MHC Matching Research Articles

Infusion of iPSC-Derived CD34 + Hematopoietic Progenitors Following Myeloablative HSC Transplantation and Assessment of Their Immunogenicity in MHC-Defined Nonhuman Primate Model

Bone marrow suppression and associated neutropenia that renders the patient more susceptible to infection is a major complication and limiting factor for current chemotherapy. Strategies aimed at neutrophil reconstitution using transfusions or accelerating neutrophil recovery with G-CSF show a limited effect in reducing the rate of infection. To overcome these limitations, administration of ex vivo expanded somatic hematopoietic progenitors (HPs) has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival following myeloablation. Recent advances in reprogramming and induced pluripotent stem cell (iPSC) technologies have created alternative platforms for off-the-shelf supply of immunologically compatible HPs, including cellular products derived from MHC homozygous superdonors which can increase the degree of MHC matching and may provide a maximum utility for stem cell banking. In these studies, we developed Mauritian cynomolgus macaque (MCM) model to evaluate the utility and safety of CD34 +CD45 + hematopoietic progenitors derived from iPSCs (iHPs) generated from MHC homozygous animals in the treatment of cytopenia following myeloablative stem cell transplantation. MCM iPSCs were generated from MHC homozygous animals and used to generate iHPs in coculture with OP9. Three groups of MCMs underwent myeloablative total body irradiation (TBI) followed by transplantation of cryopreserved autologous CD34 + HSPCs. Four days after autologous HSPC transplantation, animals received transfusion of 30x10 6/kg cryopreserved iCD34 + cells tagged with eGFP or tdTomato from homozygous MHC-matched iPSCs (group 2) or homozygous MHC mismatched iPSCs (group 3). We have demonstrated that infusion of iHPs is safe and well tolerated. No teratoma or tumor formation was observed in animals one year after infusion of iHPs. Although we detected few iPSC derived hematopoietic cells in the blood within 1-week post-infusion, we did not see any significant differences in blood counts or other peripheral blood parameters between all animal groups. Intriguingly we found footprints of iPSC-derived cells in the colon, lymph node, skin and brain specimens collected 25 days after iHP infusion from one animal. This suggests a possibility of migration and retention within these tissues of iHP and iHP-derived myeloid cells. To assess immunogenicity, MHC homozygous iHPs were infused into immunocompetent MCMs across different MHC barriers. 10x10 6/kg of cryopreserved iCD34+ from homozygous iPSCs were transfused every week for 3 weeks, into animals that were divided into 4 groups namely autologous (group I), MHC matched homozygous (group II), MHC matched heterozygous (group III) and MHC mismatch (group IV). Overall, these studies revealed low immunogenicity of MHC homozygous iHPs in MHC matched homozygous and heterozygous animals, while weak immune response was detected following iHP infusion in some MHC mismatched animals. DisclosuresSlukvin: Cynata Therapeutics: Consultancy, Current equity holder in publicly-traded company.

"HLA-10-SNP": A new qPCR-based system for rapid, effective, and accurate detection of 10 important SNPs in the HLA region.

Mastering the SNP content in the HLA region can be based on it to judiciously select unrelated donor stem cells with preferable MHC matching to lower postoperative complications. Herein, quantitative PCR-based primers and probes were designed for 10 transplants outcome-associated SNP loci with two-allelic polymorphism, and then a new detection system ("HLA-10-SNP") was established. Compared with Sanger sequencing, its accuracy has been proven to reach 100%. Additionally, fluorescent PCR typing of 10 important SNPs via this system expressed excellent repeatability (sensitivity, 20 ng). Overall, the new system achieves single-sample classification precision and easily distinguishable results, equipped with the advantages of simple, rapid, accurate, and effective, promising to acquire widespread popularization and application in clinical settings.

MHC matching fails to prevent long-term rejection of iPSC-derived neurons in non-human primates

Cell therapy products (CTP) derived from pluripotent stem cells (iPSCs) may constitute a renewable, specifically differentiated source of cells to potentially cure patients with neurodegenerative disorders. However, the immunogenicity of CTP remains a major issue for therapeutic approaches based on transplantation of non-autologous stem cell-derived neural grafts. Despite its considerable side-effects, long-term immunosuppression, appears indispensable to mitigate neuro-inflammation and prevent rejection of allogeneic CTP. Matching iPSC donors’ and patients’ HLA haplotypes has been proposed as a way to access CTP with enhanced immunological compatibility, ultimately reducing the need for immunosuppression. In the present work, we challenge this paradigm by grafting autologous, MHC-matched and mis-matched neuronal grafts in a primate model of Huntington’s disease. Unlike previous reports in unlesioned hosts, we show that in the absence of immunosuppression MHC matching alone is insufficient to grant long-term survival of neuronal grafts in the lesioned brain.

Abstract LB-071: Functional NK cells generated from human iPS cells with 3D-bioreactor for immuno-oncology

Abstract NK cells are cytotoxic cells critical for innate immune system. Early investigation using NK cells carrying CAR specific targeting tumor antigens have shown promising antitumor activities. CAR-T therapy requires either autologous or MHC matching cells to avoid graft-verse-host disease (GVHD), whereas allogeneic NK cells showed substantial anti-tumor activity without GVHD. Therefore, the “ready-to-kill” NK population is regarded as the ideal Off-The-Shelf immune cell therapy products. The development of NK-based immunotherapy, however, has been limited by unsatisfactory sources of human NK cells. Human induced pluripotent stem cells (iPSCs) offer unlimited source for manufacturing NK cells suitable for immunotherapy. Previously NK cell derivation methods from human iPS cells used feeder cells with multiple scale-up limited processes which are not suitable for industrial scale production. In this study we described a novel 3D-bioreactor platform that can continuously generate highly pure NK cells with strong cytotoxic activity for 2 weeks. First, undifferentiated human iPS cells were cultured as 3D-spheres with mesoderm inducing conditions in bioreactor, which converted 60-70% iPS cells into hemogenic endothelial progenitor (HEP, CD31+CD144+CD34+) in a week. HEP cells were then cultured under NK cell differentiation condition for up to 35 days. Starting from day 20, NK cells were started to release from these 3D-spheres which can be harvested daily by centrifugation. These iPS-NK cells can be cryopreserved for long time. Human iPS-NK cells display a distinctively homogenous morphology. Over 95% of collected cells express CD56, NKG2D, NKp44 and NKp46 markers; Approximately 30-50% of CD56+ iPS-NK cells express KIR2DL/DS1 and KIR2DS4 receptors; CD56+ PSC-NK cells do not express TCR and CD3, and CD19, which are specific markers for T and B cells, respectively. Surprisingly, we discovered that more than 80% CD56+ iPS-NK cells also expressed CD8 as compared to ≈30% of peripheral blood CD56+ NK cells, the increase of which is associated with disease regression in AIDS patients. More importantly, when iPS-NK cells and K562 leukemia cells were mixed together, even at a ratio of 1 to 1, more than 80% of leukemia cells were killed within hours as demonstrated both by flow cytometry analysis and time-lapse record. With the establishment of permanent master iPS-CAR cell lines, our novel technology platform will provide inexhaustible cell sources for the generation of truly off-the-shelf CAR-NK cells suitable for treatment of a large numbers of patients, which can revolutionize the field of immuno-oncology. Citation Format: Qiang Feng, Miao-yun Zhang, Shi-Jiang Lu. Functional NK cells generated from human iPS cells with 3D-bioreactor for immuno-oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-071.

The Advances and Challenges of CAR-NK Cells for Tumor Immunotherapy

Immunotherapies using chimeric antigen receptor (CAR)-T cells bring an encouraging vision to non-Hodgkin lymphoma patients who develop relapsed lymphoma or are unresponsive to standard chemotherapy, yet they also have limitations and drawbacks. Clinical trials have reported cases of neurotoxicity and cytokine release syndrome (CRS) accompanied by CAR-T cell therapies. To establish a more mature therapy, CAR incorporated into Natural Killer (NK) cells came into being. As a leukocyte involved in innate immunity, NK cell does not require MHC matching, making the production of allogeneic “off-the-shelf” CAR-NK cells possible. Moreover, the controllable life span of CAR-NK cells and little risk of graft-versus-host disease reduce side effects companion by CAR-T. This review provides an overview of CAR-NK design and production before delivery to patients. Different sources of NK cells are compared and the development of CAR molecule construction is introduced.

Increased MHC Matching by C4 Gene Compatibility in Unrelated Donor Hematopoietic Stem Cell Transplantation.

HLA matching is a prerequisite for successful allogeneic hematopoietic stem cell transplantation (HSCT) because it lowers the occurrence and severity of graft-versus-host disease (GVHD). However, matching a few alleles of the classic HLA genes only may not ensure matching of the entire MHC region. HLA haplotype matching has been reported to be beneficial in HSCT because of the variation relevant to GVHD risk in the non-HLA region. Because polymorphism in the MHC is highly population specific, we hypothesized that donors from the Finnish registry are more likely to be matched at a higher level for the Finnish patients than donors from other registries. In the present study we determined 25 single nucleotide polymorphisms (SNPs) of the complement component 4 (C4) gene in the γ-block segment of MHC from 115 Finnish HSCT patients and their Finnish (n = 201) and non-Finnish (n = 280) donor candidates. Full matching of HLA alleles and C4 SNPs, independently or additively, occurred more likely in the Finnish-Finnish group as compared with the Finnish-non-Finnish group (P < .003). This was most striking in cases with HLA haplotypes typical of the Finnish population. Patients with ancestral HLA haplotypes (AH) were more likely to find a full HLA and C4 matched donor, regardless of donor origin, as compared with patients without AH (P < .0001). Despite the clear differences at the population level, we could not find a statistical association between C4 matching and clinical outcome. The results suggest that screening C4 SNPs can be advantageous when an extended MHC matching or HLA haplotype matching in HSCT is required. This study also supports the need for small population-specific stem cell registries.

T Cells Derived From Human Melanoma Draining Lymph Nodes Mediate Melanoma-specific Antitumor Responses In Vitro and In Vivo in Human Melanoma Xenograft Model.

It has been established in murine models that lymph nodes draining a progressively growing tumor contain antigen-specific T cells capable of mediating protective immune responses upon adoptive transfer. However, naturally occurring human tumor-draining lymph nodes (TDLNs) have yet to be fully investigated. In this study, we analyzed TDLNs from patients with stage III melanoma who were undergoing routine lymph node dissection. Following short-term (14 d) culture activation with anti-CD3/anti-CD28 microbeads and expansion in low concentrations of IL-2, the melanoma-draining lymph node (MDLN) cells were ∼ 60% CD4-activated and ∼ 40% CD8-activated T cells. The activated MDLN cells demonstrated reactivity in response to overlapping peptides spanning the sequence of 4 different known melanoma antigens MAGEA1, Melan-A/MART-1, NY-ESO-1, and Prame/OIP4, suggesting the presence of melanoma-specific T cells. Coculture of activated MDLN T cells with cancer cells in vitro resulted in preferential apoptosis of human cancer cell lines that were cocultured with T cells with high degree of MHC matching. Adoptive transfer of MDLN T cells with high degree of MHC matching to A375 to mice-bearing human A375 melanoma xenografts resulted in dose-dependent improvement in survival. Although prior human studies have demonstrated the immune responses within melanoma vaccine-draining lymph nodes, this study presents evidence for the first time that naturally occurring human MDLN samples contain melanoma-experienced CD4 and CD8 T cells that can be readily cultured and expanded to mediate protective immune responses both in vitro and in vivo in a human melanoma xenograft model.

Behaviour of transplanted tumours and role of matching in rejection

BackgroundTumour transfer/development is one of the more serious risks associated with transplantation. The behaviour of a tumour can be unpredictable in immunosuppressed recipients. We report a highly sensitive method to monitor tumour behaviour in real time in a rodent tumour transplant model. This paper also explores the effect of MHC matching on tumour growth among control and immunosuppressed hosts. MethodsLuciferase expressing Wistar rat kidney tumour cells were transplanted into either Wistar or Lewis recipients which mimic a well and poorly matched combination to assess the effects of MHC matching on transplanted tumour cells. Experimental groups included controls with no immunosuppression and animals immunosuppressed with cyclosporine. The latter group was further divided into a continuous treatment group which received four weeks of immunosuppression and a treatment withdrawal group where immunosuppression was stopped after two weeks to assess the effects of rejection on tumour growth. ResultsAll the tumour cells were rejected in the control animals that received no immunosuppression, within 2weeks among well-matched combination and within one week in the poorly matched combination (p 0.001). The transplanted tumour cells continued to grow in both well-matched and poorly matched groups who were treated with cyclosporine, but growth was significantly faster in the well-matched combination (p 0.033). After treatment withdrawal the tumour cells were rejected in all the animals of the poorly matched group compared to 50% in well matched animals within the four-week study period (p 0.039). ConclusionIn the absence of immunosuppression the hosts reject the transplanted tumour cells, and the anti-tumour response is stronger when there is a greater mismatch in MHC with the recipient. In the presence of cyclosporine immunosuppression the tumour continues to grow, however, after withdrawal of the immunosuppression, tumour clearance is quicker in the poorly matched background. This data supports the idea of expansion of the donor pool by using kidneys after ex vivo resection of small renal tumours and that these organs should be transplanted into a less well-matched HLA recipient. We hypothesise that should a tumour recurrence occur a poorly matched recipient could clear the tumour through withdrawal of immunosuppression.

Successful Tolerance to Fully MHC Disparate Renal Allografts via Donor Hematopoietic Mixed Chimerism in Non-Human Primates

To the Editor: Rodent studies clearly show that sharing of MHC genes between donors and recipients is regularly associated with improved allograft survival. This is essentially attributed to: (1) reduction of early inflammatory direct T cell responses involved in acute allograft rejection and (2) self-MHC molecules expression on donor cells promoting regulatory immunity. The precise contribution of donor/recipient MHC gene matching to transplant rejection or acceptance in clinical transplantation has been somewhat more difficult to define due to the use of intensive immunosuppression in all human allograft recipients. Nevertheless, most authors agree that improved long-term graft survival is generally expected in patients receiving MHC-matched renal allotransplants. Rodent studies also demonstrate that transplant tolerance is easier to accomplish when allogeneic tissues express some or all of the recipient's MHC proteins. Nevertheless, tolerance of fully allogeneic transplants is regularly achieved in laboratory-bred mice using a variety of procedures including donor specific transfusion, donor hematopoietic chimerism and lymphocyte costimulation blockade. In contrast, such tolerance in primates has proven to be much more arduous due presumably to the presence in these recipients of preexisting alloreactive memory T cells (TMEM). These TMEM undergo differentiation and clonal expansion following previous exposure to alloantigens through blood transfusion, pregnancy, a previous transplantation or via cross-reactivity with microbial antigens. A recent article in AJT by Ramakrishnan et al. 1, referring to a recent report from our group 2, suggested that we had achieved tolerance of renal allografts in non-human primates via mixed chimerism only in highly MHC-matched transplant pairs. This interpretation of our experience is not fully accurate. Indeed, we have reported an inverse correlation between the level of IL-2 and γIFN memory alloreactivity and the degree of MHC class II and I gene sharing among monkey pairs 3. However, this is not an absolutely consistent observation with a substantial number of fully MHC disparate donor–recipient pairs displaying low pro-inflammatory memory responses 3. This suggests that tolerance susceptibility vs. resistance in primates depends primarily upon the frequencies of pre-existing donor-specific TMEM rather than upon donor–recipient MHC matching. In support of this view, we have observed that over one third of the monkeys that became tolerant of renal allografts had actually received a highly MHC-mismatched (>4/6 CyLA mismatches) transplant 4, 5. In summary, the beneficial effects of MHC matching with regards to transplantation tolerance are undeniable. In addition, it is likely that harnessing anamnestic T cell alloreactivity in primates will be essential to successful tolerance of allogeneic transplants across MHC barriers in non-human primates and patients. G. Benichou*, O. Nadazdin, A. B. Cosimi, and T. Kawai Transplantation Research Center, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA *Corresponding author: Gilles Benichou, gbenichou@partners.org The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Adaptive Immunity Rather Than Viral Cytopathology Mediates Polyomavirus-Associated Nephropathy in Mice

Nephropathy associated with BK polyomavirus causes kidney allograft dysfunction and failure. Understanding the pathogenesis of polyomavirus-associated allograft nephropathy (PVAN) is hampered by the species specificity of Polyomaviridae family members. Using a mouse polyomavirus (MPyV) kidney transplant model, we investigated clinically relevant variables that may contribute to PVAN. We found that the timing and source (i.e. donor vs. recipient) of MPyV infection and the titer of the viral inoculum have significant effects on the extent of allograft injury, with acute infection of the recipient by high-titer MPyV inoculums producing the most profound PVAN. In contrast, altering the degree of MHC matching or increasing ischemia/reperfusion injury by prolonging the cold ischemic time of the allograft did not affect the severity of PVAN. Survival correlated positively with serum creatinine levels, but not with viral loads in the kidney allograft. Using splenectomized alymphoplasia mice, which are unable to mount primary adaptive immune responses, we further demonstrate that persistent high viral loads in the kidney are not sufficient to cause advanced PVAN. These findings suggest that the mechanism of PVAN in mice is not a direct consequence of viral cytopathology, but rather involves interplay between viral infection and the recipient antidonor immune response.

Cornea: Window to Ocular Immunology

The ocular surface is continuously exposed to environmental agents such as allergens, pollutants, and microorganisms, which could provoke inflammation. However, an array of anatomical, physiological, and immunological features of the ocular surface conspire to limit corneal inflammation and endow the eye with immune privilege. A remarkable example of ocular immune privilege is the success of corneal allografts, which unlike all other forms of organ transplantation, survive without the use of systemic immunosuppressive drugs or MHC matching. This review describes the anatomical, physiological, and dynamic immunoregulatory processes that contribute to immune privilege.

Allogeneic adoptive cell transfer therapy as a potent universal treatment for cancer

Adoptive cell transfer (ACT), the transfer of therapeutic lymphocytes to patients, is a promising cancer treatment, with advances seen in patients with melanoma and neuroblastoma in early clinical trials [1, 2]. One type of ACT modality pioneered by our group relies on the use chimeric antigen receptors (CAR) redirected T cells [3, 4]. CARs are typically composed of single chain antibody fragments fused to T cell activating signaling motifs, endowing the T cells with MHC unrestricted tumor specificity. One advantage of CARs over conventional TCRs is that costimulatory domains could be incorporated into the CAR endowing it with supra-physiological potency. One limitation of ACT is the need to generate tumor specific lymphocytes for each individual patient, which is technically and economically challenging. We therefore sought to develop an approach for universal ACT, which would allow treatment of patients without the need for MHC matching [5]. We reasoned that because of their MHC unrestricted tumor specificity, allogeneic CAR redirected T cells could be used as ‘universal effector cells’ against cancer in all patients. However, allogeneic adoptive transfer faces the problem of the host-versus-graft response (HvG) and the danger of graft-versus-host-disease (GvHD). We hypothesized that low dose irradiation would delay the rejection of allogeneic T cells and allow them enough time to destroy the tumor on one hand while on the other hand their eventual rejection would prevent the development of GvHD. Using this approach, fully MHC mismatched allogeneic T cells redirected with a HER2/neu specific CAR were able to provide as much therapeutic benefit as syngeneic cells in a model of metastatic disease. We demonstrated that the GvH reactivity of the allogeneic T cells caused extensive proliferation, augmenting the anti-tumor response, but that the allogeneic T cells were indeed eventually rejected and no GvHD developed. Our second working hypothesis was that modulating lymphocyte migration could potentially further increase therapeutic efficacy of allogeneic ACT. Both the HvG and GvH reactions originate in the lymph nodes where T cells encounter MHC mismatched antigen presenting cells (APC), undergo massive proliferation, and then egress out of the lymph nodes into peripheral organs. By contrast ACT introduces cells directly into the blood, circumventing egress, and allowing the cells to reach the tumor directly. We therefore surmised that blocking lymphocyte egress using the lymphocyte sequestering agent FTY720[6] might inhibit the HvG and GvH reactions to a much greater extent than the graft-versus-tumor (GvT) reaction. Indeed, FTY720 enhanced the efficacy of allogeneic but not syngeneic ACT, such that allogeneic T cells provided superior therapeutic benefit over syngeneic ones with median survival of over one year and no manifestations of GvHD. In conclusion, our results suggest that MHC mismatched CAR redirected allogeneic T cells could function as ‘universal effector cells’, and could potentially be used as an ‘off-the-shelf’ cellular therapy of cancer. Blocking lymphocyte egress further increased the efficacy of the approach such that allogeneic cells were superior to syngeneic ones. This result conclusively demonstrates that we can harness the GvH reactivity of allogeneic T cells to augment the anti-tumor response without causing GvHD. While this work provides only a proof of principle, we strongly believe that this approach is widely applicable for two main reasons. First, by using other CAR, many different cancers could be targeted of both solid and hematologic malignancies [7]. Second, the HvG and GvH occur independently of the tumor, and since we were able to overcome this problem in our work, the same approach could be used regardless of tumor type. Taken together, our results suggests that with further tuning, allogeneic adoptive cell therapy may become the treatment of choice both because of its obvious technical and economical advantages and due to its greater efficacy.

Successful In Utero Hematopoietic Stem Cell Transplantation (IUHSCT) Likely Depends on Niche Maturity, Recipient/Donor MHC Compatibility and Donor Cell Origin

AbstractAbstract 574Many of the most prevalent inherited disorders currently affecting the human population could potentially be treated with IUHSCT. IUHSCT does not require myeloablation, eliminating the risks inherent to this procedure. Moreover, performing IUHSCT during the period of preimmunity removes the recipient's immune response as a barrier to successful engraftment. Unfortunately, despite numerous clinical trials over the last 20 years for a variety of disorders, IUHSCT has only been successful in treating SCID, highlighting the need for a better understanding of the factors governing engraftment following IUHSCT. We hypothesized that the success of IUHSCT likely depends upon: 1) the maturity/receptivity of the bone marrow (BM) niches at the time of transplant; 2) the degree of donor/recipient MHC compatibility; and 3) the source of donor HSC. To test our hypothesis, we began by performing confocal analyses of fetal sheep bones at gestational days 30–145, using sheep as a model based on its immunologic, physiologic and developmental similarities with the human fetus. These analyses showed that the BM microenvironment commences development with the formation of the vascular niche, beginning by day 40 of gestation. In contrast, the osteoblastic niche, which is known to be critical for the engraftment and maintenance of primitive HCS, only starts developing at roughly day 51 of gestation, and does not reach maturity until day 65. These findings thus indicate that to achieve maximal levels of engraftment following IUHSCT, the transplant should likely be performed at gestational day 65 or later, to ensure that the crucial osteoblastic niche has fully developed. Unfortunately, partial immune-competence is also achieved by roughly day 65, suggesting that immune compatibility between the donor and the recipient will also need to be addressed to maximize engraftment. This issue was addressed by transplanting 65 day-old sheep fetuses (n=21) with eGFP-transduced CD34+ cells isolated from related and unrelated BM and cord blood (CB) donors and comparing the levels of engraftment. Cell counting, PCR, and flow cytometry at 63 days post-IUHSCT revealed that the engraftment levels of the related CB HSC were nearly double those of the unrelated CB HSC in BM (1.7% vs. 0.86%) and more than 4-fold higher in PB (2.77% vs. 0.66%). Interestingly, however, there was no significant difference between the engraftment levels of related and unrelated BM-derived HSC (1.77% vs. 1.88%). We presumed that a greater degree of MHC matching could explain the higher engraftment seen with related versus unrelated CB-derived HSC. This was experimentally confirmed by performing PCR-based MHC microsatellite typing at 12 selected MHC loci on the donor CB cells and on amniotic fluid-derived recipient cells. All related donor/recipient animals exhibited 5–7 perfect matches and 1–6 half matches at the 12 MHC loci. In contrast, non-related donor/recipient animals exhibited only 1–2 perfect matches and 4–6 half matches at these loci. To evaluate the impact of the source of HSC on IUHSCT outcome, we compared these same animals without consideration to donor/recipient relatedness. These analyses demonstrated that the CB-derived HSC engrafted in PB at nearly four-fold higher levels than their BM-derived counterparts (2.75% vs. 0.93%). Conversely, the BM-derived HSC engrafted within the recipient BM at three-fold higher levels than the CB-derived HSC (0.66% vs. 2.24%). Collectively, these results suggest that transplanted HSC show a predilection for engraftment in their site of origin.In conclusion, our data collectively indicate that: 1) delaying IUHSCT until a point in gestation when the osteoblastic and vascular niches of the BM are mature is likely to improve engraftment; 2) transplanted HSC exhibit a predilection for engraftment in their site of origin; and 3) higher degrees of CB donor/recipient MHC matching doubles the engraftment levels obtained following IUHSCT, but does not seem to impact upon engraftment levels of BM-derived HSC. By simultaneously addressing these issues, it is expected that curative levels of HSC engraftment can be obtained prior to birth, making it possible to establish clinically successful and reliable IUHSCT-based treatments for a wide range of inherited disorders. Disclosures:No relevant conflicts of interest to declare.

CD28 blockade induces division-dependent downregulation of interleukin-2 receptor alpha

BackgroundBlockade of T cell costimulatory molecules represents a promising new method of attenuating donor-reactive T cell responses to promote graft survival following transplantation. However, recent studies in murine models have shown the presence of an initial high frequency of naïve donor-reactive T cells may render this strategy ineffective. MethodsIn this report, we examined the phenotypic changes associated with CD28 blockade on T cells stimulated at increasing precursor frequencies in vitro. ResultsWe found that treatment with the CD28 blocker CTLA-4 Ig resulted in downregulation of the alpha chain of the IL-2 receptor (CD25) in a division-dependent manner. Significantly, blockade of the CD28 pathway was more effective in down-regulating CD25 when the donor-reactive T cell population was present at low as compared to high precursor frequency. ConclusionsThese results imply that treatment with CD28 blockers and anti-CD25 mAbs may cooperate in promoting graft survival under conditions of low MHC matching where the donor-reactive T cell precursor frequency is high.

An MHC-Defined Primate Model Reveals Significant Rejection of Bone Marrow After Mixed Chimerism Induction Despite Full MHC Matching

In murine models, mixed hematopoietic chimerism induction leads to robust immune tolerance. However, translation to primates and to patients has been difficult. In this study, we used a novel MHC-defined rhesus macaque model to examine the impact of MHC matching on the stability of costimulation blockade-/sirolimus-mediated chimerism, and to probe possible mechanisms of bone marrow rejection after nonmyeloablative transplant. Using busulfan-based pretransplant preparation and maintenance immunosuppression with sirolimus, as well as CD28 and CD154 blockade, all recipients demonstrated donor engraftment after transplant. However, the mixed chimerism that resulted was compartmentalized, with recipients demonstrating significantly higher whole blood chimerism compared to T cell chimerism. Thus, the vast majority of T cells presenting posttransplant were recipient-rather than donor-derived. Surprisingly, even in MHC-matched transplants, rejection of donor hematopoiesis predominated after immunosuppression withdrawal. Weaning of immunosuppression was associated with a surge of antigen-experienced T cells, and transplant rejection was associated with the acquisition of donor-directed T cell alloreactivity. These results suggest that a reservoir of alloreactive cells was present despite prior costimulation blockade and sirolimus, and that the post-immunosuppression lymphocytic rebound may have lead to a phenotypic shift in these recipient T cells towards an activated, antigen-experienced phenotype, and ultimately, to transplant rejection.

Comment on ‘Tolerance Versus Immunosuppression: A Perspective’

Comment on ‘Tolerance Versus Immunosuppression: A Perspective’

Prevention of Acute GvHD during Haplo-BMT: Evaluating the Efficacy of T-Cell Costimulation Blockade Using a Novel Rhesus Macaque Transplant Model

Introduction: While bone marrow transplantation (BMT) offers curative therapy for many malignant and non-malignant hematologic diseases, it remains plagued by high morbidity and mortality, most prominently through the complications of graft-versus-host disease (GvHD). The field of GvHD research has been limited by a small number of clinically relevant model systems, which, until now, have focused on murine and canine models. However, many new human therapeutics, including those directed at T cell costimulation pathways, do not cross-react with targets in these model systems, limiting their utility to fully evaluate the new generation biologic anti-GvHD therapies.Methods: We have developed a fully pedigreed and MHC-typed rhesus macaque bone marrow transplant system, with which to model GvHD and its prevention. This model relies upon microsatellite typing of both autosomal chromosomes to create genetically-verified family trees and microsatellite-and allele-specific PCR-based MHC typing, with which to identify MHC haplotypes within each family group. By this methodology, full and half-sibling pairs can be identified with either full MHC matching, one MHC haplotype matching or full MHC disparity. This represents a significant advance compared to previous primate haplo-studies, which relied upon parent-offspring transplants, as it optimizes animal availability and colony stability as well as permiting the rigorous evaluation of the degree of MHC disparity on the non-shared chromosome 6. For the current study, we have concentrated on MHC haplo-identical BMT. Our preparative regimen consists of TBI (8.00 Gy with lung shielding to 6.00 Gy), which is performed on the day of transplant with hematopoietic stem cells. After transplant, donor engraftment is measured by microsatellite analysis, from both whole blood and from flow cytometrically sorted T and B cell populations. GvHD is graded using standard clinical grading scales. The immune phenotype after transplant is also measured by cell- and serum-based flow cytometric analyses.Results: Three animals have so far been studied. The first animal served as a control for TBI-based preparation. This animal exhibited both neutropenia (nadir = 0.015 × 103/uL at 12 days) and thrombocytopenia (nadir = 2 × 103/uL at 12 days) and prolonged anemia (hgb=5.8 g/dL on the day of sacrifice). This animal was sacrificed at day 29 due to prolonged pancytopenia. The second animal was transplanted with haploidentical hematopoietic stem cells (4.47 × 108 total nucleated cells/kg and 1.10 × 108 CD3+ T cells/kg) and was treated with only rapamycin for immunosuppression. He exhibited profuse diarrhea and necrotic skin changes within 8 days of transplant, coincident with early engraftment and was sacrificed at day 14. A full histologic analysis to determine the cause of these lesions (and the histopathologic extent of GvHD) is currently underway. The third animal was transplanted with haploidentical donor bone marrow (1.1 × 109 total nucleated cells/kg and 6.9 × 107 CD3+ T cells/kg), but was given T cell costimulation blockade with abatacept (targeting the CD28/B7 pathway) and an anti-CD40 antibody (targeting the CD40/CD154 pathway) in addition to rapamycin. Abatacept is currently clinically approved for treatment of rheumatoid arthritis, but has not been added to clinical GvHD regimens, largely due to the paucity of translational efficacy data. This animal is currently >43 days post-transplant, with full donor chimerism (including 100% T and B cell chimerism), without signs of GvHD: he has no rash, no diarrhea and is stable clinically. Additional animals are now being added to the study, to confirm these initial results.Conclusions:We have established a robust non-human primate model of GvHD using pedigreed and MHC-typed rhesus macaques.We have used this model to begin to test the efficacy of a novel agent combination, capable of preventing the onset and complications of acute GvHD.Preliminary results suggest that CD28- and CD40-directed costimulation blockade may be active agents for the prevention of GvHD. A large scale analysis of their efficacy and immune consequences is currently underway.

MHC-Matched Stem Cell Transplantation in Rhesus Macaques Using a Costimulation Blockade-Based Immunomodulation Platform.

A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. When transplants were performed between animals that were totally unmatched at the MHC, mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism in the setting of these MHC-unmatched transplants. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. In order to address the dual complications of lack of indefinite chimerism and viral reactivation, we sought to increase the level of MHC matching between donor and recipient transplant pairs. This necessitated a large-scale analysis of an NIH-sponsored rhesus macaque colony in order to determine the familial relationships and MHC-disparity between potential transplant pairs. We have analyzed over 500 animals from this colony, which has resulted in the first primate transplant series between donors and recipients with known degrees of MHC disparity. Our results suggest that viral reactivation is vastly improved when transplant occurs with even one shared MHC haplotype, but that the current immunomodulation strategy is insufficient to achieve immune tolerance in these haplo-identical transplants. However, we have observed long-term mixed chimerism in fully MHC matched transplant pairs that is stable even years after withdrawl of costimulation blockade-based immunomodulation. These results suggest that costimulation blockade may be an important addition to current immunomodulation strategies for producing stable engraftment after nonmyeloablative pre-transplant preparation, with potential application to non-malignant hematologic diseases and genetic disorders.

NK Cell Mediated Contact Sensitivity is Elicitable 1 Hour After Immunization and Depends on IFN-gamma and IL-12 Production (B101)

Abstract NK cell mediated contact sensitivity (CS) has recently been described. We confirmed CS in SCID and RAG1−/− mice but not in SCID-beige mice without NK cell function. NK mediated CS was transferred with liver mononuclear cells and unlike conventional CS not with mixed lymph node and spleen cells. Positive selection with anti-DX5 beads confirmed the NK nature of the transferring cells. Participation of NKT cells and B-1 cells was ruled out with Jα18−/− and JH−/− mice. Remarkably, NK CS was elicitable 1-hr after immunization and with an early 2-hr component 1 day after LMNC transfer, and lasted at least 7 days. Adoptive cell transfer required MHC matching and IL-12 and IFN-γ−/− were involved in the mechanism of CS. In conclusion, NK CS is a distinct entity. IL-12 and IFN-γ are involved in the effector mechanism of NK-mediated CS, similar to classical CS mediated by Th1 cells. The requirement for MHC matching may involve the specificity of the NK receptor, which in this case may be hapten altered self. In conclusion, NK cells provide a very rapid and long lasting, Ag-specific mechanism to elicit cell-mediated immunity, with features of innate and acquired immunity. Supported by NIH grants AI-59801 and AI-07174 to PWA; and the Polish Committee of Scientific Research to MS.

Haplotype matching is not an essential requirement to achieve remyelination of demyelinating CNS lesions

Transplantation of oligodendrocyte precursor cells (OPCs) results in efficient remyelination in animal models of demyelination. However, the experiments so far undertaken have not addressed the need for tissue-type matching to achieve graft-mediated remyelination. Examination of MHC expression (main determinant of allograft rejection) by OPCs showed nondetectable levels under standard culture conditions and upregulation of MHC Class I expression only upon exposure to interferon gamma. We therefore hypothesized that MHC matching of OPC grafts may not be crucial to achieve transplant-mediated remyelination. Transplant experiments performed using a nonself repairing toxin-induced demyelination model showed that, similarly to allogeneic neurons, survival of allogeneic oligodendrocyte lineage cells is influenced by donor-host haplotype combination and graft composition. Transplantation of allogeneic mixed glial cell cultures resulted in remyelination failure by 1 month postengraftment due to a rejection response targeting both myelinating oligodendrocytes and OPCs, suggesting that inflammation-induced upregulation of OPC MHC I expression results in susceptibility to cytotoxic T cell attack. In contrast, remyelination persisted for at least 2 months following transplantation of OPC-enriched cultures whose overall MHC expression level was significantly decreased. While OPC-enriched preparations elicited delayed type hypersensitivity responses in hosts sensitized to alloantigens, allografting of such preparations into a central nervous system demyelinating lesion did not result in recipient priming. We conclude that while allografted oligodendrocyte lineage cells become targets of a graft rejection response once this response has been initiated, transplantation of OPC-enriched preparations can evade priming against alloantigens and graft rejection. This finding indicates that close tissue matching may not be an essential requirement for successful transplant-mediated remyelination.