Id Peptides Research Articles

Neoantigen vaccine-induced CD4 T cells confer protective immunity in a mouse model of multiple myeloma through activation of CD8 T cells against non-vaccine, tumor-associated antigens

BackgroundCancer-associated neoantigens (neoAg) derived from tumor genomic sequencing and predictive algorithms for mutated peptides are a promising basis for therapeutic vaccines under investigation. Although these are generally designed to bind...

Neoantigen Synthetic Peptide Vaccine for Multiple Myeloma Elicits T Cell Immunity in a Pre-Clinical Model

Cancer immunotherapies such as immune checkpoint inhibitors (CI) are approved for a broad range of solid tumors and selected hematologic malignancies. Clinical response to CIs has been shown to be associated with mutation burden and is predicated on the presence of T cells specific to tumor-associated antigens. Therefore, increasing the frequency of such T cell responses should increase the efficacy of anti-tumor immunity. Individualized neoantigen peptide vaccines are a promising strategy to activate tumor-specific T cell immunity. Next generation sequencing of tumor specimens is now widely available and novel computational algorithms provide the means for the prediction and selection of neoantigens.We are conducting a preclinical study of a personalized vaccine in an animal model of multiple myeloma (MM). MM is an incurable cancer of plasma cells that resides in the bone marrow (BM) and causes lytic bone disease and other end-organ damage. MM is characterized by an intermediate level of somatic mutation that may yield mutational neoantigens for vaccine design. This investigation pursued two goals: first, to determine if neoantigens are immunogenic, and second, if they induce protective immunity against tumors. To address the immunogenicity of neoantigens, we used the MOPC.315.BM myeloma cell line in the immunocompetent BALB/c mouse background. MOPC.315.BM cells mimic human disease by homing to the BM and causing osteolytic lesions. We performed whole exome sequencing and RNAseq on MOPC.315.BM cells to identify expressed somatic mutations. Neoantigens were then selected based on predicted MHC class I and II binding and expression levels, and corresponding 27-mer peptides were synthesized and tested in vivo for their immunogenicity. Mice were immunized 3 times at day 0, 14 & 21 with pools of peptides in the presence of poly (I:C) adjuvant, and splenocytes were harvested at day 28 to measure peptide-specific T cell activation by IFNg ELISpot. Of the selected peptides, 25 of 66 induce high IFNg T cell responses in the spleen. Interestingly, 88% of these responses were CD4 T cells and 12% were CD8 T cells, consistent with other preclinical and clinical studies where neoantigens predicted to bind MHC-I molecules induced mainly CD4 T cell activation. Humoral immune response specific to the peptides were analyzed by ELISA and showed that 8 of the 25 peptides induce peptide-specific IgG antibodies in the serum.To determine whether neoantigens generate tumor protective immunity, we challenged vaccinated mice with the MOPC.315.BM cell line. We used a well-characterized antigen lambda 2315 idiotype (Id) produced by MOPC cells and known to activate CD4 T cells. Vaccines using only the Id were found to be weakly immunogenic and failed to elicit protective responses. We developed a new strategy of vaccination by adding a proprietary amino acid sequence comprising a high affinity HSP70 binding motif to the idiotype peptide. Mice vaccinated at days 0, 14 & 21 with the so-modified peptide and poly-(I:C) adjuvant had 4 times higher Id-specific IFNg CD4 T cell response in the spleen compared to mice vaccinated with the unmodified Id-peptide. When challenged with MOPC.315.BM cells after vaccination with the modified Id peptide, 60% of these mice maintained non-detectable Id level in their blood and resisted tumor growth for more than 70 days while unvaccinated mice had very high levels of circulating Id and died around 30 days after tumor injection. Id-mediated cytotoxicity is being investigated in vitro to complement these results. We are conducting tumor protection experiments with the 25 immunogenic peptides identified in the vaccination experiments described in the previous section.In summary, we showed that neoantigens derived from bioinformatic analysis of tumor genome sequencing are immunogenic and that they mainly activate CD4 T cells in this setting. Additionally, our vaccination strategy using the idiotype peptide showed that CD4 T cell immunity can be protective against tumor growth. We are currently investigating the potential of the neoantigens identified in the immunogenicity study to protect mice against tumor growth and eradicate pre-existing tumors. Our findings indicate that neoantigen vaccination is very promising and that future investigations combining this strategy with CIs and/or immunomodulatory agents may lead to the efficient generation of protective anti-tumor immunity. DisclosuresBekri:Agenus Inc: Research Funding. Cho:Ludwig Institute for Cancer Research: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Genentech: Other: advisory board, Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Agenus, Inc.: Research Funding.

Abstract B37: Mechanisms of CD4 T-cell tumor immunity in a preclinical model of multiple myeloma

Abstract The majority of immune therapies for cancer have focused on generating tumor-specific CD8+ T cells that directly engage and kill tumor cells. However, CD4+ T cells play a critical yet poorly understood role in tumor immunity. Vaccines composed of MHC class II epitopes derived from mutated neoantigens (NeoAg) conferred protection against different mouse tumor types. Clinical studies of neoAg vaccines demonstrated antitumor activity, with the majority of neoAg-specific immune responses being CD4+ T cells. These tumor Ag-specific CD4+ T cells conferred protective antitumor immunity even in the absence of CD8+ cells directed against the same antigen. These findings are even more striking because most tumor cells do not express MHC class II and CD4+ T cells cannot directly kill them. In order to understand how CD4+ T cells protect against tumors in the absence of direct cytolytic activity, we used an animal model of multiple myeloma, the MOPC315.BM (MOPC) cell line derived from BalB/C background. MOPC cells do not express MHCII and produce an IgA with a unique, mutated neoAg in the lambda-2 light chain called Idiotype lambda-2.315 (Id). This Id antigen is well characterized and known to prime CD4+ T cells; however, when used as a peptide vaccine alone, it is weakly immunogenic and fails to elicit protective immune responses. We used an Id peptide sequence fused with a high-affinity HSP70 binding site delivered with poly(I:C) that elicited strong, Id-specific CD4+ T cell immunity and protection against MOPC tumor growth. No Id-specific CD8+ T cell or B cell responses were detected, and Id-specific CD4+ cells did not directly lyse MOPC cells in vitro. These findings led to the hypothesis that Id-specific CD4+ T cells confer protective immunity by promoting cross-priming of CD8+ T cells against non-Id, MOPC-associated antigens. To investigate this hypothesis, splenocytes and bone marrow cells were harvested from mice either challenged with MOPC cells only, vaccinated with the Id peptide only, or Id vaccinated and challenged with MOPC cells with protection against tumor growth. CD4+ and CD8+ T cell activity was assessed by measuring IFNg and TNFa production by intracellular cytokine staining (ICS) after eight days of in vitro restimulation with Id peptide and irradiated MOPC cells. In the absence of Id vaccination, very little Id-specific activity was observed in the spleen and bone marrow. Id vaccination alone induced CD4+ T cells in both compartments. In mice that received both Id vax and MOPC challenge, there was induction of CD8+ T cell, with a notable trend towards high levels of CD8+ activity in the tumor microenvironment (bone marrow). These CD8+ T cells were not detected when cells from the same animals were restimulated with the Id peptide alone, indicating that these CD8+ responses were against non-Id, MOPC-associated Ags. These data support a model in which Id-specific CD4+ T cells protect against tumor by providing critical helper activity to promote cross-priming of CD8+ T cell against MOPC-associated antigens. Citation Format: Selma Bekri, Reunet Rodney-Sandy, Diana Gruenstein, Bjarne Bogen, Daniel Levey, Hearn Jay Cho. Mechanisms of CD4 T-cell tumor immunity in a preclinical model of multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B37.

B cell receptor ligation induces display of V-region peptides on MHC class II molecules to T cells

The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4+ T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id+ B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4+ T cells, extrafollicular T-B cell collaboration and some germinal center formation, and production of Id+ IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.

CD4+ T cells indirectly kill tumor cells via induction of cytotoxic macrophages in mouse models.

It is well recognized that CD4+ T cells may play an important role in immunosurveillance and immunotherapy against cancer. However, the details of how these cells recognize and eliminate the tumor cells remain incompletely understood. For the past 25years, we have focused on how CD4+ T cells reject multiple myeloma cells in a murine model (MOPC315). In our experimental system, the secreted tumor-specific antigen is taken up by tumor-infiltrating macrophages that process it and present a neoepitope [a V region-derived idiotypic (Id) peptide] on MHC class II molecules to Th1 cells. Stimulated Th1 cells produce IFNγ, which activates macrophages in a manner that elicits an M1-like, tumoricidal phenotype. Through an inducible nitric oxide synthetase (iNOS)-dependent mechanism, the M1 macrophages secrete nitric oxide (NO) that diffuses into neighboring tumor cells. Inside the tumor cells, NO-derived reactive nitrogen species, including peroxynitrite, causes nitrosylation of proteins and triggers apoptosis by the intrinsic apoptotic pathway. This mode of indirect tumor recognition by CD4+ T cells operates independently of MHC class II expression on cancer cells. However, secretion of the tumor-specific antigen, and uptake and MHCII presentation on macrophages, is required for rejection. Similar mechanisms can also be observed in a B-lymphoma model and in the unrelated B16 melanoma model. Our findings reveal a novel mechanism by which CD4+ T cells kill tumor cells indirectly via induction of intratumoral cytotoxic macrophages. The data suggest that induction of M1 polarization of tumor-infiltrating macrophages, by CD4+ T cells or through other means, could serve as an immunotherapeutic strategy.

Mechanisms of CD4 T Cell Tumor Immunity in a Preclinical Model of Multiple Myeloma

The majority of immune therapies for cancer have focused on generating tumor-specific CD8+ T cells that directly engage and kill tumor cells. However, recent data demonstrate a critical role of CD4 T cells in tumor immunity. Animal models showed that MHC class II epitopes derived from mutated tumor-associated proteins (neo-antigens, neoAg) conferred protection against different mouse tumor types. Clinical studies of neoAg vaccines demonstrated anti-tumor activity, with the majority of neoAg-specific immune responses being CD4 T cells. These tumor Ag-specific CD4 T cells could confer protective anti-tumor immunity even in the absence of CD8 T cells directed against the same antigen. These findings are even more striking because most tumor cells do not express MHC class II molecules and CD4 T cells cannot directly kill tumor cells.In order to understand how CD4 T cells eliminate tumors in the absence of direct cytolytic activity and vaccine specific CD8 T cells, we used an animal model of multiple myeloma, MOPC315.BM (MOPC) cell line derived from BalB/C background, that mimics many of the features of the human disease. MOPC cells do not express MHCII and produce an IgA with a unique mutated neoAg in the l2 light chain called Idiotype l2.315 (Id). This Id antigen is well characterized and known to activate CD4 T cells, however, when used as a peptide vaccine alone, it is weakly immunogenic and fails to elicit protective immune responses. We used an Id peptide sequence fused with a high affinity HSP70 binding site that elicited strong Id-specific CD4 T cell IFNg responses that protect against MOPC tumor growth. No Id-specific CD8 T cell or B cell responses were detected, and Id-specific CD4 cells did not directly lyse MOPC cells in vitro. These CD4 responses were specific to the neoAg, as they did not cross react with the wild type peptide fused to the same HSP70 binding sequence. These findings lead to the hypothesis that Id-specific CD4 T cells confer protective immunity by promoting cross-priming of CD8 T cells against non-Id, MOPC-associated antigens.To investigate this hypothesis, splenocytes and bone marrow cells were harvested from mice either challenged with MOPC cells only (No vax + MOPC), vaccinated with the Id peptide only (Id vax+No MOPC) or Id vaccinated and challenged with MOPC cells (Id vax + MOPC). CD4 and CD8 T cell activation was assessed by measuring IFNg and TNFa production by intracellular cytokine staining (ICS) after eight days of in vitro restimulation with Id peptide and irradiated MOPC cells. As shown in fig.1, in the absence of Id vaccination, no or very little immune activation was observed in the spleen and bone marrow. Id vaccination alone induced CD4 T cells in both compartments. In mice that received both Id vax and MOPC challenge, there was induction of CD8 T cell, with a notable trend towards high levels of CD8 activity in the tumor microenvironment (bone marrow). These CD8 T cells were not detected when cells from the same animals were restimulated with the Id peptide alone, indicating that these CD8 responses were against non-Id, MOPC-associated Ags (data not shown). All together these data support a model in which Id-specific CD4 T cell provide helper activity to promote cross-priming of CD8 T cell against MOPC-associated Ags only in the presence of tumor challenge. These responses are present both systemically and in the tumor site. We are currently studying the specificity of these CD8 T cells and the potential role of innate immune cells such as NK cells and macrophages in the anti-tumor immune response initiated by Id-specific CD4 T cells. These findings will help better understand the mechanisms of CD4 mediated anti-tumor immunity and promote the development of combination immunotherapy strategies to improve the efficacy of vaccines targeting CD4 antigens. [Display omitted] DisclosuresBekri:Agenus Inc: Research Funding. Levey:Agenus Inc: Employment, Equity Ownership. Cho:BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Agenus Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; J & J: Consultancy; Genentech Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Idiotype-specific Th cells support oligoclonal expansion of anti-dsDNA B cells in mice with lupus.

Systemic lupus erythematosus (SLE) is marked by a Th cell-dependent B cell hyperresponsiveness, with frequent germinal center reactions and hypergammaglobulinemia. The specificity of Th cells in lupus remains unclear, but B cell Ids have been suggested. A hallmark is the presence of anti-dsDNA, mutated IgG autoantibodies with a preponderance of arginines in CDR3 of the Ig variable H chain (IgVH). B cells can present V region-derived Id peptides on their MHC class II molecules to Id-specific Th cells. We show that Id-specific Th cells support the proliferation of anti-dsDNA Id(+) B cells in mice suffering from systemic autoimmune disease with SLE-like features. Mice developed marked clonal expansions of B cells; half of the IgVH sequences were clonally related. Anti-dsDNA B cells made up 40% of B cells in end-stage disease. The B cells expressed mutated IgVH with multiple arginines in CDR3. Hence, Id-driven T cell-B cell collaboration supported the production of classical anti-dsDNA Abs, recapitulating the characteristics of such Abs in SLE. The results support the concept that Id-specific Th cells may trigger the development of SLE and suggest that manipulation of the Id-specific T cell repertoire could play a role in treatment.

Escape of myeloma cells from CD4 T-cell-driven immunoprotection (P2015)

Abstract Background: CD4+ T cells can mediate efficient immunosurveillance against MHC class II-negative cancer cells through indirect presentation of tumor-specific antigens on antigen-presenting cells (APCs). A CD4+ T cell response against an epitope on the immunoglobulin light chain of the Id peptide of MOPC315 myeloma cells can mediate an antitumor immune response that induces long term tumor dormancy, followed by eventual tumor escape. Interestingly, this escape occurs despite continued secretion of the tumor-specific antigen. Objective: To identify the immunoediting mechanisms underlying the loss of T-cell-mediated immunoprotection against MOPC315 myeloma cells Method: Mice injected with fluorescently labeled MOPC315 tumor cells were monitored using in vivo imaging. Parental and escape tumor cell clones were obtained, and gene- and protein expression characterized. Results: Escape tumor cells secrete an intact M315 Id peptide, but secretion of free light chains is selectively down-regulated. This limits exposure of the inciting antigen in a form that permits efficient antigen display by tumor-infiltrating APCs, despite preserved T cell reactivity within draining lymph nodes. Our study defines a novel mechanism of tumor immunoevasion by modified secretion of a natural tumor-specific antigen, which emphasizes the importance of antigen presentation within the tumor microenvironment, and has implications for the design of effective CD4+ T cell-based immunotherapy.

CD4+ T cell-mediated protection against tumors: no bystander killing (P2114)

Abstract Background: MHC class II-restricted CD4+ T cells reactive against the Id peptide of MHC class II-negative MOPC315 murine myeloma cells can protect mice against injected tumors. This protection occurs via indirect recognition of secreted tumor-specific antigen, presented on MHC class II-molecules of tumor-infiltrating CD11b+ cells. Activated Id-specific Th1 cells produce IFNγ that activate macrophages which become cytotoxic to tumor cells. We wanted to assay the ability of such CD4+/M1 macrophage mediated protection to prevent the outgrowth of antigen-loss tumor variants. Methods: Id-specific TCR-transgenic SCID mice were challenged with a mixed population of Id-secreting and non-secreting MOPC315 cells, differentially labeled with fluorescent proteins. Tumor growth was monitored by in vivo imaging. Tumor-infiltrating macrophages were assayed for their ability to inhibit tumor growth. Results: In the presence of a large excess of antigen-secreting tumor cells, outgrowth of antigen loss-variants occurred with no evidence of bystander killing. A predominance of alternatively activated macrophages was seen in areas dominated by antigen loss-variants, whereas classic activation was the dominant phenotype in areas populated by regressing Id-secreting tumor cells. In vitro, growth of antigen loss-variants was completely inhibited by the provision of the tumor-specific antigen. This data suggests strict spatial requirements of antigen for effective macrophage-mediated protection.

SH2D2A Modulates T Cell Mediated Protection to a B Cell Derived Tumor in Transgenic Mice

BackgroundT cell specific adapter protein (TSAd), encoded by the SH2D2A gene, modulates signaling downstream of the T cell receptor (TCR). Young, unchallenged SH2D2A-deficient C57BL/6 mice exhibit a relatively normal immune phenotype. To address whether SH2D2A regulates physiologic immune responses, SH2D2A-deficient TCR-transgenic BALB/c mice were generated. The transgenic TCR recognizes a myeloma-derived idiotypic (Id) peptide in the context of the major histocompatibility complex (MHC) class II molecule I-Ed, and confers T cell mediated resistance to transplanted multiple myeloma development in vivo.Principal FindingsThe immune phenotype of SH2D2A-deficient C57BL/6 and BALB/c mice did not reveal major differences compared to the corresponding wild type mice. When challenged with myeloma cells, Id-specific TCR-transgenic BALB/c mice lacking SH2D2A displayed increased resistance towards tumor development. Tumor free TCR-transgenic SH2D2A-deficient mice had higher numbers of Id-specific single positive CD4+ thymocytes compared to TCR-transgenic wild-type mice.ConclusionOur results suggest a modulatory role for SH2D2A in T cell mediated immune surveillance of cancer. However, it remains to be established whether its effect is T-cell intrinsic. Further studies are required to determine whether targeting SH2D2A function in T cells may be a potential adjuvant in cancer immunotherapy.

Targeting Multiple Myeloma with Human Ig Light Chain Specific Cytotoxic T Lymphocytes

AbstractAbstract 3018The variable regions of Ig expressed by malignant B cells can serve as a tumor specific antigen. Clinical trials of idiotype (Id) vaccines have demonstrated humoral responses and prolonged remission duration in a recent phase III trial of follicular lymphoma patients in the first remission (Schuster et al, J Clin Oncol 27: 793S, 2009). However, the potentially immunogenic epitopes derived from Ig that stimulate CD8+ T cell immunity have been incompletely characterized. Here, we identified nine out of 14 candidate peptides derived from the Ig L-chain variable region of the human U266 myeloma line, which generated cytotoxic T lymphocytes (CTLs) from 53 HLA A2+ normal donors. These CTLs lines, as well as CTLs line isolated from myeloma patients by stimulation with autologous L-chain Id peptides, specifically produced IFN-γ in response to peptide-pulsed T2 cells and lysed U266 and autologous myeloma cell targets, respectively, but not normal blood B cells. Lysis was HLA class I-dependent, suggesting that primary myeloma cells express Id peptides on the cell surface in combination with HLA molecules. Nine CD8+ Id peptide-specific T-cell clones exhibited the effector memory phenotype and the ability of these T cell clones to eliminate U266 tumor in immune deficient mice is being tested. Finally, sequence analysis revealed shared T-cell epitopes in both framework and CDR regions of the U266 L-chain. CTLs generated against a shared U266 epitope lysed patient-derived myeloma cells expressing the shared sequence, suggesting a strategy to overcome the limitation of patient-specific Id vaccine manufacture. Our data identified novel immunogenic Id L-chain T-cell determinants and suggests that, unlike previously described Ig heavy chains, these sequences harbor common T-cell epitopes that may provide the rationale for shared Id vaccines. Disclosures:No relevant conflicts of interest to declare.

HLA-Dependent Immune Selection Pressure on the Idiotype Is Predominantly Driven by Somatic Hypermutation In Malignant Lymphoma but Also Modulates Vh Usage In Normal B Cells

AbstractAbstract 3132The idiotype (Id) of malignant lymphomas is a tumor-specific antigen and has been evaluated for active immunotherapy predominantly in follicular lymphoma (FL). We have previously described “reverse immunology” evidence that HLA class I-dependent and hence presumably T-cell mediated selection pressure restricts the growth of malignant FL clones with comparatively highly immunogenic Ids (Strothmeyer et al., Blood, epub 2010). This effect was mostly attributable to the Id CDRs. We therefore hypothesized that somatic hypermutation, a constitutively active process in FL, is the mechanism that creates immunogenic Id epitopes and enables T-cell mediated immunosurveillance. To test this hypothesis, we analyzed Id of FL that utilized the same Vh gene but arose in individuals with different HLA types (IGHV3-23*01, n=9 patients: IGHV3-7*01, n=3). For every represented HLA type, an “immunogenicity landscape” mapping was performed by plotting the ten most immunogenic peptides of the germ-line Vh sequence in comparison to FL Id peptides in the order of their HLA binding strength as predicted by the BIMAS algorithm for the respective patient. Neither changes in the order of peptides nor major differences in their binding scores were observed. Parallel plotting of Vh-related Ids from different HLA types revealed novel peptides with higher binding scores within and in addition to the ten highest ranking epitopes (Figures). Comparisons of these peptides with the germ-line and the index patient's sequence directly demonstrated that somatic hypermutation creates peptides that would have strongly bound to HLA type of the index patient but were immunologically neutral in the patient of origin. Occurrence of such highly immunogenic peptides by somatic hypermutation would hence not be tolerated by an FL patient's immune system. To explore whether this mechanism also operates in other lymphoma types, we analyzed predicted HLA-binding Id peptides from mantle cell (n=20) and marginal zone (n=12) lymphomas. Like in FL, the median sum of the 20 peptides with the highest BIMAS scores were to various degrees less immunogenic on the autologous HLA type than on allogeneic HLA types (ratio of autologous to allogeneic BIMAS sum scores: FL: 0.63; MCL: 0.78; MZL: 0.87). In subanalyses of the CDR, these differences were observed only in FL and MCL but not in MZL (CDR1: FL 0.40, MCL 0.55. MZL 1.39; CDR2: FL 0.25, MCL 0.36; MZL 1.54: CDR3: FL 0.43, MCL 0.53, MZL 1.28).To investigate whether HLA-dependent immunosurveillance acts on normal B-cell receptors, we compared BIMAS sum scores of 96 non-clonal Ids present in diagnostic biopsies of 29 FL and MCL patients. These Ids represent bona fide contaminating, healthy B cells. No significant differential immunogenicity existed for the entire Vh sequences or individual CDR regions, regardless of their mutational status. We therefore randomly sequenced B-cell receptors from two mouse strains with an identical B6 genetic background but a congenic H-2 complex (C57BL/6ByJ: H-2b; 422 sequences; B6-H2d/bByJ: H-2d; 412 sequences). In these SPF-bred mice, 61% and 71% of sequences, respectively, were unmutated. Significantly higher immunogenicity was observed in the FR1-3 regions of C57BL/6ByJ compared to B6-H2d/bByJ Ids on H-2kd (p=0.04). Also, several Vh genes were significantly (Fisher's exact test) less frequent in mice expressing the MHC type with stronger binding to Id-derived peptides of the corresponding germ-line Vh sequence. Overall, the ratio of predicted MHC binding strength of the 20 highest scoring peptides from 59 represented germ-line Vh genes correlated to the relative frequency of representation of these genes in the peripheral B-cell repertoire (p=0.016, Pearson correlation). Collectively, these results indicate that somatic hypermutation is the predominant mechanism that enables T-cell mediated control of malignant B-cell clones in FL but possibly also MCL on the basis of the HLA binding of Id-derived peptides. In addition, HLA binding strength of germ-line-encoded Vh peptides appears to exert a subtle modulation of the primary B cell repertoire by MHC class I-restricted T cells. These findings strengthen the theoretical basis of active Id-directed immunotherapy of B-cell lymphomas with mutated antigen receptors. In addition, they may also lead to a novel explanation for the association of HLA type with aberrant functions of the nonmalignant immune system. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Prediction of MHC Class I-Restricted Immunogenic Epitopes of B-Cell Receptors Indicates T Cell-Mediated Immunosurveillance of Malignant B Cell Clones.

Abstract 1949Poster Board I-972Tumor-specific immune responses can be induced in patients with indolent B cell lymphomas (iNHL) by active immunization against the individual B cell receptor (BCR) expressed by the malignant B cell clone, the so-called “idiotype” (Id). In subsequent trials of intradermal vaccination with recombinant lymphoma-derived Fab fragment in iNHL, we have studied the specificity of MHC class I-restricted anti-Id T cell responses by epitope mapping experiments with synthetic Id-derived peptides predicted to be presented by the respective patient's HLA complex. While such peptides exist in hypervariable and conserved Id regions, these assays have shown consistently that in vivo-induced T cell responses occur preferentially against individual Id epitopes located in complementarity-determining regions (CDR), whereas framework (FR) and constant region-derived epitopes are ignored (Bertinetti et al., Cancer Res. 2006; Navarrete et al., ASH 2008). These results contrast with in vitro studies showing that FR-derived peptides are excellent targets for cytotoxic T cells in iNHL patients (Trojan et al., Nat Med 2000). To gain further insight into the relative predominance and immunological role of MHC class I-restricted Id epitopes, we conducted a comprehensive reverse immunology study in follicular lymphoma (FL).Clonal and functional IgH chain transcript sequences were identified from tumor biopsies of 39 FL patients by A-PCR (Bertinetti et al., EJH 2006). The HLA-A and B haplotype of the patients was determined by conventional serological testing and high-resolution PCR genotyping. Potentially MHC-presentable peptides from all Id sequences and their corresponding germ-line (GL) VH genes were identified for the HLA haplotypes of all 39 patients by reverse immunology (bimas.cit.nih.gov). Identified peptides were ranked for each haplotype according to their predicted score, and the sum of the scores for the 20 highest ranking peptides was calculated. The sum score for any given Id was compared to the mean of the sum scores of the other 38 Ids on the respective patient's HLA haplotypes. Separate analyses were performed for CDR peptides (containing at least 2 AA in any CDR) versus non-CDR-peptides (allocated through imgt.cines.fr), Id versus GL sequences, and Id versus contaminating sporadic Ig sequences that represent bona fide normal B cells in the biopsies.72% of all peptides with BIMAS scores of ≥50 and ≥10, respectively, were located in FR, expecially in FR3. The ranked sum Id scores were lower for the patients' own tumor Id than for the mean of the allogeneic Ids (Table; Wilcoxon's matched pair test). This difference was mostly attributable to CDR-derived epitopes throughout all CDRs despite overall lower immunogenicity compared to FR. There was no evidence for differential immunogenicity between a hypermutated FL Id and the corresponding GL (p=0.58). Finally, a preliminary survey of IgH sequences from non-clonal B cells indicated similar immunogenicity compared to FL Id (p=0.31).These bioinformatic findings indicate T cell-mediated immunosurveillance against the BCR of malignant and perhaps nonmalignant B cells. T cell activity appears to be directed predominantly against individual CDR peptides despite their lesser predicted HLA binding capacity compared to FR peptides. Existing CDR epitopes are not generated during the hypermutation process of BCRs, raising the possibility that randomly generated, more immunogenic hypervariable peptides are not permitted by the immune system. In conjunction with the T cell activity observed in in vivo and in vitro studies cited above, these findings are consistent with strong peripheral tolerance to shared Id structures. On the other hand, T cell control of individual Id peptides may play a role in immunosurveillance of malignant B cells and may be exploited for active immunotherapy of lymphoma. In contrast, generic or pan-B-cell epitopes are predicted to be less effective in inducing anti-lymphoma T cell responses.Median (range) BIMASPatient IdMean of allogeneic IdscomparisonAll peptides213 (40-5920)369 (56-5520)p=0.0012FR peptides157 (20-5415)239 (18-3891)p=0.045CDR peptides74 (7-648)175 (21-1760)p<0.0001- CDR1 peptides21 (0.7-144)52 (1.9-630)p=0.0007- CDR2 peptides7.6 (0.2-345)30 (2.2-212)p=0.0089- CDR3 peptides16 (1.3-506)37 (6-980)p=0.0008 Disclosures:No relevant conflicts of interest to declare.

Lymphomas can develop from B cells chronically helped by idiotype-specific T cells

B cell lymphomas have been associated with chronic infections and autoimmunity. However, most lymphomas develop in the absence of any known chronic antigenic stimulation. B cells process their highly diversified endogenous immunoglobulin and present clonally unique variable-region idiotypic (Id) peptides on their major histocompatibility complex (MHC) class II molecules to Id-specific T cells. We show that B cells chronically helped by Id-specific Th2 cells developed into large B cell lymphomas with cytogenetic DNA aberrations. The lymphomas expressed high amounts of Id, MHC class II, CD80/86, and CD40 and bidirectionally collaborated with Th2 cells. Thus, MHC class II–presented Id peptides may represent a chronic self-antigenic stimulus for T cell–dependent lymphomagenesis. Eventually, B lymphomas grew independent of T cells. Thus, T cells do not only eliminate cancers as currently believed. In fact, Id-specific Th2 cells can induce B lymphomas.

Anti-class II antibodies, but not cytotoxic T-lymphocyte antigen 4-immunoglobulin hybrid molecules, prevent rejection of major histocompatibility complex class II-negative myeloma in T-cell receptor-transgenic mice.

We have previously shown that tumour-specific CD4+ T cells protect against subcutaneous injections of major histocompatibility complex (MHC) class II-negative MOPC315 myeloma cells. Here, we have interfered with the immunologic events that lead to successful rejection of MOPC315 challenges in T-cell receptor (TCR)-transgenic mice. The CD4+ T cells have a transgene-encoded TCR specific for a MOPC315 V-region idiotypic (Id) peptide presented on the MHC class II molecule E(d). A side-by-side comparison indicated that DNA-recombination-deficient TCR-transgenic mice were better protected against MOPC315 tumour development than recombination-sufficient counterparts, suggesting that B cells or endogenous TCR chains might facilitate tumour progression in this model. Intraperitoneal injections of E(d)-specific antibodies over a period of initial 24 days, abrogated protection against tumours in both strains of mice. By contrast, injections of anticostimulatory molecules (cytotoxic T-lymphocyte antigen 4-immunoglobulin hybrid molecules) had no effect. The findings demonstrate that tumour rejection depends on the presence of MHC class II molecules, despite the fact that MOPC315 tumour cells themselves do not express them. The results are consistent with the idea that secreted myeloma protein is processed and presented by class II+ antigen-presenting cells to Id-specific naïve CD4+ T cells that become activated and kill the myeloma cells by a bystander mechanism. While Id presentation on class II molecules is absolutely required for tumour rejection, costimulatory CD80/CD86 molecules might be dispensible in this process.

Immunotherapy in multiple myeloma: Id-specific strategies suggested by studies in animal models.

Multiple myeloma (MM) cells produce monoclonal immunoglobulin (Ig) which serves as a truly tumor-specific antigen. The tumor-specific antigenic determinants are localized in the variable (V)-regions of the monoclonal Ig and are called idiotopes (Id). We review here the evidence obtained in a T-cell receptor (TCR) transgenic mouse model that Id-specific, MHC class II-restricted CD4+ T cells play a pivotal role in immunosurveillance and eradication of MHC class II-negative MM cells. In brief, monoclonal Ig secreted by MM cells is endocytosed and processed by antigen-presenting cells (APCs) in the tumor. Such tumor-resident dendritic cell APCs in turn present Id peptide on their class II molecules to Id-specific CD4+ T cells which become activated and indirectly kill the MHC class II-negative myeloma cells. However, if the Id-specific CD4+ cells fail to eliminate the MM cells during their initial encounter, the increasing number of tumor cells secretes so much monoclonal Ig that T-cell tolerance to Id is induced. Extending these findings to MM patients, Id-specific immunotherapy should be applied at a time of minimal residual disease and when new Id-specific T cells have been educated in the thymus, like after high-dose chemotherapy and autologous stem cell transplantation.

Idiotype-specific cytotoxic T lymphocytes in multiple myeloma: evidence for their capacity to lyse autologous primary tumor cells

Multiple myeloma (MM) is a B-cell malignancy. The monoclonal immunoglobulin, secreted by myeloma plasma cells, carries unique antigenic determinants (idiotype [Id]) that can be regarded as a tumor-specific antigen. Id-based immunotherapy has been explored in myeloma patients for the purpose of enhancing or inducing Id-specific immune responses that might lead to tumor destruction. However, despite some evidence obtained from mouse plasmacytoma models, it is still unclear whether Id-specific immunity may play a role in the regulation of tumor cells in MM. In the current study, using dendritic cells (DCs) as antigen-presenting cells, autologous Id-specific cytotoxic T lymphocyte (CTL) lines containing both CD4+ and CD8+ T cells were generated from myeloma patients. The results show that Id-specific CTLs not only recognized and lysed autologous Id-pulsed DCs but also significantly killed the autologous primary myeloma cells. The cytotoxicity against the primary tumor cells was major histocompatibility complex (MHC) class I- and, to a lesser extent, class II-restricted, indicating that myeloma cells could process Id protein and present Id peptides in the context of their surface MHC molecules. Furthermore, the CTLs lysed the target cells mainly through the perforin-mediated pathway because Concanamycin A, but not Brefeldin A-the selective inhibitors for perforin- or Fas-mediated pathways-abrogated the cytolytic activity of the cells. These CTLs secreted predominantly interferon-gamma and tumor necrosis factor-alpha on antigen stimulation, indicating that they belong to the type-1 T-cell subsets. Taken together, these findings represent the first demonstration that Id-specific CTLs are able to lyse autologous tumor cells in MM and, thus, provide a rationale for Id-based immunotherapy in the disease.

Tumor-specific recognition of human myeloma cells by idiotype-induced CD8+ T cells

Immunoglobulin secreted by myeloma cells contains a unique antigenic determinant (idiotype [Id]) that may serve as a tumor-specific antigen. Although Id-protein–specific T-cell responses have been reported in patients with myeloma, it is not known whether primary myeloma tumor cells can present naturally processed Id peptides on their surface as a target. We immunized 2 healthy human stem-cell donors with Id proteins from their recipients. T cells from the immunized donors released high levels of T-helper 1–type cytokines in response to stimulation with myeloma cells from their recipients. The T-cell–mediated cytokine response to tumor cells was blocked by a major histocompatibility complex (MHC) class I monoclonal antibody, whereas the response to soluble Id protein was dependent on MHC class II. To investigate whether Id-specific CD8+ T cells can recognize and kill autologous myeloma cells, we generated T cells from peripheral blood mononuclear cells from a third patient with myeloma by means of in vitro stimulation with autologous dendritic cells pulsed with Id protein. Tumor-specific lysis of myeloma cells was demonstrated by the lack of killing of autologous nonmalignant B cells or natural killer–sensitive K562 cells. Lysis of autologous myeloma targets was restricted by MHC class I molecules. These data represent the first report of class I–restricted T-cell recognition of fresh autologous myeloma targets and formally demonstrate that human myeloma cells can serve as targets of an Id-specific T-cell response.

Tumor-specific recognition of human myeloma cells by idiotype-induced CD8+ T cells

AbstractImmunoglobulin secreted by myeloma cells contains a unique antigenic determinant (idiotype [Id]) that may serve as a tumor-specific antigen. Although Id-protein–specific T-cell responses have been reported in patients with myeloma, it is not known whether primary myeloma tumor cells can present naturally processed Id peptides on their surface as a target. We immunized 2 healthy human stem-cell donors with Id proteins from their recipients. T cells from the immunized donors released high levels of T-helper 1–type cytokines in response to stimulation with myeloma cells from their recipients. The T-cell–mediated cytokine response to tumor cells was blocked by a major histocompatibility complex (MHC) class I monoclonal antibody, whereas the response to soluble Id protein was dependent on MHC class II. To investigate whether Id-specific CD8+ T cells can recognize and kill autologous myeloma cells, we generated T cells from peripheral blood mononuclear cells from a third patient with myeloma by means of in vitro stimulation with autologous dendritic cells pulsed with Id protein. Tumor-specific lysis of myeloma cells was demonstrated by the lack of killing of autologous nonmalignant B cells or natural killer–sensitive K562 cells. Lysis of autologous myeloma targets was restricted by MHC class I molecules. These data represent the first report of class I–restricted T-cell recognition of fresh autologous myeloma targets and formally demonstrate that human myeloma cells can serve as targets of an Id-specific T-cell response.

Vaccination of multiple myeloma patients with idiotype-pulsed dendritic cells: immunological and clinical aspects.

Multiple myeloma (MM) is characterized by a clonal proliferation of malignant plasma cells in the bone marrow secreting a monoclonal immunoglobulin (paraprotein) with specific antigenic determinants, the idiotype (Id), which can be regarded as a tumour-associated antigen (TAA). In order to analyse the impact of a dendritic cell (DC)-based vaccine, 11 patients with advanced MM were treated with CD34 stem cell-derived dendritic cells that were pulsed with Id peptides. Subsequently, the patients received three boost immunizations every other week with a combination of Id and granulocyte-macrophage colony-stimulating factor (GM-CSF) (nine patients) or with Id peptide-pulsed dendritic cells again (two patients). The treatment was well tolerated with no side-effects. The present clinical study was a proof of concept analysis of dendritic cell-based vaccines in MM. The capacity of the dendritic cells to activate idiotype-specific T cells was verified by in vitro stimulation experiments before the vaccination therapy. Immunological effects of the Id vaccination were analysed by monitoring changes in anti-idiotype antibody titres and idiotype-specific T-cell activity. After vaccination, three out of 10 analysed patients showed increased anti-idiotype antibody serum titres, indicating the induction of an idiotype-specific humoral immune response. The idiotype-specific T-cell response analysed by ELISpot was increased in four out of 10 analysed patients after vaccination, and one patient had a decreased plasma cell infiltration in the bone marrow. In conclusion, five out of 11 patients showed a biological response after vaccination. Thus, our data indicate that immunotherapy with Id-pulsed DCs in MM patients is feasible and safe. DC generated from CD34+ progenitor cells can serve as a natural adjuvant for the induction of clinically relevant humoral and cellular idiotype-specific immune responses in patients suffering from advanced MM.