Durable Mixed Chimerism May Permit Subsequent Immunosuppression-Free Intestinal Transplantation - a Proof-of-Principle Study.

Intestinal transplantation (ITx) is the definitive treatment for intestinal failure but has the highest rejection rate among solid organ transplants, requiring high doses of immunosuppression with high rates of infection, graft-versus-host disease, and malignancy. Transplant tolerance would overcome the need for long-term immunosuppression. Using non-myeloablative conditioning, our laboratory has developed a novel swine model of hematopoietic stem cell transplantation (HSCT) that produces durable mixed chimerism (MC) and immune tolerance without toxicity. We investigated whether durable MC would promote tolerance of subsequently transplanted donor-matched intestinal allografts without immunosuppression. Using miniature swine with defined MHC, we performed HSCT across an MHC-Class-I haplotype mismatch. Immunosuppression was stopped by day 45. MC was evaluated by flow cytometry, and mixed lymphocyte reaction (MLR) assays were used to evaluate cellular responses. Subsequently, orthotopic ITx was performed without immunosuppression using a donor that was MHC-matched to the HSCT donor. Recipients were observed for four weeks and euthanized for tissue collection and mechanistic assays. After HSCT, the recipients developed durable multilineage MC and apparent deletional tolerance. After ITx, recipients showed no clinical or histological signs of rejection, and chimerism was unchanged. These results demonstrate the potential value of generating durable MC to achieve transplant tolerance.

Harnessing cellular therapeutics for type 1 diabetes mellitus: progress, challenges, and the road ahead.

Type 1 diabetes mellitus (T1DM) is a growing global health concern that affects approximately 8.5 million individuals worldwide. T1DM is characterized by an autoimmune destruction of pancreatic ß cells, leading to a disruption in glucose homeostasis. Therapeutic intervention for T1DM requires a complex regimen of glycaemic monitoring and the administration of exogenous insulin to regulate blood glucose levels. Advances in continuous glucose monitoring and algorithm-driven insulin delivery devices have improved the quality of life of patients. Despite this, mimicking islet function and complex physiological feedback remains challenging. Pancreatic islet transplantation represents a potential functional cure for T1DM but is hindered by donor scarcity, variability in harvested cells, aggressive immunosuppressive regimens and suboptimal clinical outcomes. Current research is directed towards generating alternative cell sources, improving transplantation methods, and enhancing cell survival without chronic immunosuppression. This Review maps the progress in cell replacement therapies for T1DM and outlines the remaining challenges and future directions. We explore the state-of-the-art strategies for generating replenishable ß cells, cell delivery technologies and local targeted immune modulation. Finally, we highlight relevant animal models and the regulatory aspects for advancing these technologies towards clinical deployment.

Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation in a pediatric-dominated cohort.

Introduction: It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of recipient gut lymphocyte populations in immunosuppressed conditions. Methods: Using polychromatic flow cytometry that includes HLA allele group-specific antibodies distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. Results: We confirm the early presence of naïve donor B cells in the circulation (donor age range: 1-14 years, median: 3 years) and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa (recipient age range at the time of transplant: 1-44 years, median: 3 years). Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection (recipient age range at the time of transplant: 1-9 years, median: 2 years) revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in deceased adult donors. In available pan-scope biopsies from pediatric recipients, we observed higher percentages of naïve recipient B cells in colon allograft compared to small bowel allograft and increased BCR overlap between native colon vs colon allograft compared to that between native colon vs ileum allograft in most cases, suggesting differential clonal distribution in large intestine vs small intestine. Discussion: Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of stabilization of the mucosal B cell repertoire in pediatric ITx patients.

Development of a large animal orthotopic intestinal transplantation model with long-term survival for study of immunologic outcomes.

Introduction: Intestinal transplantation (ITx) is the last remaining therapy for patients with intestinal failure once parenteral nutrition is no longer an option, however its use is limited by immunological complications, including high rates of rejection and morbidity associated with immunosuppression, such as infection and malignancy. We aimed to develop a large animal model of ITx with which to study the immune response to ITx and to design and test tolerance induction regimens. Methods: Learning from prior complications, we developed and progressively improved both surgical methods for the donor and recipient as well as postoperative management strategies. Methods of stoma generation, bowel positioning, vessel preparation, and fluid management were optimized. The immunosuppression strategy mirrored our clinical regimen. Results: As a result of our modifications, results improved from survival less than 1 month to consistent long-term survival with good graft function. We review several techniques that were developed to avoid pitfalls that were encountered, which can be used to optimize outcomes in this model. Discussion: Achieving long-term survival after swine orthotopic ITx permits immunological analysis and pre-clinical trials in a large animal model of ITx.

Combination cell therapy leads to clonal deletion of donor-specific T cells in kidney transplant recipients.

BACKGROUND: Induction of donor-specific tolerance is a promising approach to achieve long-term graft patency in transplantation with little to no maintenance immunosuppression. Changes to the recipient's T cell receptor (TCR) repertoire are understood to play a pivotal role in the establishment of a robust state of tolerance in chimerism-based transplantation protocols. METHODS: We investigated changes to the TCR repertoires of patients participating in an ongoing prospective, controlled, phase I/IIa trial designed to evaluate the safety and efficacy of combination cell therapy in living donor kidney transplantation. Using high-throughput sequencing, we characterized the repertoires of six kidney recipients who also received bone marrow from the same donor (CKBMT), together with an infusion of polyclonal autologous Treg cells instead of myelosuppression. FINDINGS: Patients undergoing combination cell therapy exhibited partial clonal deletion of donor-reactive CD4+ T cells at one, three, and six months post-transplant, compared to control patients receiving the same immunosuppression regimen but no cell therapy (p = 0.024). The clonality, R20 and turnover rates of the CD4+ and CD8+ TCR repertoires were comparable in both groups, showing our protocol caused no excessive repertoire shift or loss of diversity. Treg clonality was lower in the case group than in control (p = 0.033), suggesting combination cell therapy helps to preserve Treg diversity. INTERPRETATION: Overall, our data indicate that combining Treg cell therapy with CKBMT dampens the alloimmune response to transplanted kidneys in humans in the absence of myelosuppression. FUNDING: This study was funded by the Vienna Science and Technology Fund (WWTF).

Follicular helper- and peripheral helper-like T cells drive autoimmune disease in human immune system mice.

Human immune system (HIS) mice constructed in various ways are widely used for investigations of human immune responses to pathogens, transplants and immunotherapies. In HIS mice that generate T cells de novo from hematopoietic progenitors, T cell-dependent multisystem autoimmune disease occurs, most rapidly when the human T cells develop in the native NOD.Cg- Prkdc scid Il2rg tm1Wjl (NSG) mouse thymus, where negative selection is abnormal. Disease develops very late when human T cells develop in human fetal thymus grafts, where robust negative selection is observed. We demonstrate here that PD-1 + CD4 + peripheral (Tph) helper-like and follicular (Tfh) helper-like T cells developing in HIS mice can induce autoimmune disease. Tfh-like cells were more prominent in HIS mice with a mouse thymus, in which the highest levels of IgG were detected in plasma, compared to those with a human thymus. While circulating IgG and IgM antibodies were autoreactive to multiple mouse antigens, in vivo depletion of B cells and antibodies did not delay the development of autoimmune disease. Conversely, adoptive transfer of enriched Tfh- or Tph-like cells induced disease and autoimmunity-associated B cell phenotypes in recipient mice containing autologous human APCs without T cells. T cells from mice with a human thymus expanded and induced disease more rapidly than those originating in a murine thymus, implicating HLA-restricted T cell-APC interactions in this process. Since Tfh, Tph, autoantibodies and LIP have all been implicated in various forms of human autoimmune disease, the observations here provide a platform for the further dissection of human autoimmune disease mechanisms and therapies.

Tolerance in intestinal transplantation.

Intestinal transplantation (ITx) is highly immunogenic, resulting in the need for high levels of immunosuppression, with frequent complications along with high rejection rates. Tolerance induction would provide a solution to these limitations. Detailed studies of alloreactive T cell clones as well as multiparameter flow cytometry in the graft and peripheral tissues have provided evidence for several tolerance mechanisms that occur spontaneously following ITx, which might provide targets for further interventions. These include the frequent occurrence of macrochimerism and engraftment in the recipient bone marrow of donor hematopoietic stem and progenitor cells carried in the allograft. These phenomena are seen most frequently in recipients of multivisceral transplants and are associated with reduced rejection rates. They reflect powerful graft-vs-host responses that enter the peripheral lymphoid system and bone marrow after expanding within and emigrating from the allograft. Several mechanisms of tolerance that may result from this lymphohematopoietic graft-vs-host response are discussed. Transcriptional profiling in quiescent allografts reveals tolerization of pre-existing host-vs-graft-reactive T cells that enter the allograft mucosa and become tissue-resident memory cells. Dissection of the pathways driving and maintaining this tolerant tissue-resident state among donor-reactive T cells will allow controlled tolerance induction through specific therapeutic approaches.

Enhancing Kidney Transplantation and the Role of Xenografts: Report of a Scientific Workshop Sponsored by the National Kidney Foundation.

Chronic kidney disease affects an estimated 37 million people in the United States; of these,>800,000 have end-stage renal disease requiring chronic dialysis or a kidney transplant to survive. Despite efforts to increase the donor kidney supply, approximately 100,000 people are registered on the kidney transplant wait-list with no measurable decrease over the past 2 decades. The outcomes of kidney transplantation are significantly better than for chronic dialysis: kidney transplant recipients have lower rates of mortality and cardiovascular events and better quality of life, but wait-list time matters. Time on dialysis waiting for a deceased-donor kidney is a strong independent risk factor for outcomes after a kidney transplant. Deceased-donor recipients with wait-list times on dialysis of<6 months have graft survival rates equivalent to living-donor recipients with waitlist times on dialysis of>2 years. In 2021,>12,000 people had been on the kidney transplant waitlist for ≥5 years. As the gap between the demand for and availability of donor kidneys for allotransplantation continues to widen, alternative strategies are needed to provide a stable, sufficient, and timely supply. A strategy that is gaining momentum toward clinical application is pig-to-human kidney xenotransplantation. This report summarizes the proceedings of a meeting convened on April 11-12, 2022, by the National Kidney Foundation to review and assess the state of pig-to-human kidney xenotransplantation as a potential cure for end-stage renal disease.

Dynamic establishment of recipient resident memory T cell repertoire after human intestinal transplantation.

BACKGROUND: Understanding formation of the human tissue resident memory T cell (TRM) repertoire requires longitudinal access to human non-lymphoid tissues. METHODS: By applying flow cytometry and next generation sequencing to serial blood, lymphoid tissue, and gut samples from 16 intestinal transplantation (ITx) patients, we assessed the origin, distribution, and specificity of human TRMs at phenotypic and clonal levels. FINDINGS: Donor age ≥1 year and blood T cell macrochimerism (peak level ≥4%) were associated with delayed establishment of stable recipient TRM repertoires in the transplanted ileum. T cell receptor (TCR) overlap between paired gut and blood repertoires from ITx patients was significantly greater than that in healthy controls, demonstrating increased gut-blood crosstalk after ITx. Crosstalk with the circulating pool remained high for years of follow-up. TCR sequences identifiable in pre-Tx recipient gut but not those in lymphoid tissues alone were more likely to populate post-Tx ileal allografts. Clones detected in both pre-Tx gut and lymphoid tissue had distinct transcriptional profiles from those identifiable in only one tissue. Recipient T cells were distributed widely throughout the gut, including allograft and native colon, which had substantial repertoire overlap. Both alloreactive and microbe-reactive recipient T cells persisted in transplanted ileum, contributing to the TRM repertoire. INTERPRETATION: Our studies reveal human intestinal TRM repertoire establishment from the circulation, preferentially involving lymphoid tissue counterparts of recipient intestinal T cell clones, including TRMs. We have described the temporal and spatial dynamics of this active crosstalk between the circulating pool and the intestinal TRM pool. FUNDING: This study was funded by the National Institute of Allergy and Infectious Diseases (NIAID) P01 grant AI106697.

Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.

Application of enzyme-linked immunosorbent assay to detect antimicrobial peptides in human intestinal lumen.

Intestinal transplantation is the definitive treatment for intestinal failure. However, tissue rejection and graft-versus-host disease are relatively common complications, necessitating aggressive immunosuppression that can itself pose further complications. Tracking intraluminal markers in ileal effluent from standard ileostomies may present a noninvasive and sensitive way to detect developing pathology within the intestinal graft. This would be an improvement compared to current assessments, which are limited by poor sensitivity and specificity, contributing to under or over-immunosuppression, respectively, and by the need for invasive biopsies. Herein, we report an approach to reproducibly analyze ileal fluid obtained through stoma sampling for antimicrobial peptide/protein concentrations, reasoning that these molecules may provide an assessment of intestinal homeostasis and levels of intestinal inflammation over time. Concentrations of lysozyme (LYZ), myeloperoxidase (MPO), calprotectin (S100A8/A9) and ß-defensin 2 (DEFB2) were assessed using adaptations of commercially available enzyme-linked immunosorbent assays (ELISAs). The concentration of α-defensin 5 (DEFA5) was assessed using a newly developed sandwich ELISA. Our data support that with proper preparation of ileal effluent specimens, precise and replicable determination of antimicrobial peptide/protein concentrations can be achieved for each of these target molecules via ELISA. This approach may prove to be reliable as a clinically useful assessment of intestinal homeostasis over time for patients with ileostomies.

Plasticity of intragraft alloreactive T cell clones in human gut correlates with transplant outcomes.

The site of transition between tissue-resident memory (TRM) and circulating phenotypes of T cells is unknown. We integrated clonotype, alloreactivity, and gene expression profiles of graft-repopulating recipient T cells in the intestinal mucosa at the single-cell level after human intestinal transplantation. Host-versus-graft (HvG)-reactive T cells were mainly distributed to TRM, effector T (Teff)/TRM, and T follicular helper compartments. RNA velocity analysis demonstrated a trajectory from TRM to Teff/TRM clusters in association with rejection. By integrating pre- and post-transplantation (Tx) mixed lymphocyte reaction-determined alloreactive repertoires, we observed that pre-existing HvG-reactive T cells that demonstrated tolerance in the circulation were dominated by TRM profiles in quiescent allografts. Putative de novo HvG-reactive clones showed a transcriptional profile skewed to cytotoxic effectors in rejecting grafts. Inferred protein regulon network analysis revealed upstream regulators that accounted for the effector and tolerant T cell states. We demonstrate Teff/TRM interchangeability for individual T cell clones with known (allo)recognition in the human gut, providing novel insight into TRM biology.

Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation.

It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of gut lymphocyte populations. Using polychromatic flow cytometry that includes HLA allele group-specific mAbs distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. We confirm the early presence of naïve donor B cells in the circulation and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa. Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in healthy control adults. Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of establishment of a stable mucosal B cell repertoire.

Donor-derived regulatory dendritic cell infusion modulates effector CD8+ T cell and NK cell responses after liver transplantation.

Immune cell-based therapies are promising strategies to facilitate immunosuppression withdrawal after organ transplantation. Regulatory dendritic cells (DCreg) are innate immune cells that down-regulate alloimmune responses in preclinical models. Here, we performed clinical monitoring and comprehensive assessment of peripheral and allograft tissue immune cell populations in DCreg-infused live-donor liver transplant (LDLT) recipients up to 12 months (M) after transplant. Thirteen patients were given a single infusion of donor-derived DCreg 1 week before transplant (STUDY) and were compared with 40 propensity-matched standard-of-care (SOC) patients. Donor-derived DCreg infusion was well tolerated in all STUDY patients. There were no differences in postoperative complications or biopsy-confirmed acute rejection compared with SOC patients up to 12M. DCreg administration was associated with lower frequencies of effector T-bet+Eomes+CD8+ T cells and CD16bright natural killer (NK) cells and an increase in putative tolerogenic CD141+CD163+ DCs compared with SOC at 12M. Antidonor proliferative capacity of interferon-γ+ (IFN-γ+) CD4+ and CD8+ T cells was lower compared with antithird party responses in STUDY participants, but not in SOC patients, at 12M. In addition, lower circulating concentrations of interleukin-12p40 (IL-12p40), IFN-γ, and CXCL10 were detected in STUDY participants compared with SOC patients at 12M. Analysis of 12M allograft biopsies revealed lower frequencies of graft-infiltrating CD8+ T cells, as well as attenuation of cytolytic TH1 effector genes and pathways among intragraft CD8+ T cells and NK cells, in DCreg-infused patients. These reductions may be conducive to reduced dependence on immunosuppressive drug therapy or immunosuppression withdrawal.

The prospect of universal coronavirus immunity: characterization of reciprocal and non-reciprocal T cell responses against SARS-CoV2 and common human coronaviruses.

T cell immunity plays a central role in clinical outcomes of Coronavirus Infectious Disease 2019 (COVID-19) and T cell-focused vaccination or cellular immunotherapy might provide enhanced protection for some immunocompromised patients. Pre-existing T cell memory recognizing SARS-CoV-2 antigens antedating COVID-19 infection or vaccination, may have developed as an imprint of prior infections with endemic non-SARS human coronaviruses (hCoVs) OC43, HKU1, 229E, NL63, pathogens of "common cold". In turn, SARS-CoV-2-primed T cells may recognize emerging variants or other hCoV viruses and modulate the course of subsequent hCoV infections. Cross-immunity between hCoVs and SARS-CoV-2 has not been well characterized. Here, we systematically investigated T cell responses against the immunodominant SARS-CoV-2 spike, nucleocapsid and membrane proteins and corresponding antigens from α- and ß-hCoVs among vaccinated, convalescent, and unexposed subjects. Broad T cell immunity against all tested SARS-CoV-2 antigens emerged in COVID-19 survivors. In convalescent and in vaccinated individuals, SARS-CoV-2 spike-specific T cells reliably recognized most SARS-CoV-2 variants, however cross-reactivity against the omicron variant was reduced by approximately 47%. Responses against spike, nucleocapsid and membrane antigens from endemic hCoVs were significantly more extensive in COVID-19 survivors than in unexposed subjects and displayed cross-reactivity between α- and ß-hCoVs. In some, non-SARS hCoV-specific T cells demonstrated a prominent non-reciprocal cross-reactivity with SARS-CoV-2 antigens, whereas a distinct anti-SARS-CoV-2 immunological repertoire emerged post-COVID-19, with relatively limited cross-recognition of non-SARS hCoVs. Based on this cross-reactivity pattern, we established a strategy for in-vitro expansion of universal anti-hCoV T cells for adoptive immunotherapy. Overall, these results have implications for the future design of universal vaccines and cell-based immune therapies against SARS- and non-SARS-CoVs.

Leveraging the lymphohematopoietic graft-versus-host reaction (LGVHR) to achieve allograft tolerance and restore self tolerance with minimal toxicity.

Mixed allogeneic chimerism has considerable potential to advance the achievement of immune tolerance to alloantigens for transplantation and the restoration of self-tolerance in patients with autoimmune disease. In this article, I review evidence that graft-versus-host (GVH) alloreactivity without graft-vs-host disease (GVHD), termed a lymphohematopoietic graft-vs-host reaction (LGVHR), can promote the induction of mixed chimerism with minimal toxicity. LGVHR was originally shown to occur in an animal model when non-tolerant donor lymphocytes were administered to mixed chimeras in the absence of inflammatory stimuli and was found to mediate powerful graft-vs-leukemia/lymphoma effects without GVHD. Recent large animal studies suggest a role for LGVHR in promoting durable mixed chimerism and the demonstration that LGVHR promotes chimerism in human intestinal allograft recipients has led to a pilot study aiming to achieve durable mixed chimerism.

The CD58-CD2 axis is co-regulated with PD-L1 via CMTM6 and shapes anti-tumor immunity.

The cell-autonomous balance of immune-inhibitory and -stimulatory signals is a critical process in cancer immune evasion. Using patient-derived co-cultures, humanized mouse models, and single-cell RNA-sequencing of patient melanomas biopsied before and on immune checkpoint blockade, we find that intact cancer cell-intrinsic expression of CD58 and ligation to CD2 is required for anti-tumor immunity and is predictive of treatment response. Defects in this axis promote immune evasion through diminished T cell activation, impaired intratumoral T cell infiltration and proliferation, and concurrently increased PD-L1 protein stabilization. Through CRISPR-Cas9 and proteomics screens, we identify and validate CMTM6 as critical for CD58 stability and upregulation of PD-L1 upon CD58 loss. Competition between CD58 and PD-L1 for CMTM6 binding determines their rate of endosomal recycling over lysosomal degradation. Overall, we describe an underappreciated yet critical axis of cancer immunity and provide a molecular basis for how cancer cells balance immune inhibitory and stimulatory cues.

Origin, phenotype and autoimmune potential of T cells in human immune system mice receiving neonatal human thymus tissue.

Robust human immune system (HIS) mice are created using human fetal thymus tissue and hematopoietic stem cells (HSCs). A HIS mouse model using neonatal human thymus tissue and umbilical cord blood (CB) HSCs (NeoHu) was recently described. We improved the model by removing the native murine thymus, which can also generate human T cells, and demonstrated definitively the capacity of human T cells to develop in a grafted neonatal human thymus. Human T cells derived from the neonatal thymus tissue appeared in peripheral blood early post-transplantation and CB-derived T cells appeared later. Naïve T cells were demonstrated in peripheral blood but effector memory and T peripheral helper phenotypes predominated later, in association with development of autoimmunity in some animals. Treatment of thymus grafts with 2-deoxyglucose (2-DG) increased the proportion of stem cells derived from injected HSCs, delayed onset of autoimmune disease, reduced early T cell reconstitution, and reduced effector/memory T cell conversion. Younger neonatal human thymus tissue was associated with improved T cell reconstitution. While the NeoHu model bypasses the need for fetal tissue, it has yet to demonstrate equivalent reconstitution to fetal tissue, though 2-DG can improve results by removing native thymocytes prior to transplantation.