascade to dissipate the Aβ clearance and repair the brain (4, 13, 14).

It is well-known that the thymus, the primary organ for T cell generation, undergoes a profound atrophy with age, a process termed thymic involution, resulting in decreased numbers of T cells in older adults. The reduced T cell number in older adults is likely to contribute AD development and progression. Indeed, in this study, we have shown that transplantation of either APP−/− or APP+/+ mESC-TEPs enhances thymopoiesis that results in increased number of T cells, especially IFNγproducing T cells in the spleen and the CP, leading to enhanced CP activity and increased number of macrophages in the brain. In addition, these mice also have an increased number of macrophages in the spleen. It has been shown that increased IFNγ availability in the CP can enhance the CP activity (4, 13). Both APP+/+ and APP−/− mESC-TEP-transplanted mice have enhanced expression of leukocyte homing and trafficking molecules icam1, vcam1, cxcl10, and ccl2 in the CP, which may be due to the increased IFNγ availability in this compartment. It is likely that the enhanced CP activity leads to increased migration of macrophages into the brain, resulting in an increased number of macrophages in this organ. It is also possible that increased T cell numbers in the spleen aid the macrophages, increasing their number in the spleen, likewise contributing to the increase in the brain. Although the improved thymopoiesis and an increased number of immune cells in the periphery (especially macrophages in the brain) attenuate AD pathology, they are insufficient for reduction of cerebral Aβ plaque load and for improving cognitive performance as indicated by the data that transplantation of APP+/+ mESC-TEPs is less efficient than that of APP−/− mESC-TEPs.

Compared to APP+/+ mESC-TEP-transplanted mice, APP−/− mESC-TEP-transplanted mice have increased Aβinduced T cell proliferation, increased anti-Aβ Ab-producing B cells in the spleen and anti-Aβ Abs in the serum, as well as increased Aβ phagocytosing macrophages in the brain. Since TECs expressing self-antigens play a critical role in deleting autoreactive T cells specific to the antigens, transplantation of APP−/− mESC-TEPs could result in the failure to delete Aβ-specific autoreactive T cells in the thymus, leading to the presence of the autoreactive T cells in the periphery. This is supported by the data that T cells from APP−/− mESC-TEPtransplanted mice have increased proliferation in response to Aβ stimulation. The Aβ-specific autoreactive T cells may then help to produce anti-Aβ Ab-producing B cells that secret anti-Aβ Abs into the serum and to produce Aβ phagocytosing macrophages that migrate into the brain. Together, these Aβ-specific immune cells and Abs reduce the AD pathology. Our results support the notion that breaking Aβ-specific immune tolerance is a novel target for AD immunotherapy (14).

Studies have shown that adaptive–innate immunity cross talk is important in ameliorating AD progression, in which T cells are critical (19). CD4 T cells are essential in the activation of B cells to secrete antibodies to mediate humoral immune responses (73, 74). Antibody response to an antigen requires help from the antigen-specific T cells. B cell antigen receptor usually delivers an antigen to intracellular sites where it is degraded and returned to the B cell surface as the peptide bound to MHC II molecule. The peptide:MHC II complex is recognized by the antigen-specific helper T cells, inducing the B cells to develop into antibody-secreting cells. It is possible that Aβspecific autoreactive T cells generated in APP−/− mESC-TEPtransplanted AD mice recognize the Aβ peptide:MHC II on B cells, and stimulate the B cells to proliferate and differentiate into plasma cells secreting anti-Aβ antibodies. Consequently, the anti-Aβ antibodies neutralize the toxin of Aβ and/or facilitate uptake of Aβ by macrophages by coating to Aβ to enhance the recognition by Fc receptors on macrophages.

CD4 T cells are also important in activating macrophages (75). Once activated, the macrophages phagocytose the related antigens. It has been shown that recruitment of circulating monocyte-derived macrophages can modify AD pathology (16, 76–78) by removing misfolded protein including Aβ-plaques (69, 79, 80), balancing the local inflammatory milieu (71, 80), reducing gliosis (81), and protecting synaptic structures (71, 82,

83). Since activated macrophages can cause local tissue damage (84–87), it is important that the macrophage activity is strictly regulated by antigen-specific T cells. It will be of interest to determine whether Aβ-specific autoreactive T cells in APP−/− ESC-TEP-transplanted AD mice only activate the macrophages that specifically phagocytose Aβ, avoiding unnecessary local tissue damage.

In summary, we have demonstrated that transplantation of APP+/+ or APP−/− mESC-TEPs into AD mice attenuates AD pathology, which is associated with enhanced systemic IFNγproducing T cells and CP gateway activity with increased expression levels of leukocyte homing and trafficking molecules, as well as increased number of macrophages in the CNS. Furthermore, transplantation of APP−/− mESC-TEPs has significantly greater effectiveness. This is related to the generation of T cells reactive with Aβ, which accompanied by increased number of anti-Aβ Ab-producing B cells in the spleen and enhanced level of anti-Aβ Ab in the serum, as well as an increased number of Aβ phagocytosing macrophages in the brain (**Figure 8**). Our results suggest that transplantation of APP−/− human ESC-TEPs or iPSC-TEPs has the potential to be used in the prevention and treatment of AD patients.

# DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

#### ETHICS STATEMENT

The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the University of Connecticut.

# AUTHOR CONTRIBUTIONS

JZ designed experiments, performed experiments, analyzed data, and wrote the manuscript. MS performed experiments and analyzed data. YL and HL performed experiments. ZH designed experiments, analyzed data, and supervised the study. LL designed experiments, analyzed data, supervised the study and wrote the manuscript. All authors contributed to the article and approved the submitted version.

# REFERENCES

# FUNDING

This work was supported by grants from NIH (1R01AI123131, to LL), Connecticut Regenerative Medicine Research Fund (16- RMB-UCONN-02, to LL), Guizhou Medical University Graduate Student Innovation Program (Guiyi YJSCXJH [2019] 001, to JZ), National Natural Science Foundation of China (81871313, to ZH), and Guizhou Province Science and Technology Project (Qian Ke He [2016]4002, [2019]5406, to ZH).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.01781/full#supplementary-material

immune cells: local elevation of IL-10 and MMP-9. J Neurochem. (2009) 111:1409–24. doi: 10.1111/j.1471-4159.2009.06402.x

macrophages in the ischemic lesions of Alzheimer's disease patients. Neurosci Lett. (1996) 219:115–8. doi: 10.1016/S0304-3940(96)13197-9

**Conflict of Interest:** 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 © 2020 Zhao, Su, Lin, Liu, He and Lai. 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.

# When the Damage Is Done: Injury and Repair in Thymus Function

Sinéad Kinsella1 and Jarrod A. Dudakov 1,2,3 \*

*1 Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States, 2 Immunotherapy Integrated Research Center, Fred Hutchinson Cancer Research Center, Seattle, WA, United States,*

*3 Department of Immunology, University of Washington, Seattle, WA, United States*

Even though the thymus is exquisitely sensitive to acute insults like infection, shock, or common cancer therapies such as cytoreductive chemo- or radiation-therapy, it also has a remarkable capacity for repair. This phenomenon of endogenous thymic regeneration has been known for longer even than its primary function to generate T cells, however, the underlying mechanisms controlling the process have been largely unstudied. Although there is likely continual thymic involution and regeneration in response to stress and infection in otherwise healthy people, acute and profound thymic damage such as that caused by common cancer cytoreductive therapies or the conditioning regimes as part of hematopoietic cell transplantation (HCT), leads to prolonged T cell deficiency; precipitating high morbidity and mortality from opportunistic infections and may even facilitate cancer relapse. Furthermore, this capacity for regeneration declines with age as a function of thymic involution; which even at steady state leads to reduced capacity to respond to new pathogens, vaccines, and immunotherapy. Consequently, there is a real clinical need for strategies that can boost thymic function and enhance T cell immunity. One approach to the development of such therapies is to exploit the processes of endogenous thymic regeneration into novel pharmacologic strategies to boost T cell reconstitution in clinical settings of immune depletion such as HCT. In this review, we will highlight recent work that has revealed the mechanisms by which the thymus is capable of repairing itself and how this knowledge is being used to develop novel therapies to boost immune function.

Keywords: endogenous thymic regeneration, immune restoration, T cell reconstitution, thymic epithelial cells, BMP4, IL-22

# INTRODUCTION

Generation of a diverse but tolerant T cell repertoire, which is critical for adaptive immune function, is dependent on the development and maturation of T cell precursors in the thymus. The process of T cell development is reliant on the interactions with the stromal microenvironment, comprised of highly specialized thymic epithelial cells (TECs), endothelial cells (ECs), mesenchymal cells, dendritic cells (DCs) and macrophages. However, despite its importance for generating and maintaining T cells, thymic function is extremely sensitive to acute damage such as that caused by everyday insults like stress and infection, as well as more profound injuries such as that caused by cytoreductive therapies. Nevertheless, the thymus also has a remarkable capacity to regenerate itself from these acute injuries (1, 2), although until recently this phenomena has been largely unstudied. However, despite its crucial function, the ability of the thymus to facilitate efficient T cell generation deteriorates progressively with age

#### Edited by:

*Avinash Bhandoola, National Institutes of Health (NIH), United States*

#### Reviewed by:

*Dong-Ming Su, University of North Texas Health Science Center, United States Takeshi Nitta, The University of Tokyo, Japan Jennifer Elizabeth Cowan, National Institutes of Health (NIH), United States*

#### \*Correspondence:

*Jarrod A. Dudakov<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

Received: *29 April 2020* Accepted: *30 June 2020* Published: *12 August 2020*

#### Citation:

*Kinsella S and Dudakov JA (2020) When the Damage Is Done: Injury and Repair in Thymus Function. Front. Immunol. 11:1745. doi: 10.3389/fimmu.2020.01745*

**169**

(3, 4); which considerably hampers the ability of the thymus to respond to acute insults. Age-related thymic atrophy and immunosenescence are hallmarks of immune aging and ultimately lead to a constriction of the TCR repertoire (5), decreased naïve T cells and accumulation of memory T cells in the periphery; and chronic low-grade inflammation termed "inflamm-aging," all conferring insufficient protective responses to pathogens and neoantigens (6–8). Together, these acute and chronic thymic injuries underlie prolonged immune deficiency associated with multiple conditions including the conditioning required for hematopoietic cell transplantation (HCT) and cytoreductive cancer treatments such as radioand chemo-therapies.

Given the poor outcomes that are associated with deficient T cell immunity, there is a clear clinical need for therapies that can boost thymic function in periods of acute injury or reverse agerelated thymic involution. In this review, we will outline what we know about how the thymus is damaged during different modalities of insult and the work that has been done to develop therapeutic strategies to boost thymic function; either ensuing acute insult such as following HCT, or in aged individuals to boost responses to vaccines or immunotherapy (**Figure 1**).

#### ACUTE DAMAGE AND ENDOGENOUS REGENERATION IN THE THYMUS

#### Everyday Insults: Stress and Infection

Thymic involution is a routine response to acute insult incurred by multiple triggers including emotional and physical distress, malnutrition, and opportunistic bacterial and viral infections. These can be modeled using approaches such as synthetic corticosteroid treatment, such as dexamethasone (9); nutrient depletion (10); sex steroid treatment (11); and several viral and bacterial infection models. While acute thymic involution results primarily from the loss of cortical thymocytes (12, 13); in cases of chronic atrophy, such as that induced by age-related thymic decline, thymocyte loss is preceded by the loss of Foxn1+ TECs, resulting in the functional decline the TEC compartment and the initiation of age-related thymic atrophy (14, 15).

Continuous export of naïve cells from the thymus, or recent thymic emigrants (RTEs), is essential for effective immune response to acute and chronic infections (16, 17). However, most acute bacterial or viral infections result in acute thymic atrophy, largely due to intense lymphocyte depletion as a result of increased apoptosis of thymocytes and interference with thymocyte development (18–21); which can, at least partially, be attributed to the increased induction of IFNγ from activated CD8+ T cells (22) and Natural Killer (NK)-driven responses (23). Most of the studies looking at viral infection-related thymic function have concentrated on HIV, which leads to several modes of thymic dysfunction including thymic atrophy, reduced thymic output, reduced export of immature thymocytes and disruption of the thymic microenvironment (24–26). Notably, effective response to anti-retroviral therapies was found to depend on competent thymic function, with enhanced function in HIVinfected children with higher basal levels of thymic function (27), in contrast with infected adults who have a reduced thymic output and output decreased CD4+ T cells (28, 29). Moreover, in addition to viral load, quantification of CD4+ RTE has long been known as a suitable marker for HIV disease progression, and a recent study has demonstrated the use of RTE CD4+ T cells as a marker of perinatal HIV infection in infants (30); further strengthening the link between viral infection, efficient thymic function, and therapeutic implications of thymic atrophy.

Although less well studied, bacterial infections also have negative effects on thymic function, primarily by enhancing thymocyte apoptosis. Streptococcus suis infection promotes thymic atrophy specifically by inducing increased activation of pro-apoptotic pathways and apoptotic cell death in thymocytes (31); while Mycobacterium tuberculosis infection also induces thymic atrophy (32), possibly by regulating glucocorticoid levels and in this way impact on homeostatic endocrine-immune communication (33). In fact, glucocorticoids are central to many acute forms of thymic involution (34, 35), directly inducing the apoptosis of CD4+CD8+ DP thymocytes, which preferentially express the glucocorticoid receptor (36).

Metabolic distress due to lack of nutrients, primarily glucose, leads to an attenuation of thymic function, with perturbed thymopoiesis in non-obese diabetic (NOD) mice (37), and reduced thymic atrophy with glucose supplementation in models of mitochondrial dysfunction (38).

### Cytoreductive Therapies

Most therapies used in cancer treatments are cytoreductive, such as chemotherapy or radiation. One prominent example of this is that the pre-conditioning regimens required for successful HCT result in profound injury to the thymus, and, in contrast to the relatively early recovery of platelets, erythrocytes, and leukocytes involved in innate immunity, recipients of an HCT experience prolonged post-transplant deficiency in the recovery of adaptive immunity, especially T cell immunity. This delayed T cell reconstitution can last a year or more due to a delay in full recovery of function and T cell repertoire (39–41). Moreover, post-transplant T cell deficiency is associated with an increased risk of infections (39, 40, 42, 43), relapse of malignancy (44), and the development of secondary malignancies (45–50). In fact, infection and relapse account for >50% of mortality following allogeneic-HCT (allo-HCT) (51). T cell reconstitution after transplant is critically dependent on the thymus (39, 41, 47, 52–58) and thymic function pretransplant can have a significant impact on clinical outcomes. Similarly, damage caused by cytoreductive chemotherapy results in significant thymic damage and can lead to a profoundly delayed recovery of T cells (45, 59). In mouse models of chemotherapy, in addition to almost complete depletion of thymocytes, there was also a severe depletion of TECs, most prominently MHCIIhi TECs (60); likely as they are the most highly proliferating TEC subset (61, 62). Specifically, genotoxic stress caused by chemotherapy leads to senescence in the thymic stromal compartment and the induction of an inflammatory environment in the thymus with endothelial cell secretion of IL-6, generating a chemoresistant niche that is cytoprotectant to certain cancer cells, such as lymphoma and melanoma

involution can be broadly stratified into four subgroups based on their cellular or molecular targets: (1) targeting the epithelial microenvironment that supports thymopoiesis; (2) targeting the precursors that provide the supply of developing thymocytes; (3) modulation of hormones and metabolism; and (4) cellular therapies and bioengineering. However, within each of these therapeutic modalities there are key nexus points at which they act mechanistically. One approach relies on stimulating TEC function, such as IL-22, BMP4, KGF, RANKL, SSI, which act by either promoting survival, proliferation, differentiation, or expression of key thymopoietic molecules like DLL4 and KITL. In contrast, approaches such as administration of exogenous IL-7 and chemokine therapies target T cell precursors to promote their migration, proliferation, and differentiation directly. Similarly, many of the bioengineering approaches have sought to recapitulate these same functions such as providing TEC signals or a ready supply of T cell precursors. Elements of the figure were generated using Biorender.com.

(63, 64). Accompanying the damage caused by cytoreductive conditioning, the risk of further thymic damage caused by Graftvs.-Host Disease (GVHD) is significant in the context of an allo-HCT. In fact, the thymus is a particularly sensitive GVHD target organ and presents pathological features even in the context of subclinical GVHD (65–67). Furthermore, there is likely a link between acute GVHD-mediated thymic damage and the formation of chronic GVHD, which may in part be a failure for tolerance induction (68–70).

# STRATEGIES OF BOOSTING THYMIC FUNCTION I: TARGETING NON-HEMATOPOEITIC CELLS

Given the sensitivity of thymic function to negative stimuli, even everyday insults, a reparative capacity is crucially important for renewal of immune competence. In fact, this capacity of the thymus to regenerate itself has been known for longer than even the immunological function of the tissue was discovered (71, 72); however, until recently the mechanisms underlying this process have been poorly understood. One approach to developing therapies to enhance thymic function has come from exploiting these pathways of endogenous regeneration. Many of these pathways that mediate endogenous regeneration have been found to be effective for exogenous regeneration in periods of acute and profound injury such as that caused by cytoreductive chemotherapy and γ-radiation. Interestingly, many of these pathways specifically target TECs to mediate regeneration.

#### Interleukin-22

Although the phenomenon of endogenous thymic regeneration has been known for over almost a 100 years, it was not until recently that pathways mediating this regeneration have been described. The first of these was centered around the production of Interleukin-22 (IL-22), a member of the IL-10 family that typically targets non-hematopoietic cells such as epithelial cells and fibroblasts (73). In this regenerative network, acute damage to the thymus (and specifically the depletion of thymocytes) triggers the release of Interleukin-23 (IL-23) from dendritic cells, which induces the production of IL-22 by a group 3 innate lymphoid cells (2, 74–76). Expression of IL-22R in the thymus is lacking on thymocytes but detected in both cTECs and mTECs populations (2). IL-22 acts on TECs to mediate repair but the specific molecular mechanisms are not clear. In addition to the thymus, IL-22 also has a major role in the regeneration of epithelial cells in a diverse range of tissues including gut, lung, skin, breast, and kidney (77). The IL-22 receptor is a type 2 cytokine receptor, and a heterodimer formed of two subunits: IL-10 receptor 1 (IL-10R1) and IL-22 receptor A2 (IL-22RA2) (78). IL-22 receptor binding induces intracellular inactivation of the Jak1/Tyk2 complex which further allows downstream signaling and phosphorylation of Signal Transducer and Activator of Transcription (STATs) 1, 3, and 5, with a preference for STAT3 phosphorylation (79), including in TECs (2) which is consistent with the upregulation of Foxn1 concurrently with IL-22 in the thymus (76), and the importance of STAT3 for TEC maintenance (80). Furthermore, Ruxolitinib, a chemotherapeutic agent that inhibits Jak1 signaling also prevents thymic regeneration after injury (81).

Similar to other tissues (77), IL-22 is not required for the formation or maintenance of the thymus under steadystate physiological conditions; however, it has a key role in driving thymic regeneration after injury, by acting directly on TECs to induce survival and proliferation, potentially via regulation of Foxn1 expression (2, 76). Of note, both the numbers of innate lymphoid cells (ILC) 3 and IL-22 levels were decreased in the thymus and gut of mice with GVHD (74, 82), suggesting that ILCs are a target of alloreactive cells and this depletion likely causes a failure to repair after damage. Due to the diverse pathophysiological roles of IL-22, and the key role in epithelial cell regeneration, modulation of the IL-22-IL22R system is an attractive therapeutic target. In fact, a clinical trial is currently underway to assess the efficacy and safety of administration of IL-22 in combination of systemic corticosteroids to limit the effects of GVHD after hematopoietic stem cell transplantation, with secondary readouts to assess T cell reconstitution (NCT02406651).

### Bone Morphogenic Protein 4

Although the role of thymic BMP4 and the endogenous BMP4R antagonist Noggin have been well-described in thymic development (83, 84), only recently has BMP4 been described as a regulator of thymic regeneration after acute injury (85). In the thymus, the source of BMP4 is fibroblasts and endothelial cells (ECs) (85). ECs are a highly radio-resistant population of cells in the thymus (85, 86) and are unique in their ability to induce BMP4 production in response to injury. Importantly, thymic expression of both Bmpr1a and Bmpr2 were identified on TEC populations, with a higher expression of the nonredundant Bmpr2 on cTECs (85); consistent with BMP4-induced expression of FOXN1 and its downstream target delta-like 4 (DLL4) specific to cTECs (85, 87). Although the importance for FOXN1 and DLL4 for the development of TECs and thymocytes, respectively, has been well studied (88, 89), recent findings have also highlighted their importance for thymic regeneration, with intrathymic concentration of DLL4 profoundly impacting on thymic size (90), and reports suggesting that the induction of FOXN1 can counteract age-associated thymic involution (91), acute damage (85), and thymic damage post-transplantation (92). While much of the role of BMP4 seems to be mediated by induction of the FOXN1/DLL4 axis, given the requirement for BMP4 in in vitro differentiation of TECs from multipotent progenitors (93–95), it is possible that an alternate mechanism may be by stimulating bipotent progenitors present in the adult thymus (96–99). Unfortunately, the preclinical studies assessing BMP4 have yet to successfully treat mice with recombinant protein, a therapeutic strategy has been developed that utilizes a technique of allowing for the propagation and expansion of tissue-specific ECs that can be transplanted and mediate regeneration across multiple tissues (85, 100–105). In the thymus, it was found that this therapeutic cellular strategy was dependent on the expression of BMP4 by transplanted ECs (85).

#### Keratinocyte Growth Factor

Keratinocyte growth factor (KGF, also known as FGF-7), is a fibroblast growth factor and acts as a mitogen targeting TECs, inducing epithelial proliferation in several organs (106– 108). In the thymus, KGF is primarily produced by mature αβ + thymocytes and feeds back to facilitate the proliferation and expansion of mTECs via the activation of p53 and NFκB pathways (106, 108), preserving the thymic cytoarchitecture. Of note, KGF is also produced by thymic fibroblasts (108). Expression of the KGF receptor, fibroblast growth factor receptor-2 of the IIIb variant (FgfR2IIIb), is limited to TECs (109), and FgfR2-IIIb−/− mice have defective thymopoiesis and reduced cellularity, accounted for specifically by a reduction in TECs (110). KGF modulates TEC functionality by negatively regulating the levels several gatekeepers of positive selection, such as MHC-II invariant chain (Ii), and cathepsin L (CatL) (108), and acts on TECs to produce several cytokines that act directly on thymocytes to facilitate maturation, such as bone morphogenic protein 2 (BMP2), BMP4, Wnt5b, and Wnt10b (109).

Under normal physiological conditions, KGF can enhance thymic cellularity by increasing the number of early thymic progenitors (ETPs) equating to an enhanced number of engraftment niches, and increased TEC proliferation (109). Although it was shown that KGF is not essential in uninjured conditions (110, 111), studies using KGF−/− mice demonstrated the critical role of KGF on thymus function and immune reconstitution after insult, modeled by both syngeneic and allogeneic bone marrow transplant (112). The same study showed that exogenous administration of recombinant KGF enhanced thymopoiesis in young and middle-aged mice, and attenuated the negative effects of acute thymic injury, such as that caused by dexamethasone treatments, cyclophosphamide, and irradiation, highlighting an extremely attractive therapeutic approach to efficiently facilitating immunocompetence after damage. Additionally, exogenous KGF administration improved post-transplantation T cell reconstitution. Furthermore, preconditioning with KGF prior to bone marrow transplantation reduces GVHD in mouse models by protecting against epithelial injury (113). However, a recent clinical trial noted a reduction in thymopoiesis in lymphopenic patients following administration of KGF (114), highlighting that more studies need to be carried out before KGF can be used across the board as a therapeutic regulator of thymic regeneration.

#### RANKL

Receptor activator of nuclear factor kappa-B ligand (RANKL), a member of the Tumor necrosis factor (TNF) superfamily, is implicated in multiple physiological roles in the periphery, primarily in bone biology (115). RANKL has an essential role in the thymus as a potent inducer of epithelial cell differentiation by regulating the key mTEC transcription factor Aire (116). In this way, RANKL governs the maturation of Aire− mTECs to Aire+ mTECs which subsequently present MHC-II peptides that drive the elimination of self-reactive T cells during negative selection (117). RANKL is non-redundant for fetal Aire+ mTEC development, and is produced during development by ILCs, and subsequently by subsets of thymocytes (116, 118–120); although absence of RANKL postnatally can be compensated for by other factors (121). Importantly, RANKL is increased in CD4+ thymocytes and ILCs after injury from the cytoreductive conditioning required prior to HCT, suggesting that RANKL plays a role in endogenous regeneration of the thymus (2, 122).

The prominent role of RANKL in mTEC biology points to the ability of RANKL to modulate thymic regeneration and output. RANKL administration shows an enhancement of thymic function after bone marrow transplantation by boosting TEC subsets, including TEC progenitor niches (122). Moreover, systemic administration of recombinant soluble RANKL (sRANKL) improved thymic medullary architecture in RANKL deficient mice (123), and transgenic mice overexpressing human sRANKL, or mice lacking the soluble RANKL receptor OPG, have an enlarged thymic medulla with increased numbers of Aire+ mTECs (119, 124, 125), highlighting a therapeutic platform for the use of recombinant RANKL as a therapeutic for thymus regeneration.

### STRATEGIES OF BOOSTING THYMIC FUNCTION II: TARGETING HEMATOPOEITIC CELLS

Given the fact that Cell development requires the input of hematopoietic progenitors, and the fact that the supply of those progenitors is severely limited after acute injury (126, 127), one approach to promoting thymic function is to directly stimulate precursor populations; either in the BM or thymus.

#### Bone Marrow Progenitors

Several approaches have been attempted that seeks to improve thymic function by stimulating the function of bone marrow hematopoietic progenitors. For instance, preclinical studies have shown that administration of Flt3L can also enhance both thymic dependent and independent T cell reconstitution (128, 129). The effects of Flt3L are predominantly due to an expansion in Flt3+ progenitors in the BM (130). However, increases in T cell reconstitution can be at the expense of B-lymphopoiesis which is significantly declined with exogenous Flt3L administration and, in particular, its effects on the EPLM subset of BM progenitors (131, 132).

Chemokines are key regulators of thymopoiesis, facilitating thymic population and intrathymic cell migration. Importantly, as the thymus does not contain long term progenitors that would enable self-renewal, repopulation of the thymus requires continuous recruitment of T cell progenitors (133). CCL25 (with its receptor CCR9) and CCL21 (with its receptor CCR7) play an important role in thymic colonization with hematopoietic progenitors (134). Interestingly, chemokine therapy, whereby bone marrow progenitors received CCL25 and CCL21 treatment prior to transplant, rescues thymic homing of progenitors which is otherwise suppressed in irradiated mice (86).

#### Thymic T Cell Precursors

While there are several approaches that have been postulated that target thymic precursor cells the most prominent and developed of these is with the lymphopoietic cytokine interleukin-7 (IL-7). IL-7 has a non-redundant role as a survival molecule in lymphoid tissues in mice and humans, most importantly in the thymus where IL-7 is critical for appropriate thymocyte development. An elegant study by Shitara et al. (135) showed that specific deletion of IL-7 in TECs resulted in the profound reduction in αβ and γδ T cells; and mice deficient for Il-7 have a peripheral loss of γδ T cells, a significant reduction in αβ T cells (136), an absence of innate lymphoid cell subsets (137), and disorganization of lymphoid tissue (138). Moreover, mice lacking Il-7 have a reduced number of DN2 or DN3 cells (139, 140), essentially creating a thymic block and limiting the progression thymocytes to maturity.

IL-7 is produced primarily by non-hematopoietic stromal cells such as TECs and signals by binding to the heterodimeric IL-7 receptor (IL-7R), comprised of IL-7R? (also known as CD127) and the cytokine receptor γ-chain (also known as CD132), and induces an anti-apoptotic pro-survival signaling cascade via the activation of phosphoinositide 3-kinase (PI3K) and the Janus Kinase (JAK)-STAT pathway. The expression of IL-7R? on developing thymocytes occurs in a cyclical pattern, with expression seemingly dependent on the fluctuating need for IL-7 signaling at different stages of thymocyte maturation (141), demonstrated by absence in the earliest T cell progenitors, expression at later DN stages, absence at the DP stage and re-expression in SP thymocytes.

These critical roles of IL-7 in both thymocyte development and in peripheral T cell homeostasis (142) reveal IL-7 as a strong therapeutic candidate to enhance T cell development and activation. Clear evidence exists for the therapeutic potential of IL-7 administration on thymic regeneration, centered on the beneficial effects of IL-7 on increasing progenitor T cells in the thymus and subsequently expanding circulating naïve T cells in viral infection setting (143); however, IL-7 therapy only transiently increased naïve T cells in the aged setting in rhesus macaques, with a more prominent and long lasting effect in the memory T cell compartment (144). Although recombinant IL-7 immunotherapy has had some success in clinical trials for treating septic shock (145), infection (146), and cancer remission (147), along with some early promise in the setting of HCT (148), further studies are necessary to identify a strategy for thymus-dependent IL-7 therapy.

 In fact, sex steroid inhibition (SSI) has been shown to promote thymic function in young as well as old mice, and enhances reconstitution after acute insult such as chemotherapy or HCT (60, 152–154). Furthermore, given that SSI is a standard and approved therapy for prostate cancer, thymic function has been assessed in prostate cancer and after HCT and significant improvement observed (155, 156). Although whole organismal ablation of sex steroids will understandably have systemic effects, and the specific means by which SSI improves thymic function are not yet clear, several putative mechanisms have been proposed. In particular, SSI has been shown to (1) promote lymphoid potential and overall function of hematopoietic stem and progenitor cells (2, 152, 157, 158) induce the expression of CCL25 (159), which promotes the importation of hematopoietic progenitors from the circulation (3, 134, 160) induces the expression of the Notch ligand DLL4 (90). Interestingly, KGF was not required for the beneficial effects of SSI on thymus (154), and in fact combination therapies have shown great promise, with the combined KGF administration and androgen blockage with Lupron, revealing reduced epithelial damage and enhanced T cell reconstitution after bone marrow transplant in mice (161). However, it has also been reported that regrowth of the thymus can result in an increase in autoreactive T cells in the periphery, particularly in models of castration, reflecting a lack of synergy between quality and quantity of thymopoiesis (162).

In addition to sex steroids, several other hormones and metabolic components have been implicated in thymic function and their modulation has been shown to improve thymopoiesis, particularly in the aged. Administration of the appetite stimulating hormone Ghrelin led to improved thymic cellularity and thymic output in aged mice (163), and similarly oral zinc supplementation increased thymic cellularity in aged mice (164). Targeting accumulating reactive oxygen species with antioxidants has proven to be beneficial in protecting against age-related thymic atrophy, whereby treatment with the mitochondrial antioxidant SkQ1 reduced age-associated thymic atrophy and increased the number of CD4+ and CD8+ thymocytes (165). Similarly, Leptin, a peptide hormone secreted from adipose tissue, has a protective effect on thymopoiesis in LPS-treated mice and mice that had been starved, primarily due to rescue from metabolic defects including increased corticosterone levels (10, 166).

#### STRATEGIES OF THYMIC REGENERATION IV: CELL THERAPIES AND BIOENGINEERING APPROACHES

#### Hematopoietic Precursors

In addition to the use of growth factors and hormone modulation, several groups have been working on cellular therapies that may enhance thymic function. Given that some of the delay in T cell reconstitution is due to the limited supply of BM-derived progenitors (126), in addition to the time taken for development into a naïve lymphocyte from a transplanted HSC, early studies that concentrated on providing hematopoietic cells found that lymphoid precursors isolated from donor bone marrow could be used to boost thymic function when infused into a recipient at the time of HCT, giving an early boost to T cell development (167). To overcome the limited number of hematopoietic progenitors in BM, an alternate approach of using precursor T cell populations that have been expanded using ex vivo culture systems that use Notch-1 stimulation of hematopoietic precursor cells has been demonstrated (168– 173). Using this regimen, adoptive transfer of T cell precursors into lethally irradiated allogeneic HCT recipients caused a significant increase in thymic cellularity and chimerism, as well as enhanced peripheral T and NK cell reconstitution compared with recipients of allogeneic hematopoietic stem cells only (168, 174–178).

# Thymic Epithelial Cells

In addition to the use of hematopoietic cells that can act as a boost of T cell precursors, another approach is to identify and isolate populations of thymic epithelial progenitor cells (TEPC). TEPC have been successfully isolated from fetal thymi and induced to generate a new thymus in athymic recipients (179–182), and neonatal TECs, or TECs derived from pluripotent progenitors can promote enhanced thymic function (183, 184). However, while there is evidence of a bipotent TEPC in the postnatal thymus (97–99), their capacity to self-organize as a whole organ like fetal TEPCs is limited. A TEC-like progenitor

cell appropriate for this purpose has also been generated by direct conversion of embryonic fibroblasts by induced expression of the TEC transcription factor FOXN1 (185); although the efficacy of this therapy in a regeneration setting has not been investigated.

#### Artificial Thymic Niches

Finally, there are also several approaches that do not rely on the endogenous thymus at all, but rather concentrate on de novo formation of whole organs ex vivo that can be transplanted into patients as required (186, 187). Although in vivo evidence of their efficacy is still only limited, several approaches have been used to generate artificial thymuses ex vivo, including decellularizing the tissue, which has been performed in several tissues including the thymus, as well as generating synthetic matrices to support T cell development (188–190). Both of these approaches would require some cellular input to generate a functional thymus; namely the thymic epithelial microenvironment would need to be recapitulated with specific factors or, more likely, cells such as TECs derived from multipotent progenitors or reprogrammed, as above. Moreover, a recent report has demonstrated the efficacy of an artificial pre-thymic niche by implanting a scaffold with the Notch ligand DLL4 that acts as an intermediary between the BM and thymus (191). Although these approaches have shown some promise in preclinical mouse studies (192), further advances need to be made before this can be a viable therapeutic option.

#### REFERENCES

#### CONCLUSION

Enhancing the regenerative capacity of the thymus and increasing thymic output, together with the expansion of the TCR repertoire has immensely beneficial clinical implications. Although there has been extensive progress in the development of multiple therapies targeting thymic regeneration and output, a deeper understanding of key endogenous molecular mechanisms that govern involution and regeneration of the thymus are needed to further the development of clinically translatable therapies.

# AUTHOR CONTRIBUTIONS

SK and JD wrote, drafted, and edited the manuscript. All authors contributed to the article and approved the submitted version.

### FUNDING

This research was supported by National Institutes of Health award numbers R01-HL145276 (JD), Project 2 of P01-AG052359 (JD), and the NCI Cancer Center Support Grant P30-CA015704. Support was also received from a Scholar Award from the American Society of Hematology (JD), a Scholar Award from the Leukemia and Lymphoma Society (JD). SK was supported by a New investigator Award from the American Society for Transplantation and Cellular Therapy.

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**Conflict of Interest:** The authors have patents and patent applications around potential therapeutics to promote thymus regeneration, including some listed in this review (IL-22 and BMP4) and others as yet unpublished.

Copyright © 2020 Kinsella and Dudakov. 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.

# T-Cell Immunodeficiencies With Congenital Alterations of Thymic Development: Genes Implicated and Differential Immunological and Clinical Features

Giuliana Giardino, Carla Borzacchiello† , Martina De Luca† , Roberta Romano, Rosaria Prencipe, Emilia Cirillo and Claudio Pignata\*

*Department of Translational Medical Sciences, Pediatric Section, Federico II University of Naples, Naples, Italy*

#### Edited by:

*Nicolai Stanislas Van Oers, University of Texas Southwestern Medical Center, United States*

#### Reviewed by:

*Jennifer Elizabeth Cowan, National Institutes of Health (NIH), United States Naomi Taylor, National Institutes of Health (NIH), United States*

> \*Correspondence: *Claudio Pignata<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

Received: *14 April 2020* Accepted: *08 July 2020* Published: *14 August 2020*

#### Citation:

*Giardino G, Borzacchiello C, De Luca M, Romano R, Prencipe R, Cirillo E and Pignata C (2020) T-Cell Immunodeficiencies With Congenital Alterations of Thymic Development: Genes Implicated and Differential Immunological and Clinical Features. Front. Immunol. 11:1837. doi: 10.3389/fimmu.2020.01837* Combined Immunodeficiencies (CID) are rare congenital disorders characterized by defective T-cell development that may be associated with B- and NK-cell deficiency. They are usually due to alterations in genes expressed in hematopoietic precursors but in few cases, they are caused by impaired thymic development. Athymia was classically associated with DiGeorge Syndrome due to *TBX1* gene haploinsufficiency. Other genes, implicated in thymic organogenesis include *FOXN1*, associated with Nude SCID syndrome, *PAX1*, associated with Otofaciocervical Syndrome type 2, and *CHD7*, one of the genes implicated in CHARGE syndrome. More recently, chromosome 2p11.2 microdeletion, causing *FOXI3* haploinsufficiency, has been identified in 5 families with impaired thymus development. In this review, we will summarize the main genetic, clinical, and immunological features related to the abovementioned gene mutations. We will also focus on different therapeutic approaches to treat SCID in these patients.

Keywords: Thymus, FOXN1 gene, PAX1 gene, Pax 1/9, CHARGE, CHD7 gene, TBX1 gene, DiGeorge anomaly

#### INTRODUCTION

The thymus is a primary lymphoid organ which plays a pivotal role in the development of mature T cells from immature bone marrow CD34+ precursors. Together with the parathyroid glands, it develops from the 3 pharyngeal pouch (PP) (1). The epithelial components of the thymus derive from the endothelial layer, while the mesenchymal capsule derives from neural crest, originated from ectoderma (1, 2). The first stage of thymic development is independent of the transcription factor forkhead box N1 (Foxn1) expression (3). In this phase, Paired box 1 (Pax1), Eyes absent homolog 1 (Eya1), sine oculis homeobox (Six), homeobox A3 (Hoxa3), and T-box 1 (Tbx1) drive the outgrowth of the thymic epithelial anlage from the 3rd PP (1, 4, 5). Hoxa3 and Eya1 are also implicated in the development of neural crest derived mesenchymal cells (6–8). Studies suggest that chromodomain helicase DNA-binding 7 (Chd7) might be implicated in the development of both neural crest cell-derived mesenchyme and pharyngeal endoderm-derived thymic epithelial cells (TECs). Pax3 and Hoxa3 expression in mesenchymal cells allows the detachment of the thymic lobes from the pharynx (9, 10). Thymus development in early and late stages is regulated by the interactions among various cell types. The thymus three-dimensional (3D) architecture allows a proper intercellular cross talk (3). In the first phase of organogenesis, mesenchymal cells release bone morphogenetic protein 4 (Bmp4), Bmp2, fibroblast and insulin growth factors (Fgf, Igf), Wnt proteins, and retinoic acid supporting the differentiation of TECs into cortical (cTECs) and medullary (mTECs) subsets (7, 8, 11–14). In the second phase, Foxn1 induces the expression of chemokine (C-C motif) ligand 25 (CCL25), delta like canonical Notch ligand 4 (Dll4), and Hoxa3, allowing thymocyte recruitment and TECs differentiation in cTECs and mTECs. Mesenchymal cells are implicated in the recruitment of hematopoietic thymic seeding progenitors, as well (15, 16). Thymocytes participate to TECs differentiation process through the release of epidermal growth factor (Egf) and lymphotoxin factors (17–19). In support of this, mice with defective T-cell development show defective organization of the thymic medulla (20, 21), that is restored after stem cell transplantation (21, 22).

Bone marrow derived hematopoietic stem cells (HSCs) enter the thymus through cortico-medullary junction, where they proliferate (23). The V(D)J rearrangement of the double negative (DN) thymocytes T-cell receptor β (TCRβ) gene takes place in the thymic cortex (24, 25). Membrane expression of pre-TCR complex is necessary for the expression of the co-receptors CD4 and CD8, as well as V-J rearrangement of the TCRα genomic region (26). Double positive thymocytes with a functional TCRαβ receptor capable of binding to self-MHC ligands are positively selected (27–29). This process is regulated by Prss16 and β5t, which are expressed in cTECs (30–34). Into medulla, selfreactive thymocytes are deleted through the negative selection, a process mediated by dendritic cells and Aire-expressing mTECs (35, 36). Mutations in genes implicated in different steps of thymic development, including FOXN1, PAX1, TBX1, CDH7 impair T-cell development in humans (**Figure 1**). Alterations of the immune system in these conditions range from an isolated reduction of T-cell count to severe combined immunodeficiency (SCID). This review is focused on definition of the role of different genes implicated in thymus development and of primary immunodeficiencies (PIDs) due to their deficiency. Moreover, therapeutic options for PIDs with congenital athymia are discussed.

## GENE FUNCTION AND RELATED SYNDROME

#### FOXN1 Deficiency and Nude-SCID Syndrome

FOXN1 is located on chromosome 17q11.2 and it is composed of eight exons (30 kb) (37, 38). This gene is a member of the Fox gene family that comprises different Winged helix transcription factors implicated in development, metabolism, cancer, and aging (39). During fetal life, FOXN1 is expressed in mesenchymal and epithelial cells including liver, lung, intestine, kidney, and urinary tract. In postnatal life, its expression is restricted to keratinocytes and TECs (40, 41). FOXN1 is involved in development, function and maintenance of hair follicles, and TECs (**Figure 1**) (42–44). The expression of FOXN1 in TECs leads to the production of chemokines, such as C-X-C motif chemokine ligand 12 (CXCL12), implicated in the recruitment of hematopoietic progenitors and of molecules, as DLL4 notch ligand, implicated in maturation of the progenitors toward the T-cell line (**Figure 1**) (45–47). Studies in mice showed that Foxn1 plays a pivotal role in morphogenesis of the 3D architecture (48, 49) by inhibiting a basic morphogenetic pattern of tubulogenesis and inducing the expression of genes that drive TECs differentiation (50). FOXN1 also regulates Prss16 and β5t gene expression, implicated in positive selection (45, 51). Knockout of Hoxa1, Pax1, Pax9, Eya1, or Six1 affects Foxn1 expression resulting in impaired thymic function (52). Foxn1 expression is also regulated by signals from the surrounding endothelium and mesenchyme, including Bmp4 and Wnt (53, 54).

In epidermis, FOXN1 is expressed in keratinocytes of suprabasal layer, where these cells stop proliferating and start the terminal differentiation (55). In keratinocytes, FOXN1 regulates the transcription of more than 50 genes, including protein kinase B and C (PKB and PKC). PKC is an inhibitor of human hair follicle growth in vitro (56–58). It is up-regulated in Foxn1–/– mice while its activity is suppressed in mice overexpressing Foxn1 gene in which differentiation is inhibited. Studies conducted on human keratinocytes have confirmed the role of the gene in the differentiation of the epidermis (58).

Homozyous FOXN1 mutations cause Nude SCID syndrome, first observed in two Italian sisters presenting with congenital universal alopecia, nail dystrophy, and severe T-cell immunodeficiency with rudimentary thymus (59). In the last few years, neonatal screening and next generation sequencing techniques have led to the identification of subjects with novel homozygous, compound heterozygous, and heterozygous mutations (60). Homozygous patients suffer from immunodeficiency with susceptibility to pneumonia, chronic diarrhea, candidiasis or mycobacterial infections, and Omenn syndrome (59, 61–64). Most of the heterozygous patients show nail dystrophy, usually presenting as leukonychia and minor immunological changes (60, 65, 66). Recurrent infections and atopic dermatitis are observed only in a minority of the patients (60).

Immunological features in patients with homozygous FOXN1 mutations so far reported include reduction of T lymphocytes, particularly CD4+ T cells (61–64), reduction of T-cell receptor excision circles (TRECs) and naive T CD45RA+ lymphocytes, with increase of T memory CD45RO+ lymphocytes (62–64). In addition, an increase in the DN CD4-CD8- lymphocytes in the periphery (61–63), a reduction in CD31+ cells, recently emigrated from the thymus was observed in few cases (63). Proliferative response to phytohemagglutinin (PHA) is usually poor or absent and TCR repertoire is oligoclonal (59, 61–63). Although natural killer (NK) and B cells are usually numerically normal, they may be functionally compromised, with impaired production of specific antibodies. Recent studies proved that FOXN1 haploinsufficiency, caused by FOXN1 heterozygous mutations, may be associated with T cell-lymphopenia in infants showing low TRECs at newborn screening (60). A progressive normalization of CD4 count is observed in adulthood in heterozygous subjects while CD8 are usually persistently low (60). The increase in CD4 levels is associated to persistently low CD45RA levels suggesting a mechanism of homeostatic

proliferation. A more significant homeostatic expansion in CD4 than CD8 might explain the difference in CD4 and CD8 T cells. Alternatively, another hypothesis is that FOXN1 dosage plays a stricter role in the expression of CD8 development genes (60).

# PAX1 DEFICIENCY AND OTOFACIOCERVICAL SYNDROME TYPE 2

PAX1 is a member of a family of genes that encodes transcription factors implicated in embryogenesis in vertebrates (67). It is located on chromosome 20p11.22 and contains 5 exons (10 Kb) (67, 68). This gene is expressed in fetal mesenchymal cells in the body of intervertebral disks and plays an important role in formation of the segmented vertebral column in humans (69). In mice, it is also expressed in cochlea and has a role in hearing process (70). PAX1 is also implicated in thymus organogenesis by contributing to the outgrowth of the thymic epithelial anlage from the 3rd PP and to the regulation of TECs differentiation/survival balance (**Figure 1**) (71, 72). During pre-natal life, it is expressed in the 3rd PP from E9.5, while in the post-natal thymus it is only expressed in cTECs (72) (**Figure 1**). In mice, Pax1 deficiency is associated with moderate thymic hypoplasia (72) that is more severe only when it is associated with Hoxa3 haploinsufficiency (73). On the contrary, in humans, PAX1 deficiency is associated with severe thymic hypoplasia, leading to SCID (74). The difference between human and mouse phenotype may be due to a compensatory contribution by murine Pax9 when Pax1 is lacking (75).

Otofaciocervical syndrome (OTFCS) is an autosomal dominant disorder characterized by short stature, facial dysmorphism (long face, narrow mandible), shoulder girdle abnormalities, hearing loss, and mild intellectual disability (76). Two different forms of OTFCS have been described but thymus development is only affected in OTFCS2, caused by PAX1 mutations. Different biallelic deleterious PAX1 variants cause OTFCS2 and SCID, characterized by absent thymic shadow, chronic diarrhea, recurrent respiratory infections, pneumonia, and also Omenn Syndrome (74). Different severity of OTFCS2 phenotype is described in different families (70, 71, 77).

The immunological phenotype of patients with OTFC2 was described very recently in six patients who showed T-cell lymphopenia, impaired proliferative response to mitogens, and normal levels of B and NK cells. In one of them, 98% of CD4 and CD8 T cells were CD45RO+ cells, while CD45RA+CD31+ cells and TRECs were undetectable. TCRVβ analysis showed an oligoclonal spectrum. Lymph node biopsy of another patient showed absence of germinal centers and almost total absence of CD3+ T cells. Patients with Omenn Syndrome also showed eosinophilia and increased IgE levels (74).

#### DIGEORGE SYNDROME AND 22Q11.2 DELETION

DiGeorge syndrome (DGS) is usually associated with 3 or a 1.5 Mb de novo microdeletion of 22q11.2 (78, 79). In the central region of deletion maps TBX1 gene containing 9 exons (80) and encodes a Tbx transcription factor, implicated in the regulation of nearly 2,000 genes (81, 82). It is strongly expressed in the 3rd and 4th PP endoderm and in the 4th pharyngeal arch arteries (83, 84), in the otic vesicle, vertebral column, later in tooth bud and, at a lower extent, in the brain (85). It is implicated in pharyngeal arch segmentation and outgrowth of the TECs from the 3rd PP (**Figure 1**). However, it should be noted that Tbx1 is not expressed in the thymic anlage and thus it is not directly implicated in TECs development (86). On the contrary, Tbx1 enforced expression within the 3rd PP represses TECs development (86). Reduced levels of Tbx1 in 22q11.2 deletion syndrome (22q11.2DS) impair the development of neural crest-derived mesenchymal cells that surround the 3rd PP, leading to thymic hypoplasia (87). TBX1 regulates the expression of secreted Fgfs molecules, namely Fgf8 and Fgf10, implicated in the control of TECs proliferation, differentiation, migration, and survival (88). Ffg receptor IIIb (FgfR2-IIIb) regulates the cascade of Hox3 paralogs transcription factors, Pax1/Pax9, and winged helix nude (Whn) (**Figure 1**) (89). Moreover, TBX1 interferes with the ability of small mother against decapentaplegic 1 (SMAD1) to bind SMAD4, preventing effective Bmp4 signaling (82). Bmp4 also contributes to early thymus and parathyroid morphogenesis (90). Isolated TBX1 mutations may be rarely reported in patients with DGS (91) and TBX1 gain-of-function mutations can result in the same phenotypic spectrum of loss-of-function mutations (92).

An embryonic phenocopy of DGS with impaired thymus development can be observed because of the lack of retinoic acid during gestation (79, 93, 94). Retinoic acid is able to regulate the expression of Tbx1 and other molecules implicated in thymus organogenesis including Pax1, Pax9, Hoxa3, Fgf8, and Bmp4 (95). Other epigenetic factors, including maternal diabetes (93) or prenatal exposure to retinoic acid or alcohol may also explain the alteration of thymus development in DGS (96–101). Recently, we reported on a 7-year-old DGS patient born to a mother with gestational diabetes mellitus in whom a 371 Kb-interstitial deletion of 3p12.3, involving the Zinc Finger Protein 717 (ZNF717), MicroRNA-1243 (MIR-1243), and MIR-4273 genes was identified (102). MiRNAs are small, non-coding RNAs involved in the modulation of gene expression by targeting messenger RNAs for degradation, translational repression, or both (103). miRNA-4273 regulates the expression of Bmp3, a member of the transforming growth factor β superfamily, involved in thymus and kidney development (104). The expression of other miRNAs, including MIR-185, and MIR-150 can be impaired in 22q11.2DS patients (105). MIR-185 reduction increases Bruton's tyrosine kinase (Btk) expression leading to autoantibody production while MIR-185 increase leads to dose-dependent T-cell lymphopenia (105, 106). Reduced MIR-150 expression contributes to the reduction of T and B cells (107). Dysregulation of miRNA biogenesis, due to DiGeorge Critical Region Gene 8 (DGCR8) haploinsufficiency, is implicated in the pathogenesis of immunological, cardiac, endocrinological, and neurological phenotype (105, 108). Other genes implicated in the immune response are included in the deleted region. Alterations of CrK-like (CRKL), a gene encoding a 39-kDa adapter protein belonging to the Crk family, implicated in many cellular functions, including cell migration and adhesion, are associated with impaired T-cell proliferation in response to TCR triggering (109).

DGS has a prevalence of 1:4,000 newborns (79, 110). Most of these patients present with thymic and parathyroid hypoplasia, congenital heart defects, and craniofacial dysmorphisms (78, 79). Thymic development ranges from athymia in complete DGS (cDGS) to a completely normal thymus development in partial DGS (pDGS), resulting in a variable spectrum of T-cell deficiency (78, 79, 111). cDGS is reported in about 1.5% of the patients (111). Patients with DGS show a wide spectrum of T-cell alterations ranging from completely normal T-cell development to cDGS with absent thymic development (112, 113). In 22q11.2DS patients, thymus is usually small or hypoplastic. The size of the thymus does not predict the levels circulating T cells. In fact, microscopic rests of TECs may be present at aberrant locations (114). Low CD3+ Tcell percentage is the most common T-cell defect, followed by low CD3 number (78). CD4 and CD8 compartments are similarly affected (78). T-cell number and percentage tend to increase during the follow up starting from the first year of life (78). Naive CD4 and CD8 lymphocytes are lower in pDGS patients compared to controls independently of age and they decline more rapidly with age (78). The improvement of the lymphopenia with age is not due to a recovery of the thymic function but to the peripheral homeostatic expansion of the available T cells, as suggested by the evidence that T cells are predominantly or almost exclusively of a memory phenotype, TRECs are low and the repertoire is oligoclonal (78, 115, 116). T-cell proliferation, total immunoglobulins, and specific antibody response to vaccines are typically normal. IgM levels are often low, and some patients may show selective IgA deficiency (78). The study of the thymic architecture and thymocyte development in thymi obtained from pediatric pDGS patients revealed a reduction of mature CD4+ and CD8+ T cell frequency, associated with reduced proportion and function of T regulatory cells (Tregs) (117).

The majority of DGS patients suffer from chronic otitis media, which correlates with primarily conductive hearing impairment (78). Most patients have increased susceptibility to mild infections and only rarely to atypical or severe infections (78). Abnormalities of PP derivatives, predisposing to bacterial colonization, more than immune defects are implicated in this susceptibility (78). Autoimmune diseases, mainly presenting as rheumatoid diseases and idiopathic thrombocytopenia purpura, are reported in ∼10% of DGS patients (113, 116, 118). Predisposition to autoimmunity in DGS patients is partially explained by the mechanism of lymphopenia induced T-cell homeostatic proliferation together with the reduction of natural Tregs (nTregs) (117, 119).

#### CHD7 HAPLOINSUFFICIENCY AND CHARGE SYNDROME

CHARGE (coloboma, heart defects, atresia choanae, growth retardation, genital abnormalities, and ear abnormalities) syndrome is associated to haploinsufficiency in CHD7 gene, located on chromosome 8q12 (120). CHD7 is implicated in chromatin organization of mesenchymal cells, derived from the cephalic neural crest (121). Impaired CHD7 expression correlates with defects in neural crest cells, cephalic mesenchyme, pharyngeal arches, brain, otic vesicle and, in the mesoderm of the developing heart, especially in the outflow tract of the heart (122, 123). CHD7 is critical for the development of cTECs and mTECs from pharyngeal endoderm by regulating Bmp4, which in turn regulate Foxn1 expression (**Figure 1**) (124). Chd7 deficiency is also associated with down-regulation of Ikaros, Interelukin 7 receptor (Il7r), recombinase activating gene 1 (Rag1) (124). Studies suggest that CHD7 can also regulate TBX1 expression (74, 125).

CHARGE syndrome is characterized by different degrees of thymic alterations and even by complete thymic aplasia, resulting in combined immune deficiency (126). A wide spectrum of T-cell deficiency and isolated humoral immune deficiency may be observed in patients with CHARGE syndrome (126). They may show severe T-cell deficiency resembling SCID or, similarly to DGS, they may present transient lymphopenia, that usually normalizes over time. A close association between lymphopenia and hypocalcemia has been identified (115). Severity of the T-cell lymphopenia relates to the degree of thymic hypoplasia (127). CHARGE patients also show decreased naive CD4 and CD8 T-cells, peripheral T cells, and TRECs. Peripheral B-cell differentiation and immunoglobulin production are normal but in a 3rd of the patients an abnormal expression of IgM on class-switched memory B cells and diminished production of specific antibodies may be observed (127). Selective antibody deficiency tends to resolve spontaneously over time (127). In a recent paper, immunological features were compared between CHARGE and DGS patients. Total lymphocyte count was slightly lower in DGS patients compared to CHARGE patients and persistent lymphopenia was more common in DGS patients than in CHARGE. IgM levels were significantly lower in DGS patients compared to CHARGE (128).

Most of the patients have increased risk of recurrent infections, including recurrent otitis media, sinusitis, conjunctivitis, dermatitis, respiratory tract infections, pneumonia, and sepsis (127). Severe or atypical infections may also be reported including recurrent oral candidiasis, recurrent severe infections, septic shock, and chronic viral infections (129). As for partial DGS also in CHARGE syndrome the frequent need of invasive operative procedures and anatomical alterations, including extensive ear, sinus, nasal and palatal malformations, altered Eustachian tube anatomy, gastroesophageal reflux, or neurological abnormalities with compromised drainage and aspiration may help explain the susceptibility to infections (127). Immune defects including impaired thymus development and subsequent humoral deficiency may also contribute to the susceptibility to infections but at a lower extent (128). Patients with CHARGE syndrome also show increased risk of atopy, reported in 65% of the patients, usually presenting as food allergy (128). Increased susceptibility to autoimmune disorders may also feature this syndrome (129). Patients with complete athymia may present with atypical SCID (Omenn-like) phenotype (130).

# 2P11.2 MICRODELETION AND FOXI3 HAPLOINSUFFICIENCY

A microdeletion at 2p11.2 has been identified in five families presenting with DGS features, including hypocalcaemia, asymmetric crying face, low TRECs and T-cell lymphopenia, without typical facial dysmorphism, and heart abnormalities. FOXI3, a member of FOX family transcription factors, implicated in development of brachial arch-derived structures was considered the candidate gene for this phenotype (131). Early in embryonic development Foxi3 is broadly expressed in the pre-placodal ectoderm surrounding the neural plate, from which all craniofacial sensory organs derive (132, 133). Subsequently, its expression is restricted to the region from which otic and epibranchial placodes derive and finally to the ectoderm and endoderm of the pharyngeal arches. A deletion of FOXI3 gene has been recently identified in a patient with left congenital aural atresia and ipsilateral internal carotid artery agenesis (134). Foxi3 is also implicated in the differentiation of the epithelial cells within the epidermis as suggested by the identification of Foxi3 heterozygous mutations in several hairless dog breeds with hair follicle and teeth defects (135) and in thymic corticomedullary differentiation. Foxi3 is implicated in segmentation of the pharyngeal apparatus and LOF of Foxi3 alone or in combination with Tbx1 LOF, leads to failure of the pharyngeal arch segmentation due to the inability of the epithelia to properly invaginate with subsequent thymic hypoplasia/aplasia (136).

The main clinical features of the different syndromes are compared in **Table 1**.

TABLE 1 | Comparison of the main clinical features among different congenital disorders of thymic development.

*DGS, DiGeorge syndrome; cDGS, complete DGS; pDGS, partial DGS; OTFC2 syndrome, Otofaciocervical syndrome type 2; CHARGE syndrome, coloboma, heart defects, atresia choanae, growth retardation, genital abnormalities, and ear abnormalities syndrome.*

#### TREATMENT OPTIONS FOR ATHYMIC CONDITIONS

Hematopoietic stem cell transplantation (HSCT) represents the cornerstone for the treatment of SCID. However, in SCID due to genetic defects that impair development and function of the thymic epithelium, theoretically thymus transplantation would represent the most appropriate therapy. Thymic tissue is obtained from infants undergoing to median sternotomy for open heart surgery. Cultured postnatal human thymic tissue is then transplanted in thin slices into the quadriceps muscle (137). In cases with successful transplant, few months after the transplant the graft is colonized by host stem cells and is able to support normal thymopoiesis (138) leading to the development of mature naive T-cell with diverse TCRVβ repertoire and able to proliferate in response to mitogens. Tcell levels in surviving patients are usually low for age but are sufficient to respond to viral, disseminated, and other infections leading to a resolution of the immunodeficiency (139). In some cases, reduced thymic output may be explained by other comorbidities, such as heart failure and hypoxia stress, that may cause hypoperfusion of the graft (139). The success of the transplant may be also limited in patients with active viral infections since the virus itself, its treatment, or both might inhibit the thymopoiesis (139). In patients with successful transplant, naive T-cells are usually detected within 6 months after the transplant (139, 140). Antibody responses and immunoglobulin levels normalize (62) even though numbers of class-switched memory B cells may remain relatively low. The success of the transplant is not correlated with the amount of tissue transplanted, HLA matching, culture conditions or immunosuppression of the recipient (141). Immunosuppression can be used to delete reactive oligoclonal T cells and mature T cells responsible of graft-versus-host disease and graft rejection (137, 139). Overall survival in DGS is 75% and mortality is usually related to pre-transplantation morbidity, mainly viral infections, and chronic lung disease (115, 140, 142). Thymus transplantation has been recently used to treat 2 patients with FOXN1 deficiency and both survived (62, 143, 144) while its use in athymic CHARGE and OTFC2 patients has never been reported. Autoimmune disorders, including thyroiditis and severe cytopenia, represent the most common complication after thymus transplant (139, 140).

Adoptive transfer of mature T cells from human leukocyte antigen identical siblings through bone marrow transplantation represents an alternative to thymus transplant to treat SCID in athymic patients (145). However, only post-thymic T cells engraft in this case and naive T cells do not develop. Survival after matched unrelated donor and matched sibling transplantations in cDGS were reported as being 33 and 60%, respectively (145) while in CHARGE out of six patients treated with HSCT three had graft vs. host disease and three died post-transplant (125, 130, 146, 147). Long-lasting survival patients after matched sibling donor transplantation are reported (148). Four FOXN1 deficient patients underwent HSCT and two survived (59, 63, 64, 149). One of them is currently well at 22 years of age (unpublished data). The study of the T cell compartment in this patient, 5 years after HSCT showed a marked reduction of CD4CD45Ra levels with normal CD8CD45Ra levels. However, TCRVβ repertoire, was largely impaired in the CD8 subset (150). Six patients with OTFC2 were treated with allogeneic HSCT. T-cell reconstitution was not observed in any of the patients, despite successful engraftment in three of them. In one of the cases with successful engraftment all the T cells showed a memory (CD45R0+) phenotype, but no de novo generation of a polyclonal repertoire of naive T cells was observed. The remaining 2 patients showed persistent T cell lymphopenia leading to severe and recurrent infections, and death for septic shock in one patient and to severe autoimmune hemolytic anemia in the other (74). Together, these data indicate that HSCT may be unable to correct the profound T cell immunodeficiency of this disease.

#### CONCLUSIONS

In conclusion, in this review we described different pathways involved in thymus development and the clinical phenotypes associated with their impairment. We also summarized the outcome related to different therapeutic approaches to these disorders. We highlighted the clinical importance of the early detection of a defect in the pathways involved in T-cell development. With the recent introduction of newborn screening programs a timely identification of patients affected with defects of the T-cell development before the onset of the symptoms

#### REFERENCES

is now possible, and this prompted the definitive treatment with HSCT. This has been proven effective in improving the prognosis. However, in some cases HSCT is not required for the management of infants with T-cell lymphopenia at birth, since the T-cell development tend to improve with age (prematurity, FOXN1 haploinsufficiency). In other cases, HSCT may not be curative since the defect involves thymus development. However, genome-wide association studies have shown that a large proportion of variants likely to cause human disease are located outside of the protein-coding domains, so whole genome wide approaches might lead to proper identification and thus correct treatment plans for immunodeficiency disorders resulting from aberrant expression of some of the genes discussed in the present review.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. GG, CB, MD, EC, and RR reviewed the literature, organized and wrote the manuscript. CP wrote the manuscript and supervised all the work. RP did the picture. All authors reviewed and approved the manuscript.

### FUNDING

This work was partially supported by a public grant overseen by Italian Ministry of Health as part of the project 'Ricerca Finalizzata' (reference: RF-2016-02364303).

development of thymic medullary epithelial cells. PLoS Genet. (2016) 12:e1005776. doi: 10.1371/journal.pgen.1005776

gene born to a mother with gestational diabetes mellitus. Am J Med Genet A. (2017) 7:1913–8. doi: 10.1002/ajmg.a.38242

**Conflict of Interest:** 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 © 2020 Giardino, Borzacchiello, De Luca, Romano, Prencipe, Cirillo and Pignata. 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.

# Thymic Engraftment by in vitro-Derived Progenitor T Cells in Young and Aged Mice

Jastaranpreet Singh1 , Mahmood Mohtashami 2 , Graham Anderson3 and Juan Carlos Zúñiga-Pflücker 1,2 \*

1 Department of Immunology, University of Toronto, Toronto, ON, Canada, 2 Sunnybrook Research Institute, Toronto, ON, Canada, 3 Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, United Kingdom

#### Edited by:

Nicolai Stanislas Van Oers, University of Texas Southwestern Medical Center, United States

#### Reviewed by:

Katsuto Hozumi, Tokai University School of Medicine, Japan Koji Yasutomo, Tokushima University, Japan

\*Correspondence:

Juan Carlos Zúñiga-Pflücker<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology

Received: 05 May 2020 Accepted: 09 July 2020 Published: 18 August 2020

#### Citation:

Singh J, Mohtashami M, Anderson G and Zúñiga-Pflücker JC (2020) Thymic Engraftment by in vitro-Derived Progenitor T Cells in Young and Aged Mice. Front. Immunol. 11:1850. doi: 10.3389/fimmu.2020.01850 T cells play a critical role in mediating antigen-specific and long-term immunity against viral and bacterial pathogens, and their development relies on the highly specialized thymic microenvironment. T cell immunodeficiency can be acquired in the form of inborn errors, or can result from perturbations to the thymus due to aging or irradiation/chemotherapy required for cancer treatment. Hematopoietic stem cell transplant (HSCT) from compatible donors is a cornerstone for the treatment of hematological malignancies and immunodeficiency. Although it can restore a functional immune system, profound impairments exist in recovery of the T cell compartment. T cells remain absent or low in number for many months after HSCT, depending on a variety of factors including the age of the recipient. While younger patients have a shorter refractory period, the prolonged T cell recovery observed in older patients can lead to a higher risk of opportunistic infections and increased predisposition to relapse. Thus, strategies for enhancing T cell recovery in aged individuals are needed to counter thymic damage induced by radiation and chemotherapy toxicities, in addition to naturally occurring age-related thymic involution. Preclinical results have shown that robust and rapid long-term thymic reconstitution can be achieved when progenitor T cells, generated in vitro from HSCs, are co-administered during HSCT. Progenitor T cells appear to rely on lymphostromal crosstalk via receptor activator of NF-κB (RANK) and RANK-ligand (RANKL) interactions, creating chemokine-rich niches within the cortex and medulla that likely favor the recruitment of bone marrow-derived thymus seeding progenitors. Here, we employed preclinical mouse models to demonstrate that in vitro-generated progenitor T cells can effectively engraft involuted aged thymuses, which could potentially improve T cell recovery. The utility of progenitor T cells for aged recipients positions them as a promising cellular therapy for immune recovery and intrathymic repair following irradiation and chemotherapy, even in a post-involution thymus.

Keywords: T cell progenitors, T cell development, thymus regeneration, thymic hypoplasia, thymic involution, aged thymus

# INTRODUCTION

T cells are essential mediators of antigen-specific, longterm adaptive immunity. The thymus is responsible for the development of self-tolerant, immunocompetent T cells, but given a lack of self-renewing cells, is continually reliant on replenishment of new T cell progenitors derived from bone marrow (BM) hematopoietic stem cells (HSCs). Subsequent maturation of T cells occurs through a series of tightly regulated and directed differentiation stages that are dependent on signals from the specialized thymic microenvironment. These processes lead to the generation of mature CD4+CD8− and CD4−CD8+ single positive (SP) T cells that exit the thymus and seed the peripheral organs (spleen, lymph nodes), where they can subsequently encounter antigen, undergo expansion, and acquire effector or memory functions (1).

T cell immunodeficiency, or T lymphopenia, can result from perturbations to the thymic microenvironment due to age-related thymic involution, irradiation required for cancer treatment or from infections that directly target T cells (2). One of the major causes of T cell deficiency is primary immunodeficiency, which is often inherited, leading to an early disease onset. Extrinsic factors can also adversely affect the immune response. In particular, the use of chemotherapeutic agents such as cyclophosphamide prior to hematopoietic stem cell transplantation (HSCT) can cause rapid transient involution of the thymus (3–5). While these drugs are critically required for eradication of the cancer cells, they also harm healthy dividing cells, including cells of the hematopoietic compartment and thymic epithelial cells (TECs) that constitute part of the thymic microenvironment. As a result, there is a delay in T cell recovery following HSCT, and a paucity of de novo T cell generation from HSC-derived progenitors (5–9), which can be particularly problematic for aged patients that are concomitantly undergoing age-related thymic involution (**Figure 1**). The end result is dramatic changes in the T cell compartment of patients including a decline in naïve T cell output, reduced T cell diversity, and increased susceptibility to infection, autoimmune diseases and cancer (10). Therefore, altered thymic architecture is a key trigger for the deterioration of T cell-related immune function in the aged, and insight into strategies that enhance thymic function in adults is of critical importance. Here, we explore the challenges of T cell recovery and thymic regeneration following myeloablative and irradiation treatments, and leading approaches in the field to overcome these issues. We focus on recent advances that take advantage of cell-based treatments, such as progenitor T cell engraftment, for overcoming periods of immunodeficiency following HSCT, particularly in aged individuals.

# T CELL RECONSTITUTION AFTER MYELOABLATIVE TREATMENTS

Allogeneic HSCT is a mainstay for the treatment of a large number of diseases of the hematopoietic system. A number of modifications to HSCT procedures, including T cell depletion, CD34+ hematopoietic stem/progenitor cell selection, and the use of irradiation and chemotherapeutic drugs, have greatly improved post-transplant clinical outcomes (11, 12). Nevertheless, T cell repopulation post-transplantation remains a major hurdle (5). T cell recovery is often delayed by months and it may take years to fully restore normal numbers of T cells and functionality, if at all (13, 14). Furthermore, there appears to be an inverse correlation between time to post-HSCT T cell recovery and the age of the recipient (15). While it may take up to 6 months to 1 year in the young to recover T cells with a wide T cell receptor (TCR) repertoire, it may take years in adult patients to witness evidence of new T cell generation, if ever (16–18). With an increasingly aging population, there is an imminent need for a dependable way to reconstitute all blood cells, including T cells, in cancer patients that have received chemotherapy and irradiation. Otherwise, patients remain susceptible to a variety of complications that can result in mortality due to general weakened immunity that renders them vulnerable to opportunistic infections and potential cancer relapse (19).

To mitigate cancer relapse, many clinics maintain the standard practice of not depleting donor T cells from HSCT grafts. While donor T cells contained within the graft can mount anti-tumor responses against the relapsed cancer, they can also induce graft vs. host disease (GvHD) against vital organs, even in human leukocyte antigen (HLA)-matched transplants (20). The complications of GvHD are, in turn, managed by defined doses of prescribed immunosuppressants, which aim to balance the antitumor effects of the graft with the suppression of GvHD (21). However, this is an unlikely long-term solution for patients, as the vast majority of patients succumb to GvHD or cancer relapse due to immunosuppression within 5 years post-transplant. To this end, an extensive understanding of the phenotypic changes and microenvironmental cues facilitating the transition of HSCs in the BM to T cells in the thymus may be useful for development of strategies to improve T cell outcomes post-HSCT.

## The Intrathymic Intricacies of T Cell Development

T cell precursors arise from HSCs in the adult BM (**Figure 1**). Thymus seeding progenitors (TSPs) arrive in the thymus in small numbers, where upon interaction with the thymic epithelial cells (TECs), give rise to early thymic progenitors (ETPs). ETPs in both human and mice differentiate through successive CD4−CD8− double negative (DN) stages that require Notch receptor and Delta-like-4 (Dll4) ligand interactions. Targeted deletions of either Notch1 receptor in hematopoietic cells, or Dll4 in TECs, result in the abrogation of T cell development (22– 25). Notch signaling mediates cell fate restriction, which begins within the BM TSP subset, and continues following entry into the thymus (26).

Proper stage-specific maturation of T cell precursors is dependent on thymic structure and the diverse cells present in thymic stroma. The thymus is differentiated into an outer cortical and an inner medullary region, which contain cortical and medullary TECs (cTECs and mTECs), respectively. Their maintenance, in turn, is governed by a complex interplay between lymphoid and stromal compartments within the thymus (termed

lymphostromal thymic crosstalk) (27). Studies in the postnatal mouse have demonstrated that TSPs express CCR7 and CCR9, and enter the thymus at the junction between the cortex and medulla (1). Thymic entry is governed by the chemokines CCL21 and CCL25, which are expressed primarily by mTECs and cTECs, respectively. As they differentiate, T cell precursors migrate outwards toward the subcapsular zone of the cortex in response to a CCL25 chemotactic gradient (1, 28, 29). In the subcapsular zone, the CD4−CD8− DN thymocytes proliferate and subsequently differentiate into CD4+CD8+ DP cells that up-regulate CCR7 on their cell surface and migrate toward the medulla (mediated by CCR7 ligands, CCL19 and CCL21) where the final stages of T cell development take place (30). These chemokine-receptor interactions constitute one of the pivotal crosstalk signals dictating strict migration of developing thymocytes through the thymus, and they are conserved in humans (31, 32).

Thymic crosstalk also induces proliferation and ensures selection of a T cell repertoire that is self-tolerant, but reactive to foreign challenges. Relatively little is known of the involvement of crosstalk in the development and function of the thymic cortex, although the notion of crosstalk is well-accepted (27). Mouse models showing a block in early T cell development (CD3ε transgenic mice, CD117−/−CD132−/− double-mutant mice) lack demarcation of cortical and medullary areas, and show marked reductions in thymocyte numbers compared to wildtype (WT) mice, implying that the early stages of T cell development are important in TEC maturation (27). Thymic crosstalk during positive and negative selection events have been better defined. Low/intermediate affinity interactions between TCR and major histocompatibility complex (MHC)/peptide complexes on TECs prevent cell death during positive selection. Positively selected thymocytes then localize to the medulla, where high affinity interactions between the TCR and MHC/peptide complexes on thymic dendritic cells result in apoptosis via negative selection of self-reactive cells (1, 28). Self-tolerance by negative selection is established through the expression of the transcription factors autoimmune regulator (Aire) and Fezf2 by mTECs (33). These two independent factors are regulated by distinct signaling pathways, the RANK/CD40 pathway and the LTβR pathways, respectively, are non-redundant and promote the expression of peripheral tissue-restricted antigens (TRA) on mTECs (33, 34). T cells displaying a strong self-reactivity toward TRA are deleted.

The non-canonical nuclear factor-κB (NF-κB) pathway has a well-established role in thymic crosstalk during selection. Lymphotoxin-β receptor (LTβR), RANK, and CD40 are the key tumor necrosis factor (TNF) superfamily members that signal through the non-canonical NF-κB pathway in the context of thymic crosstalk and self-tolerance (35). In mice, LTβR deletions led to defective medullary development, and a reduced or absent medulla. The reduction of Aire+ mTECs was relatively mild (35, 36), although Fezf2-dependent TRA expression on mTECs was drastically decreased (33). The involvement of CD40 ligand (CD40L)-CD40 interactions has also been described (35). Overexpression of CD40L in the thymus resulted in abrogated thymus cortex formation and medullary expansion, whereas analysis of CD40L-deficient mice revealed no obvious defect in medullary organization by immunohistochemistry. RANK- and RANKL-deficient mice exhibit a severe reduction in Aire+ mTECs and overall mTEC numbers, although the formation of a thymic medulla is still detectable. Furthermore, RANKL-RANK and CD40L-CD40 signaling exhibit a cooperative role in mTEC development and self-tolerance (35). Notably, the defects seen in these deficient mice do not affect Fezf2-restricted TRA expression and are milder compared to mutant mice deficient in the NF-κB activation pathway such as aly/aly (NF-κB-inducing kinase mutation), and RelB-deficient mice (35–37). Although the expression and function of these molecules on thymocyte and TEC subsets is well-understood in mice, this has yet to be elucidated in the human context. Furthermore, harnessing insights from these developmental processes could be important for restoring the thymus and T cell-mediated immunity in the aged following HSCT.

#### AGE-RELATED THYMIC INVOLUTION

Defects in both the hematopoietic and thymic stromal components contribute to decreased thymic output in aged individuals. The fundamental reason for the late or absent T cell recovery in aged patients is the natural and progressive decline of the immune system, especially of the thymus, in a process called thymic involution. Thymic involution starts to occur before puberty, but is at its peak during puberty and the decline continues steadily with age (38). Although thymus involution in the aged was noted many years before the function of thymus was discovered (39), the evolutionary advantage of this physiological process remains a mystery. Thymic involution, which results in its several fold size reduction, is not perceptible with physical examination, but it is concomitant with physiological changes that can be measured. Multidetector computed tomography (CT) scans can detect increasing replacement of soft tissue that is mainly occupied by thymocytes and TECs by a less dense, X-ray permissive fatty tissue (40, 41). These changes impact both the thymocyte and TEC compartments of the thymus, having detrimental impacts on the establishment of T cell-based immunity in the adult.

#### Impairments in T Cell Development

Since the thymus requires continuous seeding by BM HSCderived progenitors, the chance of T cell generation is further diminished since aged HSCs have a propensity to differentiate toward the myeloid fate (42) in both mice (43– 47) and humans (48, 49). These alterations in HSC function inevitably affect the lymphoid compartment, and different groups have shown that aged mice not only have less ETPs, but that the ETPs also have reduced expansion and differentiation potential (50, 51). Furthermore, HSCs from old mice do not efficiently repopulate lymphoid cells in young recipients (46).