Minor Histocompatibility Ags Research Articles

Prophylactic Infusion of Multi-Antigen Specific T-Cell Products to Prevent Complications after T Cell Depleted Allogeneic Stem Cell Transplantation — a Phase I/II Study

Viral infections and disease relapses are major complications between T cell depleted allogeneic stem cell transplantation (TCD alloSCT) and donor lymphocyte infusion (DLI). The prophylactic infusion of selected donor T cells may be an effective method to restore anti-viral and anti-tumor immunity early after TCD alloSCT. In this phase I/II study, we aimed to prevent these complications by the infusion of donor-derived T cell products containing CD8+ T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) antigens (Ag), tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) 6-8 weeks after TCD alloSCT. The feasibility of multi-Ag specific T cell product generation and safety of early administration were investigated. Furthermore, clinical outcome and immune reconstitution were assessed.HLA-A2+ patients treated for a hematological malignancy with a ≥9/10 HLA-matched TCD graft from a CMV+ and/or EBV+ (un)related donor were eligible for inclusion. Donor T cells directed against HLA-A2-restricted peptides of CMV, EBV, AdV, and TAA NY-ESO-1, WT-1, RHAMM, PRAME and proteinase 3 were simultaneously isolated under GMP conditions in 1 day, using MHC I-Streptamers and CliniMACS selection out of the naïve and memory T cell compartment from 2*109 PBMC. Depending on patient/donor HLA-typing, additional MHC I-Streptamers targeting viral peptides in HLA-A1, -A24, -B7, or -B8 were added to the procedure, as well as the HLA-A2/HA-1h MHC I-Streptamer in case of MiHA disparity in the GVL direction. The incidence of GVHD, mortality, disease relapses and viral reactivations was monitored until 6 months after alloSCT. In addition, follow-up samples were stained with tetramers to analyze in vivo reconstitution of target-Ag specific T cells.Products were generated for 27/28 included patients that showed stable engraftment after TCD alloSCT; 1 patient died early after alloSCT. All donors were EBV+, 14/27 donors were CMV+. 26/27 products were successfully generated and contained a median of 5.2*106 cells (range 0.4 - 26.5*106) with a median purity of target-Ag specific T cells within the CD3+ compartment of 80.4% (range 46.0-99.9). Target-Ag specific T cells consisted for 99% of virus-specific memory T cells, while the remaining 1% included TAA, MiHA and naïve virus-specific T cells. 24 patients received their product (median 58 days after alloSCT; range 51-107) without infusion related complications; in 2 patients the infusion was cancelled due to acute skin GVHD on the day of infusion. During follow-up, 1 patient experienced skin GVHD requiring immunosuppressive therapy. One patient died due to complications of a nephrotic syndrome, probably unrelated to product infusion. Four patients showed disease relapse and were treated accordingly; coinciding expansion of TAA- or MiHA-specific T cells was not observed. None of the patients experienced AdV reactivations; in 2 patients AdV-specific T cells expanded after infusion. Four patients had detectable EBV loads; in one of these patients the EBV reactivation progressed to EBV-PTLD and he was treated with rituximab followed by DLI. In 2 patients, the rise in EBV load led to expansion of EBV specific T cells in vivo. 4/5 CMV+ patients with a CMV- donor had ongoing CMV reactivations at the moment of product infusion, CMV specific T cells could already be detected just before product infusion and reactivations were cleared within 7-84 days. Of the 8 CMV+ patients with a CMV+ donor, 3 patients remained free of CMV reactivations, 4 had already a reactivation at the moment of product infusion and 1 patient developed a CMV reactivation during follow up. One patient had progressed to CMV pneumonia within 2 weeks after infusion, requiring ganciclovir. In all 5 CMV reactivating patients, T cells directed against 2-3 CMV-specific epitopes expanded significantly and viral loads declined. TCR sequencing of the CDR3 region illustrated in vivo persistence and expansion of target-Ag specific T cells present in the product.In conclusion, we have shown that MHC I-Streptamer isolation based generation and adoptive transfer of donor-derived multi Ag-specific T cell products is safe and feasible. Moreover, efficacy of the prophylactic infusion is illustrated by expansion of target-Ag specific T cells in patients coinciding viral reactivations and the prevention of viral complications in the majority of patients between TCD alloSCT and DLI. DisclosuresGermeroth:Juno Therapeutics: Employment, Equity Ownership.

MTORC2 deficiency in cutaneous dendritic cells potentiates CD8+ effector T cell responses and accelerates skin graft rejection.

Mechanistic target of rapamycin (mTOR) complex (mTORC)1 and mTORC2 regulate the differentiation and function of immune cells. While inhibition of mTORC1 antagonizes dendritic cell (DC) differentiation and suppresses graft rejection, the role of mTORC2 in DCs in determining host responses to transplanted tissue remains undefined. Using a mouse model in which mTORC2 was deleted specifically in CD11c+ DCs (TORC2DC-/- ), we show that the transplant of minor histocompatibility Ag (HY)-mismatched skin grafts from TORC2DC-/- donors into wild-type recipients results in accelerated rejection characterized by enhanced CD8+ T cell responses in the graft and regional lymphoid tissue [Correction added on January 9, 2019, after first online publication: in the previous sentence, major was changed to minor]. Similar enhancement of CD8+ effector T cell responses was observed in MHC-mismatched recipients of TORC2DC-/- grafts. Augmented CD8+ T cell responses were also observed in a delayed-type hypersensitivity model in which mTORC2 was absent in cutaneous DCs. These elevated responses could be ascribed to an increased T cell stimulatory phenotype of TORC2DC-/- and not to enhanced lymph node homing of the cells. In contrast, rejection of ovalbumin transgenic skin grafts in TORC2DC-/- recipients was unaffected. These findings suggest that mTORC2 in skin DCs restrains effector CD8+ T cell responses and have implications for understanding of the influence of mTOR inhibitors that target mTORC2 in transplant.

PD-L1 Prevents the Development of Autoimmune Heart Disease in Graft-versus-Host Disease.

Effector memory T cells (TEM) are less capable of inducing graft-versus-host disease (GVHD) compared with naive T cells (TN). Previously, in the TS1 TCR transgenic model of GVHD, wherein TS1 CD4 cells specific for a model minor histocompatibility Ag (miHA) induce GVHD in miHA-positive recipients, we found that cell-intrinsic properties of TS1 TEM reduced their GVHD potency relative to TS1 TN Posttransplant, TS1 TEM progeny expressed higher levels of PD-1 than did TS1 TN progeny, leading us to test the hypothesis that TEM induce less GVHD because of increased sensitivity to PD-ligands. In this study, we tested this hypothesis and found that indeed TS1 TEM induced more severe skin and liver GVHD in the absence of PD-ligands. However, lack of PD-ligands did not result in early weight loss and colon GVHD comparable to that induced by TS1 TN, indicating that additional pathways restrain alloreactive TEM TS1 TN also caused more severe GVHD without PD-ligands. The absence of PD-ligands on donor bone marrow was sufficient to augment GVHD caused by either TEM or TN, indicating that donor PD-ligand-expressing APCs critically regulate GVHD. In the absence of PD-ligands, both TS1 TEM and TN induced late-onset myocarditis. Surprisingly, this was an autoimmune manifestation, because its development required non-TS1 polyclonal CD8+ T cells. Myocarditis development also required donor bone marrow to be PD-ligand deficient, demonstrating the importance of donor APC regulatory function. In summary, PD-ligands suppress both miHA-directed GVHD and the development of alloimmunity-induced autoimmunity after allogeneic hematopoietic transplantation.

Prevention of Viral Infections after T Cell Depleted Allogeneic Stem Cell Transplantation By Infusion of Multi-Antigen Specific T Cell Products

Opportunistic viral infections and relapse are major complications in patients after T cell depleted allogeneic stem cell transplantation (TCD alloSCT). Since the application of unmodified donor lymphocyte infusion (DLI) early after alloSCT results in a high risk of graft versus host disease (GVHD), infusion of selected populations of virus-specific donor T cells can be an effective approach to safely restore anti-viral immunity early after alloSCT. As part of the EU FP7 consortium T Control, in this phase I/II study the feasibility and safety of the generation and administration of selected populations of donor-derived T cells targeting multiple antigens (Ag) is assessed. The multi Ag-specific T cell products contained T cells targeting cytomegalovirus (CMV), Epstein Bar virus (EBV) and adenovirus (AdV) as well as T cells targeting tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) to boost the graft versus leukemia (GVL) reactivity. To assess efficacy, in-vivo appearance or expansion of Ag-specific T cells, and the effect on viral reactivations and/or disease relapse was evaluated for 20 weeks after infusion until regular DLI was applied.HLA-A*02+ patients treated for a hematological malignancy with an HLA-matched TCD alloSCT from a CMV and/or EBV seropositive donor were included in this study. 6-8 weeks after alloSCT, T-cells directed against HLA-A*02-restricted peptides of CMV, EBV and AdV, and the TAA NY-eso, WT-1, RHAMM, PRAME and proteinase 3 were isolated using the reversible streptamer-nanobead technology (Juno) by cliniMACS selection out of the naïve and/or memory T cell compartment from 2*10^9 donor PBMC. Depending on the HLA-typing of the patient/donor additional streptamers targeting viral peptides in A*01, A*24, B*07 or B*08 were added to the selection procedure. In case of patient/donor MiHA disparity in the GvL direction, the HLA-A*0201/HA-1h streptamer was also added. This procedure allows purification of T cells under GMP conditions in 1 day.20 multi Ag-specific T cell products were generated of which 19 met the release criteria. These products consisted of 0.5-12*10^6 cells containing purities of 46,0-94,3% target Ag-specific CD8+ T cells within the T cell compartment. In all products CMV and/or EBVas well as AdV virus-specific memory T cells were isolated comprising 99% of the target Ag-specific CD8+ T cells, while the other 1% included the TAA and MiHA specificities or CMV specific T cells from seronegative donors. 17 products were administered without infusion-related complications or GVHD; 2 patients experienced GVHD before infusion and did not receive their product.Of the 14 patients evaluated at this stage, 13 completed the follow-up period until DLI and 1 patient died during follow-up. No product-related adverse events were reported. In all 5 CMV- patients and in 3/9 CMV+ patients no CMV reactivation and no expansion of CMV-specific T cells was observed. The other 6 CMV+ patients experienced CMV reactivations. In 2/6 patients who received the product from a CMV+ donor CMV-specific T cells were detected with tetramer analysis and CMV was cleared. From the other 4 reactivating patients with CMV- donors 3 had circulating CMV-specific T cells already at the moment of infusion, whereas in 1 patient CMV-specific T cells clearly expanded after infusion, resulting in viral clearance in all 4 patients.All 14 donors were EBV+. In 7 patients EBV reactivations were observed, which coincided in 2/7 patients with the appearance of EBV-specific T cells and subsequent clearance of the virus. In 4/7 patients EBV reactivation was cleared without clear expansion of EBV-specific T cells. However, 1 patient required treatment for an EBV-PTLD, although ultimately EBV-specific T cells appeared and EBV was cleared.In none of the patients AdV DNA loads were detected in the follow-up period, while in 1 patient expansion of AdV-specific T cells was observed.Expansion of TAA and MiHA-specific T cells could not be demonstrated in-vivo using tetramer staining. More sensitive techniques will be required to visualize these cells. 2 patients showed relapse of their malignancy before DLI infusion.In this clinical study, we have shown that the reversible streptamer technology based generation and adoptive transfer of donor-derived multi Ag-specific T cell products is feasible and safe and can be used as a strategy to prevent viral infections in the interval between TCD alloSCT and DLI. DisclosuresGermeroth:Juno Therapeutics: Employment.

T-bet Promotes Acute Graft-versus-Host Disease by Regulating Recipient Hematopoietic Cells in Mice.

Beyond its critical role in T cells, T-bet regulates the functions of APCs including dendritic cells and B cells, as well as NK cells. Given that recipient APCs are essential for priming allogeneic T cells and recipient NK or T cells are able to reject allogeneic donor cells, we evaluated the role of T-bet on the host in acute graft-versus-host disease (GVHD) using murine models of allogeneic bone marrow transplantation. T-bet(-/-) recipients developed significantly milder GVHD than their wild type counterparts in MHC-mismatched or CD4-dependent minor histocompatibility Ag-mismatched models. Allogeneic donor T cells, in particular, CD4 subset, significantly reduced IFN-γ production, proliferation and migration, and caused less injury in liver and gut of T-bet(-/-) recipients. We further observed that T-bet on recipient hematopoietic cells was primarily responsible for the donor T cell response and pathogenicity in GVHD. T-bet(-/-) dendritic cells expressed higher levels of Trail, whereas they produced lower levels of IFN-γ and IL-12/23 p40, as well as chemokine CXCL9, resulting in significantly higher levels of apoptosis, less priming, and infiltration of donor T cells. Meanwhile, NK cells in T-bet(-/-) hosts partially contribute to the decreased donor T cell proliferation. Furthermore, although T-bet on hematopoietic cells was required for GVHD development, it was largely dispensable for the graft-versus-leukemia effect. Taken together with our previous findings, we propose that T-bet is a potential therapeutic target for the control of GVHD through regulating donor T cells and recipient hematopoietic cells.

Minor histocompatibility Ags: identification strategies, clinical results and translational perspectives.

Allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusion are effective treatment modalities for various hematological malignancies. Their therapeutic effect, the graft-versus-tumor (GvT) effect, is based mainly on an alloimmune response of donor T cells directed at tumor cells, in which differences in the expression of minor histocompatibility Ags (mHags) on the cells of the patient and donor have a crucial role. However, these differences are also responsible for induction of sometimes detrimental GvHD. As relapse and development of GvHD pose major threats for a large proportion of allotransplanted patients, additional therapeutic strategies are required. To augment the GvT response without increasing the risk of GvHD, specific mHag-directed immunotherapeutic strategies have been developed. Over the past years, much effort has been put into the identification of therapeutically relevant mHags to enable these strategies for a substantial proportion of patients. Currently, the concept of mHag-directed immunotherapy is tested in clinical trials on feasibility, safety and efficacy. In this review, we will summarize the recent developments in mHag identification and the clinical data on mHag-specific immune responses and mHag-directed therapies in patients with hematological malignancies. Finally, we will outline the current challenges and future prospectives in the field.

UGT2B17 minor histocompatibility mismatch and clinical outcome after HLA-identical sibling donor stem cell transplantation

Minor histocompatibility Ags (mHags) have been implicated in the pathogenesis of GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Uridine diphospho-glucuronosyltransferase 2B17 (UGT2B17) gene deletion may act as a mHag and its association with acute GVHD (aGVHD) has been described. We retrospectively studied the clinical impact of a UGT2B17 mismatch in a cohort of 1127 patients receiving a HSCT from an HLA-identical sibling donor. UGT2B17 mismatch was present in 69 cases (6.1%). Incidence of severe aGVHD was higher in the UGT2B17 mismatched pairs (22.7% vs 14.6%), but this difference was not statistically significant (P: 0.098). We did not detect differences in chronic GVHD, overall survival, relapse-free survival, transplant-related mortality or relapse. Nevertheless, when we analyzed only those patients receiving grafts from a male donor (616 cases), aGVHD was significantly higher in the UGT2B17 mismatched group (25.1% vs 12.8%; P: 0.005) and this association was confirmed by the multivariate analysis (P: 0.043; hazard ratio: 2.16, 95% confidence interval: 1.03-4.57). Overall survival was worse for patients mismatched for UGT2B17 (P: 0.005). We conclude that UGT2B17 mismatch has a negative clinical impact in allogeneic HSCT from HLA-identical sibling donors only when a male donor is used. These results should be confirmed by other studies.

HY-Specific Induced Regulatory T Cells Display High Specificity and Efficacy in the Prevention of Acute Graft-versus-Host Disease.

Naturally derived regulatory T cells (Tregs) may prevent graft-versus-host disease (GVHD) while preserving graft-versus-leukemia (GVL) activity. However, clinical application of naturally derived regulatory T cells has been severely hampered by their scarce availability and nonselectivity. To overcome these limitations, we took alternative approaches to generate Ag-specific induced Tregs (iTregs) and tested their efficacy and selectivity in the prevention of GVHD in preclinical models of bone marrow transplantation. We selected HY as a target Ag because it is a naturally processed, ubiquitously expressed minor histocompatibility Ag (miHAg) with a proven role in GVHD and GVL effect. We generated HY-specific iTregs (HY-iTregs) from resting CD4 T cells derived from TCR transgenic mice, in which CD4 cells specifically recognize HY peptide. We found that HY-iTregs were highly effective in preventing GVHD in male (HY(+)) but not female (HY(-)) recipients using MHC II-mismatched, parent→F1, and miHAg-mismatched murine bone marrow transplantation models. Interestingly, the expression of target Ag (HY) on the hematopoietic or nonhematopoietic compartment alone was sufficient for iTregs to prevent GVHD. Furthermore, treatment with HY-iTregs still preserved the GVL effect even against pre-established leukemia. We found that HY-iTregs were more stable in male than in female recipients. Furthermore, HY-iTregs expanded extensively in male but not female recipients, which in turn significantly reduced donor effector T cell expansion, activation, and migration into GVHD target organs, resulting in effective prevention of GVHD. This study demonstrates that iTregs specific for HY miHAgs are highly effective in controlling GVHD in an Ag-dependent manner while sparing the GVL effect.

Cutting Edge: Roles for Batf3-Dependent APCs in the Rejection of Minor Histocompatibility Antigen-Mismatched Grafts.

In transplantation, a major obstacle for graft acceptance in MHC-matched individuals is the mismatch of minor histocompatibility Ags. Minor histocompatibility Ags are peptides derived from polymorphic proteins that can be presented by APCs on MHC molecules. The APC subtype uniquely responsible for the rejection of minor Ag-mismatched grafts has not yet been identified. In this study, we examined graft rejection in three mouse models: 1) mismatch of male-specific minor Ags, 2) mismatch of minor Ags distinct from male-specific minor Ags, and 3) skin transplant. This study demonstrates that in the absence of pathogen-associated molecular patterns, Batf3-dependent dendritic cells elicit the rejection of cells and grafts expressing mismatched minor Ags. The implication of our findings in clinical transplantation may be significant, as minor Ag reactivity has been implicated in the pathogenesis of multiple allograft tissues.

Altered Peptide Ligands Revisited: Vaccine Design through Chemically Modified HLA-A2–Restricted T Cell Epitopes

Virus or tumor Ag-derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.

Rejection of Leukemic Cells Requires Antigen-Specific T Cells with High Functional Avidity

In a context where injection of antigen (Ag)-specific T cells probably represents the future of leukemia immunotherapy, identification of optimal target Ags is crucial. We therefore sought to discover a reliable marker for selection of the most potent Ags. To this end, (1) we immunized mice against 8 individual Ags: 4 minor histocompatibility Ags (miHAs) and 4 leukemia-associated Ags (LAAs) that were overexpressed on leukemic relative to normal thymocytes; (2) we assessed their ability to reject EL4 leukemic cells; and (3) we correlated the properties of our Ags (and their cognate T cells) with their ability to induce protective antileukemic responses. Overall, individual miHAs instigated more potent antileukemic responses than LAAs. Three features had no influence on the ability of primed T cells to reject leukemic cells: (1) MHC-peptide affinity; (2) the stability of MHC-peptide complexes; and (3) epitope density at the surface of leukemic cells, as assessed using mass spectrometry. The cardinal feature of successful Ags is that they were recognized by high-avidity CD8 T cells that proliferated extensively in vivo. Our work suggests that in vitro evaluation of functional avidity represents the best criterion for selection of Ags, which should be prioritized in clinical trials of leukemia immunotherapy.

Discovery of T Cell Epitopes Implementing HLA-Peptidomics into a Reverse Immunology Approach

T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry-based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.

Graft-versus-Host Disease Impairs Vaccine Responses through Decreased CD4+ and CD8+ T Cell Proliferation and Increased Perforin-Mediated CD8+ T Cell Apoptosis

Tumor-targeted vaccines represent a strategy to enhance the graft-versus-leukemia effect after allogeneic blood and marrow transplantation (BMT). We have previously shown that graft-versus-host disease (GVHD) can negatively impact quantitative responses to vaccines. Using a minor histocompatibility Ag-mismatched BMT (B6 → B6 × C3H.SW) followed by adoptive transfer of HY-specific T cells and HY-expressing dendritic cells, we assessed whether GVHD induced by donor lymphocyte infusion (DLI) affects the persistence, proliferation, and survival of vaccine-responding, nonalloantigen reactive T cells. Both CD8(+) and CD4(+) HY-specific T cells undergo less vaccine-driven proliferation in allogeneic recipients with GVHD. Although vaccine-responding CD8(+) T cells show decreased IFN-γ and CD107a production, CD4(+) T cells exhibit increased programmed death 1 and T cell Ig mucin-like domain 3 expression. In addition, the degree of apoptosis in vaccine-responding CD8(+) T cells was higher in the presence of GVHD, but there was no difference in CD4(+) T cell apoptosis. Using Fas ligand-deficient or TRAIL-deficient DLI had no impact on apoptosis of HY-specific T cells. However, perforin-deficient alloreactive DLI induced significantly less apoptosis of vaccine-responding CD8(+) T cells and resulted in enhanced tumor protection. Thus, diminished vaccine responses during GVHD result from impaired proliferation of CD8(+) and CD4(+) T cells responding to vaccination, with an additional contribution from perforin-mediated CD8(+) T cell apoptosis. These results provide important insights toward optimizing vaccine responses after allogeneic BMT.

Transmaternal cell flow leads to antigen-experienced cord blood

AbstractUmbilical cord blood (UCB) is used for HSCT. It is known that UCB can comprise Ag-specific T cells. Here we question whether solely transmaternal cell flow may immunize UCB. Twenty-three female UCB samples were collected from healthy mothers and analyzed for minor histocompatibility Ag HY-specific responses. Forty-two of 104 tetramerpos T-cell clones, isolated from 16 of 17 UCB samples, showed male-specific lysis in vitro. Male microchimerism was present in 6 of 12 UCB samples analyzed. In conclusion, female UCB comprises HY-specific cytotoxic T cells. The immunization is presumably caused by transmaternal cell flow of male microchimerism present in the mother. The presence of immune cells in UCB that are not directed against maternal foreign Ags is remarkable and may explain the reported clinical observation of improved HSCT outcome with younger sibling donors.

Two Preferentially Expressed Proteins Protect Vascular Endothelial Cells from an Attack by Peptide-Specific CTL

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.

Induction of acute GVHD by sex-mismatched H-Y antigens in the absence of functional radiosensitive host hematopoietic–derived antigen-presenting cells

AbstractIt is currently thought that acute GVHD cannot be elicited in the absence of Ag presentation by radiosensitive host hematopoietic-derived APCs after allogeneic BM transplantation. Because clinical data suggest that sex-mismatched H-Y Ags may be important minor histocompatibility Ags for GVH responses, we directly tested their relevance and ability to initiate GVHD when presented by either the hematopoietic- (host or donor) or the nonhematopoietic-derived APCs. H-Y minor Ag incompatibility elicited both CD4+ and CD8+ T-cell driven GVHD lethality. Studies with various well-established BM chimera recipients, in contrast to the current views, have reported that in the absence of functional radiosensitive host hematopoietic-derived APCs, H-Y Ag presentation by either the donor hematopoietic-derived or the host nonhematopoietic-derived APCs is sufficient for inducing GVHD. Our data further suggest that infusion of sufficient numbers of alloreactive donor T cells will induce GVHD in the absence of radiosensitive host hematopoietic-derived APCs.

Novel myeloma-associated antigens revealed in the context of syngeneic hematopoietic stem cell transplantation

AbstractTargets of curative donor-derived graft-versus-myeloma (GVM) responses after allogeneic hematopoietic stem cell transplantation (HSCT) remain poorly defined, partly because immunity against minor histocompatibility Ags (mHAgs) complicates the elucidation of multiple myeloma (MM)–specific targets. We hypothesized that syngeneic HSCT would facilitate the identification of GVM-associated Ags because donor immune responses in this setting should exclusively target unique tumor Ags in the absence of donor-host genetic disparities. Therefore, in the present study, we investigated the development of tumor immunity in an HLA-A0201+ MM patient who achieved durable remission after myeloablative syngeneic HSCT. Using high-density protein microarrays to screen post-HSCT plasma, we identified 6 Ags that elicited high-titer (1:5000-1:10 000) Abs that correlated with clinical tumor regression. Two Ags (DAPK2 and PIM1) had enriched expression in primary MM tissues. Both elicited Ab responses in other MM patients after chemotherapy or HSCT (11 and 6 of 32 patients for DAPK2 and PIM1, respectively). The index patient also developed specific CD8+ T-cell responses to HLA-A2–restricted peptides derived from DAPK2 and PIM1. Peptide-specific T cells recognized HLA-A2+ MM-derived cell lines and primary MM tumor cells. Coordinated T- and B-cell immunity develops against MM-associated Ags after syngeneic HSCT. DAPK1 and PIM1 are promising target Ags for MM-directed immunotherapy.

Primary Vascularization of the Graft Determines the Immunodominance of Murine Minor H Antigens during Organ Transplantation

Grafts can be rejected even when matched for MHC because of differences in the minor histocompatibility Ags (mH-Ags). H4- and H60-derived epitopes are known as immunodominant mH-Ags in H2(b)-compatible BALB.B to C57BL/6 transplantation settings. Although multiple explanations have been provided to explain immunodominance of Ags, the role of vascularization of the graft is yet to be determined. In this study, we used heart (vascularized) and skin (nonvascularized) transplantations to determine the role of primary vascularization of the graft. A higher IFN-γ response toward H60 peptide occurs in heart recipients. In contrast, a higher IFN-γ response was generated against H4 peptide in skin transplant recipients. Peptide-loaded tetramer staining revealed a distinct antigenic hierarchy between heart and skin transplantation: H60-specific CD8(+) T cells were the most abundant after heart transplantation, whereas H4-specific CD8(+) T cells were more abundant after skin graft. Neither the tissue-specific distribution of mH-Ags nor the draining lymph node-derived dendritic cells correlated with the observed immunodominance. Interestingly, non-primarily vascularized cardiac allografts mimicked skin grafts in the observed immunodominance, and H60 immunodominance was observed in primarily vascularized skin grafts. However, T cell depletion from the BALB.B donor prior to cardiac allograft induces H4 immunodominance in vascularized cardiac allograft. Collectively, our data suggest that immediate transmigration of donor T cells via primary vascularization is responsible for the immunodominance of H60 mH-Ag in organ and tissue transplantation.

Prediction of Reactivity to Noninherited Maternal Antigen in MHC-Mismatched, Minor Histocompatibility Antigen-Matched Stem Cell Transplantation in a Mouse Model

The immunologic effects of developmental exposure to noninherited maternal Ags (NIMAs) are quite variable. Both tolerizing influence and inducing alloreaction have been observed on clinical transplantation. The role of minor histocompatibility Ags (MiHAs) in NIMA effects is unknown. MiHA is either matched or mismatched in NIMA-mismatched transplantation because a donor of the transplantation is usually limited to a family member. To exclude the participation of MiHA in a NIMA effect for MHC (H-2) is clinically relevant because mismatched MiHA may induce severe alloreaction. The aim of this study is to understand the mechanism of NIMA effects in MHC-mismatched, MiHA-matched hematopoietic stem cell transplantation. Although all offsprings are exposed to the maternal Ags, the NIMA effect for the H-2 Ag was not evident. However, they exhibit two distinct reactivities, low and high responder, to NIMA in utero and during nursing depending on the degree of maternal microchimerism. Low responders survived longer with less graft-versus-host disease. These reactivities were correlated with Foxp3 expression of peripheral blood CD4(+)CD25(+) cells after graft-versus-host disease induction and the number of IFN-γ-producing cells stimulated with NIMA pretransplantation. These observations are clinically relevant and suggest that it is possible to predict the immunological tolerance to NIMA.

The Immunological Impact of Genetic Drift in the B10.BR Congenic Inbred Mouse Strain

The MHC-matched, minor histocompatibility Ag (miHA)-mismatched B10.BR-->CBA strain combination has been used to elucidate the immunobiology of graft-vs-host disease (GVHD) following allogeneic bone marrow transplantation. Studies conducted in the 1980s had established that B10.BR CD8+ T cells were capable of mediating GVHD in the absence of CD4+ T cells, and that CD4+ T cells were unable to induce lethal disease. In more recent studies with this GVHD model, we detected etiological discrepancies with the previously published results, which suggested that genetic drift might have occurred within the B10.BR strain. In particular, there was increased allorecognition of CBA miHA by B10.BR CD4+ T cells, as determined by both TCR Vbeta spectratype analysis and the induction of lethal GVHD in CBA recipients. Additionally, alloreactivity was observed between the genetically drifted mice (B10.BR/Jdrif) and mice rederived from frozen embryos of the original strain (B10.BR/Jrep) using Vbeta spectratype analysis and IFN-gamma ELISPOT assays, suggesting that new miHA differences had arisen between the mice. Furthermore, T cell-depleted B10.BR/Jdrif bone marrow cells were unable to provide long-term survival following either allogeneic or syngeneic bone marrow transplantation. Gene expression analysis revealed several genes involved in hematopoiesis that were overexpressed in the lineage-negative fraction of B10.BR/Jdrif bone marrow, as compared with B10.BR/Jrep mice. Taken together, these results suggest that genetic drift in the B10.BR strain has significantly impacted the immune alloreactive response in the GVHD model by causing altered expression of miHA and diminished capacity for survival following transplantation into lethally irradiated recipients.