MHC-matched Bone Marrow Research Articles

Transplantation of MHC-mismatched mouse embryonic stem cell-derived thymic epithelial progenitors and MHC-matched bone marrow prevents autoimmune diabetes

BackgroundType 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-secreting islet β cells by autoreactive T cells. Non-obese diabetic (NOD) mice are the widely used animal model for human T1D. Autoimmunity in NOD mice is associated with particular major histocompatibility complex (MHC) loci and impaired islet autoantigen expression and/or presentation in the thymus, which results in defects in both central and peripheral tolerance. It has been reported that induction of mixed chimerism with MHC-mismatched, but not MHC-matched donor bone marrow (BM) transplants prevents the development T1D in NOD mice. We have reported that mouse embryonic stem cells (mESCs) can be selectively induced in vitro to generate thymic epithelial progenitors (TEPs) that further develop into thymic epithelial cells (TECs) in vivo to support T cell development.MethodsTo determine whether transplantation of MHC-mismatched mESC-TEPs could prevent the development of insulitis and T1D, NOD mice were conditioned and injected with MHC-mismatched B6 mESC-TEPs and MHC-matched BM from H-2g7 B6 mice. The mice were monitored for T1D development. The pancreas, spleen, BM, and thymus were then harvested from the mice for evaluation of T1D, insulitis, chimerism levels, and T cells.ResultsTransplantation of MHC-mismatched mESC-TEPs and MHC-matched donor BM prevented insulitis and T1D development in NOD mice. This was associated with higher expression of proinsulin 2, a key islet autoantigen in the mESC-TECs, and an increased number of regulatory T cells.ConclusionsOur results suggest that embryonic stem cell-derived TEPs may offer a new approach to control T1D.

Close To Clinics: Acute Gvhd In a Novel Chemotherapy Based Minor Mismatch Transplantation Model

GVHD (graft-vs-host disease) is one of the major complications after allogeneic hematopoietic stem cell transplantation leading to substantial morbidity and mortality. Several murine models are used to investigate this systemic inflammatory disease in vivo and to identify possible targets for clinical treatment. However, current well-established murine GVHD models have their clinical limitations as they are based on major histocompatibility mismatches and only lethal irradiation as conditioning. Development of new treatment options would be forwarded by the availability of a more clinically relevant murine model.To reflect the clinical situation of transplantation and GVHD, we established a new murine GVHD model which is based on Chemotherapy conditioning and MHC-matched bone marrow transplantation (BMT). In LP/J -> C57BL/6 mouse model, our chemotherapy transplantation protocol resulted in stable full donor chimerism and development of acute GVHD.C57BL/6 recipients were conditioned with a 7-day busulfan-cyclophosphamide Chemotherapy protocol. Mice received for 5 days 20 mg/kg/day busulfan intraperitoneally (i.p.) and the last three days additionally 100 mg/kg/day cyclophosphamide. After a two-day resting phase, recipients were transplanted with 1.5 x 107 bone marrow and 2 x 106 splenic T cells from LP/J donors. Recipients showed good engraftment with already 80 % engraftment in peripheral blood at day 15 after BMT. 90 % donor chimerism in peripheral blood and bone marrow could be shown already at day 30 after BMT.LP/J and C75BL/6 mice share the same haplotype (H2b) with only mismatches at different minor histocompatibility loci. As in clinical BMT, GVHD still arises after MHC-matched transplantation because of multiple minor mismatches. We could show in our LP -> C57BL/6 model typical features of acute GVHD. Allogeneic transplanted animals showed significant decreased survival compared to syngeneic transplanted animals (Figure 1A). GVHD animals exhibited GVHD-typical weight loss (Figure 1B) and a significant increased clinical GVHD index consisting of five clinical parameters: weight loss, posture, activity, fur and skin (Figure 1C). Our model showed a strong skin phenotype, especially between day 15 and 25 after BMT extensive scurf spread over the whole body combined with fur loss. Pronounced weight loss and highest clinical GVHD index were monitored between day 15 and 25, implicating this period as acute phase of GVHD. Histopathologic analysis of GVHD organs (large bowel, liver and skin) confirmed acute GVHD symptoms in our model as histopathological scores after Lerner (1974,Transplant Proc 6:367-371) were significantly increased in GVHD animals compared to BMT recipients without GVHD (Figure 1D). [Display omitted] We next characterized systemic inflammation in our model by determining the number of circulating inflammatory cells in peripheral blood at different time points by flow cytometry. CD8+ T cells were significantly increased in GVHD animals in acute phase (day 15 after BMT), whereas CD4+ T cells were elevated in late phase (day 50-60 after BMT). These data suggests, that in our model two inflammatory phases occur, an early acute CD8-driven and a late CD4-driven phase.Finally, we could demonstrate GVHD-typical T cell infiltration in GHVD target organs in acute phase (Figure 2). In large and small bowel, liver and skin CD4 and CD8 infiltration was significantly elevated in GVHD animals, however, CD8 infiltration was more prominent. We conclude that CD8 T cells play a prominent role in acute phase of GVHD in our model. [Display omitted] In summary, we could establish a novel chemotherapy based minor mismatch GVHD model which shows good engraftment, typical features of acute GVHD and systemic and target organ specific inflammation. Our murine GVHD model closely resembles the clinical situation of MHC-matched stem cell transplantation and may help to better understand pathogenic mechanisms in GVHD and develop new treatment options. Disclosures:No relevant conflicts of interest to declare.

Donor Immunization with WT1 Peptide Augments Anti-Leukemic Activity After MHC-Matched Bone Marrow Transplantation

Abstract 1896The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part due to immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilm’s Tumor 1 (WT1) peptides, induces a T cell population that is tumor antigen specific. We determined whether BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent anti-tumor activity when combined with allotransplantation. WT1 peptide vaccinations of healthy syngeneic or allogeneic donor mice with a 9-mer WT1 peptide (amino acids 126–134, the WT1 9-mer which has the highest binding affinity for H-2Db) and Incomplete Freund’s Adjuvant induced CD8+ T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that compared to vaccination with IFA alone, four weekly WT1 vaccinations induced an increased percentage of WT1-tetramer+CD8 T-cells (0.15% vs. 1%) in the peripheral blood 28 days following the first vaccination (Figure A *p<.001). CD8 T-cells producing IFN-γ+ after co-culture with tumor cells were similarly increased (0.11% vs. 13.6%) at this timepoint (Figure B *p<.001). They were CD44hi suggesting a memory phenotype, specifically reactive to WT1-expressing tumor (FBL3 and not H11), and increased in a vaccination dose-dependent fashion (Figure A and B). Four weekly WT1 vaccinations prevented tumor growth in donors following intravenous leukemia challenge. In contrast, in tumor-bearing mice, WT1 vaccinations failed to induce WT1-tetramer+ or IFN-γ+ CD8 T-cells and were ineffective as a therapeutic vaccine based on intensity of bioluminescence from luciferase-labeled FBL3 leukemia and mortality. BMT from WT1 vaccinated MHC-matched donors including LP/J and C3H.SW, but not C57BL/6 syngeneic donors, into C57BL/6 recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of luciferase-labeled FBL3 leukemia and survival of 70–90% of mice. Interestingly, the transfer of total CD8+ T cells from immunized donors was more effective than the transfer of WT1-tetramer+CD8+ T cells, likely as a result of alloreactive and tumor-antigen reactive T cells contained with the donor total CD8+ T cells. Total and tetramer+CD8+ T cells required CD4+ T cell help for maximal anti-tumor activity, which was equivalent in efficacy from immunized or unimmunized CD4+ T cell donors. Total CD4+ T cells, alone, from immunized donors provided no anti-tumor activity. The infused donor LP/J or C3H.SW CD8+ T cells collected from cured C57BL/6 recipients, were highly reactive against WT1-expressing FBL3 leukemia cells (14% IFN-γ+) compared to non-WT1-expressing H11 leukemia cells (5% IFN-γ+). The circulating, WT1-tetramer+CD8+ T cell population expanded in cured recipients, peaking at 3.5% on day 50 and contracting through day 100 post-BMT to 0.56%. These findings show that peptide vaccination of donor mice with a tumor antigen dramatically enhances GvT activity and is synergistic with allogeneic BMT. This novel and broadly applicable approach, using leukemia-associated antigen immunization to enhance GvT by creating an “educated” donor T cell graft for allogeneic transplantation of patients with acute myeloid leukemia and myelodysplastic syndrome, is currently being translated to a Phase 1 clinical trial at our institution. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Donor immunization with WT1 peptide augments antileukemic activity after MHC-matched bone marrow transplantation

AbstractThe curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part because of immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilms Tumor 1 (WT1) peptides, induces a T-cell population that is tumor antigen specific. We determined whether allogeneic BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent antitumor activity than either therapy alone. WT1 peptide vaccinations of healthy donor mice induced CD8+ T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that peptide immunization was effective as a prophylactic vaccination before tumor challenge, yet was ineffective as a therapeutic vaccination in tumor-bearing mice. BMT from vaccinated healthy MHC-matched donors, but not syngeneic donors, into recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of FBL3 leukemia. The transfer of total CD8+ T cells from immunized donors was more effective than the transfer of WT1-tetramer+CD8+ T cells and both required CD4+ T-cell help for maximal antitumor activity. These findings show that WT1 peptide vaccination of donor mice can dramatically enhance GvT activity after MHC-matched allogeneic BMT.

Recipient lymphocyte infusion in MHC-matched bone marrow chimeras induces a limited lymphohematopoietic host-versus-graft reactivity but a significant antileukemic effect mediated by CD8+ T cells and natural killer cells

Challenge of MHC-mismatched murine bone marrow chimeras with recipient-type lymphocytes (recipient lymphocyte infusion) produces antileukemic responses in association with rejection of donor chimerism. In contrast, MHC-matched chimeras resist eradication of donor chimerism by recipient lymphocyte infusion. Here, we investigated lymphohematopoietic host-versus-graft reactivity and antileukemic responses in the MHC-matched setting, which is reminiscent of the majority of clinical transplants. We challenged C3H→AKR radiation chimeras with AKR-type splenocytes (i.e. recipient lymphocyte infusion) and BW5147.3 leukemia cells. We studied the kinetics of chimerism using flowcytometry and the mechanisms involved in antileukemic effects using in vivo antibody-mediated depletion of CD8(+) T and NK cells, and intracellular cytokine staining. Whereas control chimeras showed progressive evolution towards high-level donor T-cell chimerism, recipient lymphocyte infusion chimeras showed a limited reduction of donor chimerism with delayed onset and long-term preservation of lower-level mixed chimerism. Recipient lymphocyte infusion chimeras nevertheless showed a significant survival benefit after leukemia challenge. In vivo antibody-mediated depletion experiments showed that both CD8(+) T cells and NK cells contribute to the antileukemic effect. Consistent with a role for NK cells, the proportion of IFN-γ producing NK cells in recipient lymphocyte infusion chimeras was significantly higher than in control chimeras. In the MHC-matched setting, recipient lymphocyte infusion elicits lymphohematopoietic host-versus-graft reactivity that is limited but sufficient to provide an antileukemic effect, and this is dependent on CD8(+) T cells and NK cells. The data indicate that NK cells are activated as a bystander phenomenon during lymphohematopoietic T-cell alloreactivity and thus support a novel type of NK involvement in anti-tumor responses after post-transplant adoptive cell therapy.

Mixed Chimerism with MHC-Matched Bone Marrow Transplantation Results in Dominant Protection From Autoimmune Diabetes (83.8)

Abstract Allogeneic mixed chimerim can prevent disease onset in Type 1 diabetes. While bone marrow (BM) allografts from MHC-mismatched donors can prevent disease in NOD mice, levels of mixed chimerism can be variable and unstable. Also, MHC-mismatched BM usually involve donor T cell depletion, increasing risk for graft-versus-host reactivity. Therefore, we tested the ability of MHC-matched donors to protect NOD mice from disease. Young female NOD mice were irradiated (2 x 450R) and grafted with the following BM donors: (1) Disease-prone NOD (H-2g7), (2) MHC-matched, disease-resistant C57Bl/6.H-2g7 (B6.H-2g7), (3) MHC-mismatched, disease-resistant C57Bl/6 (B6), or (4) Mixtures of disease-prone + disease-resistant BM. Importantly, donor BM was depleted of mature T and B cells. Recipients of only NOD BM recapitulated diabetes in 31/33 (94%) recipients by 30 weeks. Conversely, recipients of B6.H-2g7 BM alone did not develop diabetes (0/26). Mixed NOD + B6.H-2g7 BM grafts resulted in marked disease protection in 27/30 recipients (p &amp;lt;.001). However, recipients of mixed NOD + B6 BM grafts rejected the MHC-mismatched B6 donor and developed disease in 9/10 hosts (90%). Importantly, mixed NOD + B6.H-2g7 BM transplants resulted in remarkably stable, multi-lineage chimerism (&amp;gt;40-50% NOD) for ≥ 30 weeks. Thus, MHC-matched BM allografts permits dominant disease protection and stable macro-chimerism in NOD mice. Also, disease-prone MHC is not an intrinsic barrier to autoimmune regulation.

Transfusion of minor histocompatibility antigen–mismatched platelets induces rejection of bone marrow transplants in mice

Bone marrow transplantation (BMT) represents a cure for nonmalignant hematological disorders. However, compared with the stringent conditioning regimens used when performing BMT to treat hematological malignancies, the reduced intensity conditioning regimen used in the context of nonmalignant hematological disorders leads to substantially higher rates of BMT rejection, presumably due to an intact immune system. The relevant patient population typically receives transfusion support, often including platelets, and the frequency of BMT rejection correlates with the frequency of transfusion. Here, we demonstrate that immunity to transfused platelets contributes to subsequent BMT rejection in mice, even when the BMT donor and recipient are MHC matched. We used MHC-matched bone marrow because, although immunity to transfused platelets is best characterized in relation to HLA-specific antibodies, such antibodies are unlikely to play a role in clinical BMT rejection that is HLA matched. However, bone marrow is not matched in the clinic for minor histocompatibility antigens, such as those carried by platelets, and we report that transfusion of minor histocompatibility antigen-mismatched platelets induced subsequent BMT rejection. These findings indicate previously unappreciated sequelae of immunity to platelets in the context of transplantation and suggest that strategies to account for minor histocompatibility mismatching may help to reduce the chance of BMT rejection in human patients.

Surviving Host CD4+CD25+Foxp3+ Cells Following Ablative Conditioning Expand and Comprise the Major Component of the Treg Compartment during the Lymphoid Reconstitution Period Following HCT.

The capacity of CD4+CD25+Foxp3+ (Treg) cells to regulate adaptive and innate immune responses has led to studies investigating their application to regulate allogeneic T cell responses arising during hematopoietic stem cell transplants (HCT). With respect to HCT, a fundamental clinical concern is the reconstitution of the lymphoid compartment in recipients, particularly T cells since this process can be exceedingly delayed. We have previously found that host Treg cells can regulate resistance to engraftment following HCT, demonstrating that such cells survive and function at least transiently in recipients. The present studies investigated the residual host Treg compartment following HCT. Utilizing varying levels of total body irradiation (5.0 – 14Gy), we observed:1.recipient CD4+CD25+Foxp3+ cells can survive ablative as well as reduced intensity conditioning,2.the surviving, i.e. residual Treg cells undergo expansion as assessed by BrdU uptake and cell numbers, and3.these cells comprise the majority of the Treg compartment in recipients for several months post-HCT during which time donor derived Treg cells gradually arise and cede this compartment.Residual Tregs also dominated the compartment following allogeneic HCT of MHC-matched bone marrow depleted of T cells. To assess the functional capacity of the residual Treg cell compartment, the development of autoimmune disease following transplant of IL-2Rβ −/− (CD122−/−) bone marrow into syngeneic recipients with and without residual Tregs was examined. Autoimmune disease symptoms and T cell alterations were prevented in B6-wt but not T cell deficient recipients. Interestingly, the failure to transfer autoimmune disease following IL-2Rβ −/− HCT into lethally conditioned B6-CD4−/− recipients was associated with the presence of a peripheral CD8+FoxP3+ population not detected in B6-wt mice or the B6-wt mice transplanted with IL-2Rβ −/− BM. This finding indicates that in the genetic absence of CD4+ T cells, a CD8 regulatory population appears to emerge. In total, our observations support the notion that functioning host Tregs initially occupy a niche in the transplant recipient permitting lymphopenic expansion and an extended period of contribution to this compartment. Notably, this contribution reflected much greater levels than conventional T cell populations - even in aggressively conditioned recipients. Finally, these findings imply that the presence of host regulatory cells may be important to consider with respect to eliciting anti-tumor responses and vaccination in recipients during the early period post -hematopoietic cell transplantation.

Use of anti-CD3 epsilon F(ab')2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation.

The use of T cell-specific mAb in vivo for prevention and treatment of graft-vs-host disease (GVHD) and its impact on graft-vs-leukemia (GVL) reactivity was examined in a murine model of MHC-matched bone marrow transplantation (BMT). F(ab')2 fragments of a CD3 epsilon-specific mAb were administered to irradiated AKR (H-2k) hosts after transplantation of BM plus spleen cells from B10.BR donors (BMS chimeras). The effects on GVH and GVL reactivity were Ab dose- and schedule-dependent. A short course of mAb (qe2d, days 0 to 8) prevented clinical evidence of GVHD and mortality. Anti-CD3 F(ab')2 mAb reversed clinical symptoms of acute GVHD when delayed up to 18 days post-transplant. Anti-host (Mls-1a)-specific V beta 6+ cells were absent from the spleens of GVH-negative control mice, but persisted in Ab-treated BMS chimeras despite the absence of GVHD. Leukemic mice given 16.7 micrograms of Ab on days 0, 2, and 4 survived leukemia-free without developing severe GVHD. A longer course of Ab completely prevented GVHD, but led to leukemia relapse in tumor-bearing hosts, despite engraftment of donor T cells. The GVL effect was quantitatively stronger when Ab was used for GVH therapy as compared with GVH prevention. Some Ab-treated, GVH-free chimeras relapsed with lymphomas in unusual sites, suggesting that occult tumor cells may persist in nonlymphoid tissues. These experiments demonstrate that T cell-specific mAb can be used successfully in vivo to avoid severe GVHD, but that excessive or ill-timed administration of Ab may eliminate GVL reactivity.

A decrease in graft-vs.-host disease without loss of graft-vs.-leukemia reactivity after MHC-matched bone marrow transplantation by selective depletion of donor NK cells in vivo.

It is thought that natural killer cells may play a role in graft-vs.-host reactions after allogeneic bone marrow transplantation, but the use of NK cell-specific reagents has been limited. In this report, an NK allele-specific monoclonal antibody, anti-NK 1.1, was used to study the impact of in vivo donor NK cell depletion on GVH disease, graft-vs.-leukemia (GVL) reactivity and donor T cell chimerism after allogeneic murine BMT. AKR/J (H-2k) recipient mice were preconditioned with suboptimal irradiation (9 Gy = LD50) and transplanted with major histocompatibility complex-matched B10.BR (H-2k) BM cells with or without added spleen cells as a source of T cells. The addition of increasing numbers of spleen cells to the BM inoculum produced GVHD of varying intensities. The beneficial effect of NK depletion on GVHD was dependent on the intensity of the GVH reaction. Donor NK cell depletion had no effect on the survival of mice with severe GVHD after MHC-matched BMT (B10.BR into AKR) or after MHC-mismatched BMT (B10.BR into DBA/2; H-2k into H-2d). However, donor NK depletion increased survival of AKR hosts given sufficient B10.BR splenic T cells to induce mild-to-moderate GVHD. Ex vivo depletion of donor CD8+ T cells also reduced GVH-associated mortality, but the use of both CD8 and NK depletion offered no improvement over either alone, suggesting an interaction between CD8+ and NK 1.1+ cells. In contrast to CD8 depletion, donor NK depletion did not compromise the rapid and complete establishment of donor T cell chimerism nor the ability of chimeras to mount an effective GVL reaction. Thus, elimination of donor NK cells provides an alternate strategy for reducing GVHD without loss of GVL reactivity following MHC-matched allogeneic BMT.