Cornelis A.M. van Bergen

High-Throughput Characterization of 10 New Minor Histocompatibility Antigens by Whole Genome Association Scanning

Patients with malignant diseases can be effectively treated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Polymorphic peptides presented in HLA molecules, the so-called minor histocompatibility antigens (MiHA), play a crucial role in antitumor immunity as targets for alloreactive donor T cells. Identification of multiple MiHAs is essential to understand and manipulate the development of clinical responses after allo-SCT. In this study, CD8+ T-cell clones were isolated from leukemia patients who entered complete remission after allo-SCT, and MiHA-specific T-cell clones were efficiently selected for analysis of recognition of a panel of EBV-transformed B cells positive for the HLA restriction elements of the selected T-cell clones. One million single nucleotide polymorphisms (SNP) were determined in the panel cell lines and investigated for matching with the T-cell recognition data by whole genome association scanning (WGAs). Significant association with 12 genomic regions was found, and detailed analysis of genes located within these genomic regions revealed SNP disparities encoding polymorphic peptides in 10 cases. Differential recognition of patient-type, but not donor-type, peptides validated the identification of these MiHAs. Using tetramers, distinct populations of MiHA-specific CD8+ T cells were detected, demonstrating that our WGAs strategy allows high-throughput discovery of relevant targets in antitumor immunity after allo-SCT.

Identification of 4 new HLA-DR-restricted minor histocompatibility antigens as hematopoietic targets in antitumor immunity

Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+ T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I-restricted mHags have been identified, the first 2 autosomal HLA-class II-restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II-restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+ T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

HLA class II upregulation during viral infection leads to HLA-DP–directed graft-versus-host disease after CD4+ donor lymphocyte infusion

CD8+ T cell-depleted (TCD) donor lymphocyte infusion (DLI) after TCD allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with a reduced risk of graft-versus-host disease (GVHD) while preserving conversion to donor hematopoiesis and antitumor immunity, providing a rationale for exploring CD4+ T cell-based immunotherapy for hematologic malignancies. Here, we analyzed the clinical course and specificity of T cell immune responses in 2 patients with acute myeloid leukemia (AML) who converted to full-donor chimerism but developed severe acute GVHD after prophylactic CD4+ DLI after 10/10-HLA-matched, but HLA-DPB1-mismatched TCD-alloSCT. Clonal analysis of activated T cells isolated during GVHD demonstrated allo-reactivity exerted by CD4+ T cells directed against patient-mismatched HLA-DPB1 molecules on hematopoietic cells and skin-derived fibroblasts only when cultured under inflammatory conditions. At the time of CD4+ DLI, both patients contained residual patient-derived T cells, including cytomegalovirus (CMV)-specific T cells as a result of CMV reactivations. Once activated by CMV antigens, these CMV-specific T cells could stimulate HLA-DPB1-specific CD4+ T cells, which in turn could target nonhematopoietic tissues in GVHD. In conclusion, our data demonstrate that GVHD after HLA-DPB1-mismatched CD4+ DLI can be mediated by allo-reactive HLA-DPB1-directed CD4+ T cells and that ongoing viral infections inducing HLA class II expression on nonhematopoietic cells may increase the likelihood of GVHD development. This trial is registered at http://www.controlled-trials.com/ISRCTN51398568/LUMC as #51398568.

Allo-HLA reactive T cells inducing graft versus host disease are single peptide specific

T-cell alloreactivity directed against non-self-HLA molecules has been assumed to be less peptide specific than conventional T-cell reactivity. A large variation in degree of peptide specificity has previously been reported, including single peptide specificity, polyspecificity, and peptide degeneracy. Peptide polyspecificity was illustrated using synthetic peptide-loaded target cells, but in the absence of confirmation against endogenously processed peptides this may represent low-avidity T-cell reactivity. Peptide degeneracy was concluded based on recognition of Ag-processing defective cells. In addition, because most investigated alloreactive T cells were in vitro activated and expanded, the previously determined specificities may have not been representative for alloreactivity in vivo. To study the biologically relevant peptide specificity and avidity of alloreactivity, we investigated the degree of peptide specificity of 50 different allo-HLA-reactive T-cell clones which were activated and expanded in vivo during GVHD. All but one of the alloreactive T-cell clones, including those reactive against Ag-processing defective T2 cells, recognized a single peptide allo-HLA complex, unique for each clone. Down-regulation of the expression of the recognized Ags using silencing shRNAs confirmed single peptide specificity. Based on these results, we conclude that biologically relevant alloreactivity selected during in vivo immune response is peptide specific.

Lectins from opportunistic bacteria interact with acquired variable-region glycans of surface immunoglobulin in follicular lymphoma

B-cell antigen receptor (BCR) expression is a key feature of most B-cell lymphomas, but the mechanisms of BCR signal induction and the involvement of autoantigen recognition remain unclear. In follicular lymphoma (FL) B cells, BCR expression is retained despite a chromosomal translocation that links the antiapoptotic gene BCL2 to the regulatory elements of immunoglobulin genes, thereby disrupting 1 heavy-chain allele. A remarkable feature of FL-BCRs is the acquisition of potential N-glycosylation sites during somatic hypermutation. The introduced glycans carry mannose termini, which create potential novel binding sites for mannose-specific lectins. Here, we investigated the effect of N-linked variable-region glycosylation for BCR interaction with cognate antigen and with lectins of different origins. N-glycans were found to severely impair BCR specificity and affinity to the initial cognate antigen. In addition, we found that lectins from Pseudomonas aeruginosa and Burkholderia cenocepacia bind and stimulate FL cells. Human exposure to these bacteria can occur by contact with soil and water. In addition, they represent opportunistic pathogens in susceptible hosts. Understanding the role of bacterial lectins might elucidate the pathogenesis of FL and establish novel therapeutic approaches.

Patient HLA-DP-specific CD4+ T cells from HLA-DPB1-mismatched donor lymphocyte infusion can induce graft-versus-leukemia reactivity in the presence or absence of graft-versus-host disease.

Clinical studies have demonstrated that HLA-DPB1-mismatched allogeneic stem cell transplantation (allo-SCT) is associated with a decreased risk of disease relapse and an increased risk of graft-versus-host disease (GVHD) compared with HLA-DPB1-matched SCT. In T cell-depleted allo-SCT, mismatching of HLA-DPB1 was not associated with an increased risk of severe GVHD, but a significant decreased risk of disease relapse was still observed. To investigate whether patient HLA-DP-specific CD4(+) T cell responses were frequently induced after T cell-depleted HLA-DPB1-mismatched allo-SCT and donor lymphocyte infusion (DLI), we developed a method to screen for the presence of HLA-DP-specific CD4(+) T cells using CD137 as an activation marker and analyzed 24 patient-donor combinations. The patients suffered from various B cell malignancies, multiple myeloma, and myeloid leukemias. Patient HLA-DP-specific CD4(+) T cells were detected after DLI in 13 of 18 patients who exhibited a clinical response to DLI, compared with only 1 of 6 patients without a clinical response to DLI. Eight patients developed significant GVHD. These data show that patient HLA-DP-specific CD4(+) T cells frequently occur after HLA-DPB1-mismatched T cell-depleted allo-SCT and DLI, and are associated with graft-versus-leukemia reactivity both in the presence and absence of GVHD.

Autosomal Minor Histocompatibility Antigens: How Genetic Variants Create Diversity in Immune Targets.

Allogeneic stem cell transplantation (alloSCT) can be a curative treatment for hematological malignancies. Unfortunately, the desired anti-tumor or graft-versus-leukemia (GvL) effect is often accompanied with undesired side effects against healthy tissues known as graft-versus-host disease (GvHD). After HLA-matched alloSCT, GvL and GvHD are both mediated by donor-derived T-cells recognizing polymorphic peptides presented by HLA surface molecules on patient cells. These polymorphic peptides or minor histocompatibility antigens (MiHA) are produced by genetic differences between patient and donor. Since polymorphic peptides may be useful targets to manipulate the balance between GvL and GvHD, the dominant repertoire of MiHA needs to be discovered. In this review, the diversity of autosomal MiHA characterized thus far as well as the various molecular mechanisms by which genetic variants create immune targets and the role of cryptic transcripts and proteins as antigen sources are described. The tissue distribution of MiHA as important factor in GvL and GvHD is considered as well as possibilities how hematopoietic MiHA can be used for immunotherapy to augment GvL after alloSCT. Although more MiHA are still needed for comprehensive understanding of the biology of GvL and GvHD and manipulation by immunotherapy, this review shows insight into the composition and kinetics of in vivo immune responses with respect to specificity, diversity, and frequency of specific T-cells and surface expression of HLA–peptide complexes and other (accessory) molecules on the target cell. A complex interplay between these factors and their environment ultimately determines the spectrum of clinical manifestations caused by immune responses after alloSCT.

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Background Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease. Design and Methods We analyzed in detail CD8+ T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8+ T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens. Results Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones. Conclusions Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.

Selective graft-versus-leukemia depends on magnitude and diversity of the alloreactive T cell response

Patients with leukemia who receive a T cell–depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD.

Identification of Biological Relevant Minor Histocompatibility Antigens within the B-lymphocyte–Derived HLA-Ligandome Using a Reverse Immunology Approach

Purpose: T-cell recognition of minor histocompatibility antigens (MiHA) not only plays an important role in the beneficial graft-versus-leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) but also mediates serious GVH complications associated with allo-SCT. Using a reverse immunology approach, we aim to develop a method enabling the identification of T-cell responses directed against predefined antigens, with the goal to select those MiHAs that can be used clinically in combination with allo-SCT. Experimental Design: In this study, we used a recently developed MiHA selection algorithm to select candidate MiHAs within the HLA-presented ligandome of transformed B cells. From the HLA-presented ligandome that predominantly consisted of monomorphic peptides, 25 polymorphic peptides with a clinically relevant allele frequency were selected. By high-throughput screening, the availability of high-avidity T cells specific for these MiHA candidates in different healthy donors was analyzed. Results: With the use of MHC multimer enrichment, analyses of expanded T cells by combinatorial coding MHC multimer flow cytometry, and subsequent single-cell cloning, positive T-cell clones directed to two new MiHA: LB-CLYBL-1Y and LB-TEP1-1S could be demonstrated, indicating the immunogenicity of these two MiHAs. Conclusions: The biologic relevance of MiHA LB-CLYBL-1Y was demonstrated by the detection of LB-CLYBL-1Y–specific T cells in a patient suffering from acute myeloid leukemia (AML) that experienced an anti-leukemic response after treatment with allo-SCT. Clin Cancer Res; 21(9); 2177–86. ©2015 AACR.