Host Minor Histocompatibility Antigens Research Articles

Ex Vivo Generation and Infusion of Anti-Minor Histocompatibility Antigen Expanded T Cells for Patients Who Relapse after Allogeneic HLA-Matched Stem Cell Transplantation

Rationale Long-term clinical remissions of hematologic malignancies after allogeneic hematopoietic stem cell transplantation (HSCT) largely rely on the graft-versus-leukemia effect (GVL). In a fully HLA-matched setting, the GVL effect is mediated primarily by T cell immune responses against host minor histocompatibility antigens (MiHAs) found on the surface of malignant cells. Upon relapse of leukemia following HSCT, donor lymphocyte infusions (DLI) have unfortunately failed to exert efficient GVL activity. This is likely due to the insufficient number of anti-MiHA T cells within DLIs and to the risk of graft-vs-host disease (GVHD) that prevents the administration of high numbers of T cells. To generate potent and selective anti-leukemia activity, we have developed a strategy for the ex vivo selection and expansion of T cells targeting MiHAs preferentially expressed on hematopoietic cells (Perreault, Leukemia 2016). Thus, these cells should display augmented GVL immune reactivity while harboring a low risk of causing GVHD. To our knowledge, this is the first clinical study targeting several different molecularly defined MiHAs and on different HLA molecules. Patients and Methods In an open-label, multi-center phase 1 clinical trial (CR-MIHA-001; NCT 03091933), patients with hematologic malignancies who relapsed after an allogeneic HLA-matched HSCT were treated with sibling donor-derived, ex vivo- expanded T cells selected and expanded for reactivity toward tissue-restricted recipient MiHAs (GLIDE: Guided Lymphocyte Immunopeptide Derived Expansion). After donor cell collection, patients received fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2 for 3 days followed by infusion of GLIDE, consisting of donor T-lymphocytes expanded ex vivo following exposure to dendritic cells coated with immunogenic peptides (MiHAs) present on the surface of host hematopoietic cells, at a single target dose of 4x107 viable T-cells/m2. A second dose of GLIDE, aiming at 3-5 times the original dose, could be administered to patients at a minimum of 6 weeks after the first infusion. GLIDE cell characteristics were evaluated using multi-parameter flow cytometry and TCR sequencing. Patients were followed up for one year post-GLIDE infusion for toxicity and clinical outcome measures. Results Ten patients, median age of 58 (range 33-69), 4 females/6 males with AML (n=7), NHL (1), CLL (1), and myelofibrosis (1) were treated with GLIDE. These patients had relapsed at a median of 12 months (range 5-126 mo) after allogeneic HSCT. GLIDE cells were personalized to target single MiHAs in 9 patients and two MiHAs in 1 patient. Overall, GLIDE cell products were directed at 9 different MiHAs on 6 different HLA-class 1 molecules (HLA-A02:01, A24:02, B07:02, B18:01, B44:02, and B44:03). A first dose of GLIDE was administered at 4.0x107 CD3+ cells/m2 to 8 patients, and 2 patients received slightly lower doses (2.6 and 3.7 x 107 CD3+ cells/m2). Final GLIDE products consisted mainly of T-cells (median: 98,7%), with CD8>CD4>NKT>NK cells. Most CD8+ T cells were effector memory and central memory T cells (Figure 1). At the end of the expansion period, CD8+ T cells expressed activation markers (CD25+ = 67%) with low levels of PD1+, PDL1+, CD57+ and KLRG1+ as markers of exhaustion. Not surprisingly, GLIDE cells had a more restricted TCR β chain profile than initial lymphopheresis cells. Six patients received a second dose of GLIDE (mean: 12 x 107 CD3+ cells/m2) at 53 days after the first infusion (median, range: 48-118 days). There were no infusional toxicities, and cells were well-tolerated by patients. Only one patient developed thrombocytopenia and subsequent chronic GVHD. There were no other adverse events potentially related to GLIDE. According to TCR β sequencing data, T cells present in the adoptively transferred anti-MiHA GLIDE product persisted in peripheral blood up to one year after infusion. Survival at one year was observed in 4 patients with 3 patients in sustained remission (Figure 2). Conclusion This pilot study demonstrates the feasibility of anti-MiHA T cell ex vivo generation (GLIDE) and safety of its administration to patients relapsing after allogeneic HSCT, as well as the clinical potential of these cells. The persistent remissions and low occurrence of GVHD support further development of GLIDE to target acute myeloid leukemia and other hematologic malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Human regulatory T cells against minor histocompatibility antigens: ex vivo expansion for prevention of graft-versus-host disease

AbstractAlloreactive donor T cells against host minor histocompatibility antigens (mHAs) cause graft-versus-host disease (GVHD) after marrow transplantation from HLA-identical siblings. We sought to identify and expand regulatory CD4 T cells (Tregs) specific for human mHAs in numbers and potency adequate for clinical testing. Purified Tregs from normal donors were stimulated by dendritic cells (DCs) from their HLA-matched siblings in the presence of interleukin 2, interleukin 15, and rapamycin. Male-specific Treg clones against H-Y antigens DBY, UTY, or DFFRY-2 suppressed conventional CD4 T cell (Tconv) response to the specific antigen. In the blood of 16 donors, we found a 24-fold (range, 8-fold to 39-fold) excess Tconvs over Tregs reactive against sibling mHAs. We expanded mHA-specific Tregs from 4 blood samples and 4 leukaphereses by 155- to 405-fold. Cultured Tregs produced allospecific suppression, maintained demethylation of the Treg-specific Foxp3 gene promoter, Foxp3 expression, and transforming growth factor β production. The rare CD4 T conv and CD8 T cells in the end product were anergic. This is the first report of detection and expansion of potent mHA-specific Tregs from HLA-matched siblings in sufficient numbers for application in human transplant trials.

Inhibition of Cathepsin S Reduces Allogeneic T Cell Priming but Not Graft-versus-Host Disease Against Minor Histocompatibility Antigens

Cathepsin (Cathepsin) S, L, and B proteases mediate antigen presentation on major histocompatibility complex (MHC) class II by degrading the invariant chain Ii, which blocks peptide loading. The ability of the Cathepsin S inhibitor LHVS (morpholinurea-leucine-homophenylalanine-vinylsulfone phenyl) to impede antigen presentation has led its development as a therapy for autoimmune diseases. There is substantial evidence that donor T cell recognition of host minor histocompatibility antigens (miHA) and subsequent destruction of host tissue mediates graft-versus-host disease (GVHD). We hypothesized that enzymes involved in antigen presentation may play a role in the development of GVHD. Using the C57BL/6 → BALB.B minor mismatch acute GVHD (aGVHD) model, we found that the cathepsin S activity of spleens from allogenetically transplanted mice were significantly increased 1 week after transplantation compared with syngeneic mice. Although LHVS decreased T cell priming responses against both single OVA antigen and miHA invitro, LHVS did not reduce the severity of aGVHD. In fact, LHVS exacerbated a CD4(+)-T cell-dependent model of GVHD similar to chronic GVHD. This suggests that cytokines rather than T cells may mediate much of the damage in the aGVHD model and that therapeutics based on inhibition of antigen presentation for GVHD must be approached with caution.

Mechanisms of antigen presentation to T cells in murine graft-versus-host disease: cross-presentation and the appearance of cross-presentation

AbstractRecipient antigen-presenting cells (APCs) initiate GVHD by directly presenting host minor histocompatibility antigens (miHAs) to donor CD8 cells. However, later after transplantation, host APCs are replaced by donor APCs, and if pathogenic CD8 cells continue to require APC stimulation, then donor APCs must cross-present host miHAs. Consistent with this, CD8-mediated GVHD is reduced when donor APCs are MHC class I−. To study cross-presentation, we used hosts that express defined MHC class I Kb-restricted miHAs, crossed to Kb-deficient backgrounds, such that these antigens cannot be directly presented. Cross-priming was surprisingly efficient, whether antigen was restricted to the hematopoietic or nonhematopoietic compartments. Cross-primed CD8 cells were cytolytic and produced IFN-γ. CD8 cells were exclusively primed by donor CD11c+ cells, and optimal cross-priming required that they are stimulated by both type I IFNs and CD40L. In studying which donor APCs acquire host miHAs, we made the surprising discovery that there was a large-scale transfer of transmembrane proteins from irradiated hosts, including MHC class I–peptide complexes, to donor cells, including dendritic cells. Donor dendritic cells that acquired host MHC class I–peptide complexes were potent stimulators of peptide-specific T cells. These studies identify new therapeutic targets for GVHD treatment and a novel mechanism whereby donor APCs prime host-reactive T cells.

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.

Donor APCs Promote Gvhd in MHC-Mismatched Transplants by Indirectly Presenting Host Minor Histocompatibility Antigens.

Abstract 689In MHC-mismatched allogeneic bone marrow/stem cell transplantation (alloBMT), T cells that at least in-part recognize the allogeneic MHC are likely the dominant mediators of acute GVHD. This would explain why far fewer T cells induce acute GVHD in MHC-mismatched as opposed to MHC-matched alloBMT. However, despite this difference, in human recipients of haploidentical allografts, the organ distribution and histologic appearance of GVHD, especially chronic GVHD, are similar. Moreover, late-onset GVHD in MHC-mismatched alloBMT occurs when recipient antigen presenting cells (APCs) have been replaced by donor APCs. We therefore investigated how indirect presentation of host minor histocompatibility antigens (miHAs) by donor APCs contributes to GVHD. To test this, we employed the MHC/miHA mismatched B6 (H-2b) (right arrow) BALB/c (H-2d) strain pairing. We compared GVHD in irradiated BALB/c mice that were reconstituted with B6 CD4 cells and either B6 BM or B6 MHCII-/- BM. We reasoned that if BALB/c miHAs presented by donor B6 APCs were important, GVHD would be reduced in recipients of MHCII-/- BM. As measured by weight loss and survival, this was indeed the case. However, it was possible that receiving MHCII-/- donor BM reduced GVHD for reasons unrelated to the presentation of BALB/c miHAs. To address this we compared GVHD in irradiated B6.C (H-2d) recipients of B6 CD4 cells and either B6 or B6 MHCII-/- BM. This strain pairing shares the same MHC mismatch as does the B6(right arrow)BALB/c model, but lacks miHAs except those encoded by the congenic H-2d region. In contrast to the prior experiments, B6.C recipients of MHCII-/- or wild type BM developed similar GVHD. To further define indirect-presentation of miHAs in MHC/miHA-mismatched alloBMT, we used B6 Marilyn CD4+ T cell receptor transgenic T cells that recognize an IAb-restricted peptide derived from the male DBY protein. Male or female BALB/c mice were irradiated and reconstituted with female B6 BM and female polyclonal B6 CD4 cells along with graded numbers of Marilyn T cells. At day 7 and 14 post transplant Marilyn cells expanded at least 1000-fold greater in male than in female recipients. Marilyn T cell expansion was similar when male antigen was restricted to hematopoietic or nonhematopoietic cells. There was little expansion of Marilyn cells in male BALB/c mice if donor BM was MHCII-/-, confirming that expansion of Marilyn T cells depended on donor APCs presenting DBY. Indirectly primed Marilyn cells were functional as measured by IFN-γ production after restimulation by the DBY peptide in vitro. As we saw reduced GVHD in the B6 (right arrow) BALB/c model in recipients of MHCII-/- BM, we reasoned we should be able to detect polyclonal CD4 cells responding to host miHA presented by donor APCs. That was the case as we found an increase in DBY-reactive polyclonal CD4 cells by ELISPOT in male recipients as compared to female recipients at day 7 (1.5 fold for IFN-γ and TNF-αa). This difference was lost when donor BM was MHCII-/-. In summary, indirect presentation of host miHAs by donor APCs in MHC-mismatched alloBMT is surprisingly efficient and alloreactive CD4 cells that target host miHAs presented by donor APCs contribute to GVHD in MHC/miHA-disparate alloBMT. The generation of T cells that target host miHAs presented by donor APCs allows GVHD to persist even when host APCs that prime T cells that recognize the allogeneic MHC have been eradicated. This provides a mechanistic explanation for why late onset GVHD, including chronic GVHD, in MHC-mismatched alloBMT resembles that in MHC-matched alloBMT as both may target host miHAs indirectly presented by donor-derived APCs. These data also provide further rationale for targeting donor-derived APCs as a method for preventing and treating GVHD. Disclosures:No relevant conflicts of interest to declare.

Simulataneous Vaccination and Delayed Lymphocyte Infusion for Enhancement of Immune Reactivity Against Acute Lymphoblastic Leukemia or Lymphoma.

BACKGROUND: Delayed lymphocyte infusion (DLI) can be employed as an antileukemia immune therapy when patients with leukemia relapse after allogeneic hematopoietic stem cell transplantation (HSCT). While effective in patients with chronic myelogenous leukemia, it is generally ineffective with relapse of acute lymphoblastic leukemia (ALL). This ineffectiveness may be multifactorial and possible reasons include rapid leukemia cell growth in ALL, reduced propensity to apoptosis signals, and poor presentation of antigens present on lymphoblasts resulting in failure to generate a significant immune response. If the failure of malignant lymphoblasts in marrow and lymphoid tissues to elicit meaningful T cell responses plays a role in the ineffectiveness of DLI, it is possible that therapeutic vaccines may be able to ameliorate this barrier. We have recently demonstrated that vaccination immediately prior to HSCT enhances antigen specific T cell responses in the post-transplant immune repertoire (Bone Marrow Transplantation, 35:793–801, 2005.)HYPOTHESIS: Vaccination with leukemia related antigens at the time of DLI will increase donor T cell responses to leukemia related antigens without exacerbation of GVHD responses to ubiquitous host minor histocompatibility antigens (mHA).METHODS: We employ a murine MHC matched allogeneic HSCT model in which 6 antigens are molecularly characterized. C3.SW mice are donors and C57BL/6 mice are recipients. In this model the H7 mHA is the immunodominant antigen, while alloresponses to H3 and H13 are also present. By using spontaneously arising lymphoblastic leukemia/lymphoma cells from Ig-cmyc transgenic male C57BL/6 as the model malignancy in a female to female transplant the male HY associated antigens Uty, Dby and Smcy can be used as antigens restricted to the lymphoid malignancy. Four weeks after allogeneic HSCT mice were vaccinated with 107 cells expressing the HY antigens and the C57BL/6 H7, H3 and H13 minor antigens. The cells used for pre-DLI vaccination were either irradiated male C57BL/6 spleen cells or irradiated male malignant lymphoblasts. One day later mice received iv infusion of 107 spleen cells from C3.SW female donor mice previously primed against HY antigens by immunization with male C3.SW cells. Ten days interferon gamma Elispot assays were performed on HSCT recipient spleen cells using the peptide-defined antigens.RESULTS: Vaccination of HSCT recipients with male spleen cells 1 day prior to DLI from HY immune donors significantly increased T cell responses to HY peptide epitopes. Recipients of DLI alone harbored 40 interferon secreting cells per 106, while combination of vaccination and DLI yielded 94 per 106(p < 0.05). In contrast, there was no change in the number of T cells responding to the H7, H3 and H13 mHA. DLI only exhibited 26 per 106 while DLI plus vaccine exhibited 27 per 106 (p < 0.05). Whole cell irradiated lymphoblasts were not as effective as irradiated splenocytes in augmenting the HY responses, although they were equally effective in producing T cell responses in normal, nontransplanted mice.CONCLUSIONS: Simultaneous vaccination and DLI can lead to significant expansion of donor cells potentially reactive with antigens present on malignant lymphoblasts without exacerbation of T cell responses to ubiquitous mHA associated with GVHD. Current work is exploring methods by which indirect presentation of antigens from lymphoblasts can be enhanced, since it is unlikely that vaccines relying on direct antigen presentation by lymphoblasts will be effective.

Cellular tumor vaccines administered after T cell-depleted allogeneic bone marrow transplantation induce effective anti-tumor immune responses

In allogeneic hematopoietic stem cell (HSC) transplantation, graft vs. tumor (GVT) activity contributes to the cancer cure. It is closely associated with graft vs. host disease (GVHD), an immune response initiated by transplanted donor T-cell responses against host minor histocompatibility antigens (mHAgs). GVHD is prevented by T-cell depletion of the donor graft, but T-cell depletion also abrogates curative GVT. We wished to test the hypothesis that cellular tumor vaccines administered after T cell-depleted HSC transplant can induce significant GVT effects, despite the absence of transplanted mature donor T cells. In this investigation, a murine model of major histocompatibility complex (MHC)-matched, mHAg-mismatched allogeneic HSC transplant was studied. T cell-depleted or normal T cell-containing grafts were given to myeloablated recipients. Following reconstitution the recipients were vaccinated with tumor vaccines. GVT responses were measured in vitro by T-cell function assays and flow cytometry, and in vivo by tumor burden or survival. Post-transplant tumor vaccines induced effective anti-tumor responses in recipients of T cell-depleted transplants, producing cytolytic and cytokine responses, reduced tumor burden and prolonged survival. Recipients of T cell-depleted transplants that still have significant thymic function may be suitable subjects for post-transplant vaccine therapy.

Graft-vs.-Lymphoma Effect in Various Histologies of Non-Hodgkin's Lymphoma

Generally, there is a significantly lower risk of lymphoma relapse following allogeneic than after autologous stem cell transplant. Factors contributing to this lower risk of relapse include an absence of the use of ablative conditioning, with a tumor-free graft, and the generation of a graft-vs.-tumor (GVT) immune response. Allogeneic transplantation, however, has the possibility of graft-vs.-host disease (GVHD). The use of autologous and conventional allogeneic hematopoietic stem cell transplantation (HSCT) in follicular lymphoma, diffuse large cell lymphoma, chronic lymphocytic leukemia and multiple myeloma is discussed. Due to a 1-year transplant-related mortality of 30 – 40% and complications caused by GVHD, conventional, myeloablative, allogeneic transplantation is a high-risk option for low-grade lymphoproliferative disorders. Novel applications of allogeneic HSCT are described that take advantage of a GVT effect while reducing the risk of GVHD. Minimally myelotoxic pretransplant conditioning regimens allow host antigen-presenting cells to persist, enabling presentation of host minor histocompatibility antigens to donor T cells, causing a GVT response. Although complications may arise due to GVHD, non-myeloablative HSCT can be offered to patients previously ineligible for conventional high-dose treatment. A protocol developed in Seattle using a low-dose total body irradiation (TBI)-based conditioning regimen with immunosuppression using mycophenolate mofetil in combination with cyclosporin has been used in a multicenter trial. To overcome the problem of graft rejection fludarabine was later added to the protocol. A second protocol from a smaller trial used a preparative, conventional-dose regimen of fludarabine, given with cyclophosphamide. Rituximab was also given to provide synergistic action with the chemotherapy to enhance tumor control in the early post-transplant period to allow time for the establishment of the GVT effect. Following transplantation, GVHD prophylaxis was given using tacrolimus with methotrexate. A trial of a further variation of allogeneic HSCT, tandem auto/allo transplants, is described. First, high-dose therapy with autologous PBSC rescue was used to cytoreduce the disease. This was followed by a reduced-intensity or non-myeloablative allogeneic graft. This procedure was devised to take advantage of high-dose therapy and allogeneic HSCT. Results for non-myeloablative allogeneic HSCT are particularly promising in low-grade NHL and the GVT effect may augment response and delay or prevent relapse. However, for aggressive disease, non-myeloablative regimens are only indicated for patients with minimal disease, as the non-myeloablative regimens are unable to control the tumor before the generation of a GVT effect, and/or lack the ability to control rapidly proliferating disease. Patients with relapsed disease may require a higher-dose regimen or tandem transplant approach.

Analysis of immunodominance among minor histocompatibility antigens in allogeneic hematopoietic stem cell transplantation.

In major histocompatibility complex (MHC)-matched allogeneic hematopoietic stem cell transplantation (HSCT), donor responses are directed against multiple host minor histocompatibility antigens (mHAgs), producing graft-versus-host disease (GVHD) and graft-versus-tumor (GVT) effects. We studied MHC-matched, mHAg-mismatched C3H.SW>C57BL/6 HSCT in which three mHAg are molecularly defined (B6dom1, H3, H13) to determine if there is a hierarchy of immunodominance among the mHAgs and to learn the contribution of each to GVHD. We found that B6dom1 was the immunodominant mHAg. B6dom1 did not block responses to the subdominant mHAgs H3 and H13. The mechanism of immunodominance was not mHAg avidity or affinity for class I. B6dom1 elicited a broader variety of Vbeta clonotypes than either H3 or H13. Severe GVHD could occur in the absence of a strong B6dom1 response. Alloreactivity to isolated B6dom1, H3 or H13 differences did not produce severe GVHD. We concluded that immunodominance is explained by both mHAg density on host cells and the repertoire of donor T cells capable of responding to the mHAgs. Clinically significant GVHD requires donor responses to multiple mHAgs. Modulation of responses to a single immunodominant mHAg is insufficient for the prevention of GVHD, while immunotherapies directed against isolated mHAgs may not provoke severe GVHD.

Adoptive cancer immunotherapy: discovering the best targets.

Numerous animal and clinical studies have shown that injection of T lymphocytes from a major histocompatibility complex matched donor can cure subjects with chemotherapy-resistant hematological malignancies. This graft-versus-tumor effect, which represents the most conclusive evidence that the immune system can cure cancer in humans, is mediated primarily if not exclusively by T cells specific for host minor histocompatibility antigens. Since minor histocompatibility antigens are present on all tissues and organs, injection of unselected donor T cells also causes graft vs. host disease, which drastically limits the use and benefits of this treatment. Recent studies in mice have shown that adoptive transfer of primed T cells targeted to a single major histocompatibility complex class I restricted immunodominant minor histocompatibility antigen can eradicate leukemia cells without causing any toxicity to the host. We present the promises and caveats of this and other new approaches for adoptive T cell immunotherapy of cancer.

Oligoclonal expansion of T cell receptor V beta 2 and 3 cells in the livers of mice with graft-versus-host disease

The nonsuppurrative destructive cholangitis lesions in the B10.D2 (donor) into BALB/c (host) mouse graft-versus-host disease (GVHD) model are dependent on CD4 T cells that use a T cell receptor-β chain variable region (Vβ) repertoire, which is heavily biased toward Vβ2 and Vβ3 usage. We hypothesized that liver Vβ2 + and Vβ3 + CD4 T cells originate from donor mice and recognize BALB/c minor histocompatibility alloantigens and BALB/c endogenous retroviral superantigen-6, respectively. To test this hypothesis, we determined the donor:host chimera status of infiltrating liver lymphocytes and the clonal states of liver Vβ2 + and liver Vβ3 + CD4 cells isolated from GVHD mice. A limited donor TCR Vβ repertoire composed of Vβ1 +, 2 +, 3 +, 4 +, 6 +, and 8 + cells infiltrated the livers of GVHD mice on day 3. Consistent with a response to immunodominant host minor histocompatibility antigens, we detected oligoclonal liver Vβ2 + T cells in 40% of GVHD mice studied on day 3 and in 100% of GVHD mice studied on day 14. Typical of superantigen stimulation, extremely polyclonal liver Vβ3 + T cells were detected in 100% of GVHD mice studied on day 3 and 40% of GVHD mice studied on day 14. Yet, the liver Vβ3 + T cells in 60% of the day 14 GVHD mice were oligoclonal, pointing to a response to minor histocompatibility antigens. (H EPATOLOGY 2002;35:23-29.)

Graft-host tolerance in bone marrow transplant chimeras. Absence of graft-versus-host disease is associated with unresponsiveness to minor histocompatibility antigens expressed by all tissues

Because bone marrow (BM) transplantation is used with increasing frequency, it is important to elucidate the mechanisms involved in the establishment of tolerance to host minor histocompatibility antigens (MiHA) in recipients transplanted with T-cell-undepleted marrow grafts. We have previously shown that BM chimeras transplanted across MiHA barriers showed specific unresponsiveness to MiHA expressed on recipient-type concanavalin A blasts. Because expression of many MiHA is tissue-specific, we wanted to determine if chimera T lymphocytes would be tolerant to MiHA expressed by all host tissues and organs. To investigate this issue, we measured in vivo proliferation of lymphoid cells from normal C57BL/10 (B10) mice and (B10--&gt;LP) chimeras in tissues and organs of lethally irradiated syngeneic and allogeneic recipients. Donor B10 cells were either untreated, or depleted with anti-Thy-1.2, anti-CD4, or anti-CD8 antibodies. Transplantation of B10 cells in LP recipients triggered an important T-cell-dependent 125I- dUrd uptake in several organs that involved both CD4+ and CD8+ cells. Using Thy-1-congeneic mice we showed that in long-term chimeras practically all CD4+ and CD8+ T lymphocytes were derived from hematopoietic progenitors and not from mature T cells present in the BM graft. When (B10--&gt;LP) BM chimera cells were injected to secondary recipients, no proliferation was observed in any organ of LP hosts whereas normal proliferation was seen in H-2k allogeneic hosts. Thus, in these BM chimeras, tolerance encompasses MiHA expressed by all organs.

Graft-Host Tolerance in Bone Marrow Transplant Chimeras. Absence of Graft-Versus-Host Disease Is Associated With Unresponsiveness to Minor Histocompatibility Antigens Expressed by All Tissues

Because bone marrow (BM) transplantation is used with increasing frequency, it is important to elucidate the mechanisms involved in the establishment of tolerance to host minor histocompatibility antigens (MiHA) in recipients transplanted with T-cell-undepleted marrow grafts. We have previously shown that BM chimeras transplanted across MiHA barriers showed specific unresponsiveness to MiHA expressed on recipient-type concanavalin A blasts. Because expression of many MiHA is tissue-specific, we wanted to determine if chimera T lymphocytes would be tolerant to MiHA expressed by all host tissues and organs. To investigate this issue, we measured in vivo proliferation of lymphoid cells from normal C57BL/10 (B10) mice and (B10-->LP) chimeras in tissues and organs of lethally irradiated syngeneic and allogeneic recipients. Donor B10 cells were either untreated, or depleted with anti-Thy-1.2, anti-CD4, or anti-CD8 antibodies. Transplantation of B10 cells in LP recipients triggered an important T-cell-dependent 125I-dUrd uptake in several organs that involved both CD4+ and CD8+ cells. Using Thy-1-congeneic mice we showed that in long-term chimeras practically all CD4+ and CD8+ T lymphocytes were derived from hematopoietic progenitors and not from mature T cells present in the BM graft. When (B10-->LP) BM chimera cells were injected to secondary recipients, no proliferation was observed in any organ of LP hosts whereas normal proliferation was seen in H-2k allogeneic hosts. Thus, in these BM chimeras, tolerance encompasses MiHA expressed by all organs.

Tolerance to host minor histocompatibility antigens after allogenic bone marrow transplantation. Specific donor-host unresponsiveness is maintained by peripheral tolerizing cells.

The development of graft-host tolerance after allogeneic bone marrow (BM) transplantation is more demanding than the acquisition of self-tolerance because both donor-derived mature T cells and immature thymocytes encounter host Ag. The mechanism involved in tolerization of mature T cells, contained in unmanipulated BM, remains undefined. In previous experiments, we showed in vivo unresponsiveness to host minor histocompatibility Ag (MiHA) in immunocompetent chimeras obtained after MiHA-incompatible BM transplantation. In this study, we wanted to determine: first, what was the specificity of this graft-host unresponsiveness, and second, whether peripheral cells were involved in tolerization? LP recipients were irradiated (9, 5 Gy), injected with 10(7) undepleted BM cells from B10 donors and studied 100 to 150 days later. (B10-->LP) chimeras were immunized in vivo and restimulated in vitro with cells displaying one or multiple incompatible MiHA. In bulk culture experiments, chimeras demonstrated specific CTL unresponsiveness to host MiHA but responded normally to third party MiHA. In limiting dilution analysis conditions, chimeras showed a profound deficit, but not a complete absence of anti-host CTL precursor. Studies with congenic stimulators/targets showed that graft-host tolerance was induced against both immunodominant (e.g., H-3.2) and nonimmunodominant (e.g., H-8.2) MiHA although at the CTL precursor level, it was more complete against the former. Furthermore, chimera spleen cells inhibited the generation of CTL activity against host- and donor-type MiHA but not against third party Ag. This specific suppressor activity was not T cell dependent, and was mediated by radiosensitive cells that are not found in freshly explanted organs from normal mice. Taken together, our results suggest that peripheral tolerization can be a remarkably efficient process to maintain tolerance to MiHA after BM transplantation. Thus, peripheral tolerizing mechanisms may contribute not only to the induction of tolerance to Mls superantigens or to the product of transgenes (if expressed at high levels) but also to a wide array of MiHA.

Mls-1a-induced peripheral tolerance to host minor histocompatibility antigens in radiation bone marrow chimeras. Modification of T cell repertoire associated with active suppression and permanent presentation of host antigens.

Minor histocompatibility Ag (mHAg) can be responsible for the development of graft vs host reaction (GVHR) after bone marrow transplantation. In a mouse model, B10.D2 donor immunization against Mls-1a prevents lethal GVHR developed by CD4+ T cells against DBA mHAg in irradiated (DBA/2 x B10.D2)F1 hosts. Such F1 hosts become 100% chimeric and show long term survival (LS mice). The cellular mechanisms underlying the tolerance in LS mice was investigated. It was found that a state of tolerance can be induced in thymectomized F1 hosts. Although spleen cells from LS mice are able to initiate lethal GVHR in third-party H-2k-incompatible hosts, no GVHR is observed in secondary hosts incompatible for specific DBA/2 mHAg. Mixed lymphocyte experiments in vitro confirm that T cells from LS mice are unresponsive toward specific DBA/2 mHAg, although they are able to proliferate in response to H-2 or Mls-1a Ag. The responsiveness to Mls-1a correlates with the presence of V beta 6+ cells in LS mice, probably derived from mature T cells present in the donor inoculum. The tolerance in LS mice is not due to the lack of DBA/2 mHAg presentation; instead, permanent presentation of Ag (Ag I and Ag II) previously described as being responsible for lethal GVHR is consistently observed. A significant protection against GVHR is obtained by transferring normal B10.D2 cells together with spleen cells from LS mice, clearly indicating the contribution of active suppression in the state of tolerance; this is further confirmed by in vitro results obtained in limiting dilution assays. It is concluded that tolerance in chimeric LS mice 1) is due to a peripheral (thymus-independent) mechanism; 2) is specific for mHAg; 3) correlates with unresponsiveness of the repertoire to host mHAg, without alteration of the repertoire for H-2 and Mls-1a Ag; and 4) is associated with an active suppression and with a permanent presentation of at least two mHAg responsible for GVHR mortality.

Evaluation of in vitro cytotoxic T lymphocyte assays as a predictive test for the occureence of graft vs host disease

The potential value of in vitro cytotoxic T lymphocyte (CTL) assays for predicting the occurrence of graft vs host disease (GVHD) following allogeneic bone marrow transplantation was evaluated in 12 mouse donor-host combinations associated with various degrees of GVHD. These donor-host combinations were selected after evaluation of GVHD triggered by minor histocompatibility antigens (MiHA) in 24 allogeneic strain combinations derived from six strains of H-2b mice. Recipients (n = 475), previously submitted to total body irradiation (9.5 Gy), were transplanted with 10(7) bone marrow cells along with 5 x 10(7) spleen cells. While lethal GVHD was observed in half of the strain combinations, it was possible to select 12 donor-host combinations characterized by severe, mild, or absent GVHD. When levels of anti-host CTL activity were assessed following in vivo priming and in vitro boosting, strong CTL-mediated cytotoxicity was observed in all combinations whether they developed GVHD or not. CTL frequency measured by limiting dilution analysis (LDA) ranged from 1/16880-1/306. The Spearman rank test revealed no positive correlation between GVHD intensity and donor anti-host CTL activity assayed either in bulk culture experiments or in LDA conditions. These results indicate that MiHA capable of triggering potent CTL responses in vitro do not necessarily initiate GVHD, and that in vitro measurement of donor CTL activity against host-type Con A blasts is not a predictive assay for anti-MiHA GVHD. However, the possibility to recruit CTL populations targeting host MiHA expressed specifically on hematopoietic cells suggests a novel therapeutic strategy for the cure of hematopoietic malignancies. Indeed, transplantation of donor hematopoietic stem cells supplemented with T cells aimed at MiHA specifically expressed by host hematopoietic cells, could possibly potentiate the desirable graft vs leukemia effect without increasing the risk of GVHD.

Adoptive immunotherapy of metastatic B16 melanoma with allogeneic immune cells sensitized to minor histocompatibility antigens.

Host mice bearing pulmonary metastases of B16 melanoma were treated by adoptive immunotherapy with allogeneic donor lymphocytes. Rejection of the allogeneic donor cells by the host was delayed by pretreatment immunosuppression of the host with cyclophosphamide and selection of donors that were matched at the major histocompatibility complex (MHC) but disparate for background minor histocompatibility genes. Adoptively transferred normal nonimmune donor cells exhibited no therapeutic activity. However, allogeneic MHC-matched donor cells that were primed in vivo and secondarily sensitized in vitro to host minor histocompatibility antigens expressed on normal lymphocytes were cytotoxic to B16 tumor cells in vitro and mediated a dose-dependent antitumor effect in vivo following i.v. infusion. The therapeutic activity of sensitized allogeneic cells, which presumably reflected recognition of minor histocompatibility antigens expressed both on normal host tissues and on malignant B16 tumor cells, was not associated with any detectable toxicity to these transiently immunosuppressed tumor-bearing hosts.