Nominal Antigen Research Articles

Abstract LB344: Complement-mediated signaling during cross-presentation of tumor antigen augments T cell responses

Abstract Production of complement C3a and C5a locally at the immune synapse by activated dendritic cells (DCs) enhances T cell responses in multiple experimental models. Complement signaling through cognate C3a receptor (C3aR) and C5a receptor (C5aR) modulate effector T cell stability, proliferation and differentiation. We have previously shown that complement C3 plays a mechanistic role in driving synergistic anti-tumor effects of blocking indoleamine 2,3-dioxygenase (IDO) during radiation and chemotherapy in a murine brain tumor model that expresses the gp10025-33 peptide antigen. We hypothesize that, when anti-tumor responses are allowed to happen, complement C3 production by local DCs acts as a costimulation factor, operating through C3aR and C5aR on the T cell surface to allow optimal T cell activation and proliferation. To study the role of complement C3 as a costimulation factor during T cell responses against a nominal tumor antigen (gp10025-33 peptide, expressed by B16F10 melanoma), we used T cells from syngeneic pmel-1 mice (TCR transgenic with CD8 T cells that recognize the cross-presented gp10025-33 peptide) co-cultured with gp10025-33 peptide and dendritic cells from either wild-type (WT) or C3-defiecient (C3-KO) mice. In addition, we used in vitro culture conditions where gp10025-33 tumor peptide was in stringent limited supply to model homeostatic conditions of the tumor microenvironment. We found that, when stimulated by DCs from C3-KO mice, pmel-1 CD8 T cells had decreased activation marker expression, proliferation, and cytokine effector function relative to stimulation by DCs from WT mice. Next, we bred pmel-1 mice onto a syngeneic background deficient in receptors for both C3a and C5a complement factors (C3aR-KO/C5aR-KO double knock-out mice). We used CD8 T cells from the resulting mouse strain (pmel-1/C3aR-KO/C5aR-KO) co-cultured with WT DCs to study the role of complement signaling in T cells during responses to tumor antigen. Using the same stringent antigen conditions, CD8 T cells from the pmel-1/C3aR-KO/C5aR-KO mice had decreased activation marker expression and proliferation, and differences in cytokine function compared to CD8 T cells from the pmel-1 mice. These findings suggest that lack of local complement production by the DCs in the tumor microenvironment may inhibit anti-tumor immune responses, and that complement signaling through C3aR and C5aR on T cells may play an important role during such anti-tumor responses. Citation Format: Caryn L. Bird, Gabriela A. Pacholczyk, Theodore S. Johnson. Complement-mediated signaling during cross-presentation of tumor antigen augments T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB344.

Design and characterization of lipid nanocarriers for oral delivery of immunotherapeutic peptides.

The use of therapeutic proteins and peptides is of great interest for the treatment of many diseases, and advances in nanotechnology offer a path toward their stable delivery via preferred routes of administration. In this study, we sought to design and formulate a nanostructured lipid carrier (NLC) containing a nominal antigen (insulin peptide) for oral delivery. We utilized the design of experiments (DOE) statistical method to determine the dependencies of formulation variables on physicochemical particle characteristics including particle size, polydispersity (PDI), melting point, and latent heat of melting. The particles were determined to be non-toxic in vitro, readily taken up by primary immune cells, and found to accumulate in regional lymph nodes following oral administration. We believe that this platform technology could be broadly useful for the treatment of autoimmune diseases by supporting the development of oral delivery-based antigen specific immunotherapies.

The role of complement-mediated signaling during antigen presentation

Abstract Local production of complement C3a and C5a at the immune synapse by activated dendritic cells (DCs) enhances T cell responses in a variety of experimental systems. We have shown that complement C3 plays a mechanistic role in driving synergistic anti-tumor effects of blocking indoleamine 2,3-dioxygenase (IDO) during radiation and chemotherapy in a murine brain tumor model. We hypothesize that, when anti-tumor responses are allowed to happen, locally produced complement C3 acts as a costimulation factor for activation of T cells. To study the role of complement C3 in promoting T cell responses against a nominal tumor antigen (gp10025–33 peptide expressed by B16F10 melanoma), we used T cells from syngeneic pmel-1 mice (TCR transgenic with CD8 T cells that recognize the cross-presented gp10025–33 peptide). We found florid deposition of complement C3 in B16F10 tumors when IDO-blockade was added to a pmel-1/gp10025–33 based vaccine. To model homeostatic conditions of the tumor microenvironment, we used an in vitro co-culture system with gp10025–33 peptide in stringent limited supply. Under these conditions, pmel-1 CD8 T cells stimulated by DCs from complement C3 knock-out (C3-KO) mice had decreased activation marker expression, proliferation, and cytokine effector function, relative to pmel-1 CD8 T cells stimulated by DCs from wild-type (WT) mice. These findings suggest that suppression of local complement production by DCs in the tumor microenvironment may inhibit anti-tumor immune responses. Supported by grants from Alex's Lemonade Stand Foundation, Press On Foundation, and Hyundai Hope On Wheels.

Distinct roles but cooperative effect of TLR3/9 agonists and PD-1 blockade in converting the immunotolerant microenvironment of irreversible electroporation-ablated tumors.

Irreversible electroporation (IRE) is a new cancer ablation technology, but methods to improve IRE-induced therapeutic immunity are only beginning to be investigated. We developed a mouse model bearing large primary (300 mm3) and medium distant (100 mm3) EG7 lymphomas engineered to express ovalbumin (OVA) as a nominal tumor antigen. We established experimental protocols including IRE alone and IRE combined with Toll-like receptor (TLR)3/9 agonists (poly I:C/CpG) (IRE + pIC/CpG), PD-1 blockade (IRE + PD-1 blockade), or both (IRE + Combo) to investigate therapeutic effects on primary and distant EG7 tumors and conversion-promoting effects on the immunotolerant tumor microenvironment (TME). We demonstrated that IRE alone simulated very weak OVA-specific CD8+ T cell responses and did not inhibit primary tumor growth. IRE + pIC/CpG synergistically stimulated more efficient OVA-specific CD8+ T cell responses and primary tumor growth inhibition than IRE + PD-1 blockade. IRE + pIC/CpG played a major role in the modulation of immune cell profiles but a minor role in the downregulation of PD-L1 expression in the TME and vice versa for IRE + PD-1 blockade. IRE + Combo cooperatively induced potent OVA-specific CD8+ T cell immunity and rescued exhausted intratumoral CD8+ T cells, leading to eradication of not only primary tumors but also untreated concomitant distant tumors and lung metastases. IRE + Combo efficiently modulated immune cell profiles, as evidenced by reductions in immunotolerant type-2 (M2) macrophages, myeloid-derived suppressor-cells, plasmacytoid dendritic cells, and regulatory T cells and by increases in immunogenic M1 macrophages, CD169+ macrophages, type-1 conventional dendritic cells, and CD8+ T cells, leading to conversion of immunotolerance in not only primary TMEs but also untreated distant TMEs. IRE + Combo also showed effective therapeutic effects in two breast cancer models. Therefore, our results suggest that IRE + Combo is a promising strategy to improve IRE ablation therapy in cancer.

A Disquisition on MHC Restriction and T Cell Recognition in Five Acts.

The seminal discovery in the early 1970s, credited to Peter Doherty and Rolf Zinkernagel, of major histocompatibility complex (MHC) restriction exhibited by cytotoxic T cells represented a major conceptual advance in understanding antigen recognition by conventional T cells. This advance also led to other major new insights into the ontogeny and immunobiology of T cells and catalyzed a renaissance in viral immunology. In this commentary in honor of Peter Doherty, I offer five brief reflections on different aspects of the phenomenon of MHC restriction and the process by which it was discovered and explained. In the first of these sections, I offer a reinterpretation of MHC restriction that reframes the constraints on self-MHC recognition in terms of the probabilities of recognizing a given nominal antigen peptide in the context of an MHC molecule that is nonself on the basis of differing in amino acid sequence from the self-restriction element at one or more positions. Subsequent sections address: (i) the ways in which general ideas, developed subsequent to the discovery of MHC restriction, about the intricacies of antigen recognition by antibodies apply to T cell receptors binding to MHC/peptide complexes; (ii) how to reconcile the existence of MHC restriction with the impressive magnitude of T cell responses to nonself MHC antigens; (iii) the possible relevance to MHC restriction and immune system function of ideas from mathematical logic that relate to the consequences of self-reference; and (iv) the implications for the philosophy of science of MHC restriction and the processes of its discovery and acceptance within the immunology research community.

Antibody Conditioning Enables MHC-Mismatched Hematopoietic Stem Cell Transplants and Organ Graft Tolerance.

Hematopoietic cell transplantation can correct hematological and immunological disorders by replacing a diseased blood system with a healthy one, but this currently requires depleting a patient's existing hematopoietic system with toxic and non-specific chemotherapy, radiation, or both. Here we report an antibody-based conditioning protocol with reduced toxicity and enhanced specificity for robust hematopoietic stem cell (HSC) transplantation and engraftment in recipient mice. Host pre-treatment with six monoclonal antibodies targeting CD47, Tcells, NK cells, and HSCs followed by donor HSC transplantation enabled stable hematopoietic system reconstitution in recipients with mismatches at half (haploidentical) or all major histocompatibility complex (MHC) genes. This approach allowed tolerance to heart tissue from HSC donor strains in haploidentical recipients, showing potential applications for solid organ transplantation without immune suppression. Fully mismatched chimeric mice developed antibody responses to nominal antigens, showing preserved functional immunity. These findings suggest approaches for transplanting immunologically mismatched HSCs and solid organs with limited toxicity.

CD20-Mimotope Peptides: A Model to Define the Molecular Basis of Epitope Spreading.

Antigen-mimicking peptide (mimotope)-based vaccines are one of the most promising forms of active-immunotherapy. The main drawback of this approach is that it induces antibodies that react poorly with the nominal antigen. The aim of this study was to investigate the molecular basis underlying the weak antibody response induced against the naïve protein after peptide vaccination. For this purpose, we analyzed the fine specificity of monoclonal antibodies (mAb) elicited with a 13-mer linear peptide, complementary to theantigen-combining site of the anti-CD20 mAb, Rituximab, in BALB/c mice. Anti-peptide mAb competed with Rituximab for peptide binding. Even so, they recognized a different antigenic motif from the one recognized by Rituximab. This explains their lack of reactivity with membrane (naïve) CD20. These data indicate that even on a short peptide the immunogenic and antigenic motifs may be different. These findings highlight an additional mechanism for epitope spreading and should be taken into account when designing peptides for vaccine purposes.

Salivary gland immunization via Wharton's duct activates differential T-cell responses within the salivary gland immune system.

Salivary glands are a major component of the mucosal immune system that confer adaptive immunity to mucosal pathogens. As previously demonstrated, immunization of the submandibular gland with tissue culture-derived murine cytomegalovirus (tcMCMV) or replication-deficient adenoviruses expressing individual murine cytomegalovirus (MCMV) genes protected mice against a lethal MCMV challenge. Here, we report that salivary gland inoculation of BALB/cByJ mice with tcMCMV or recombinant adenoviruses differentially activates T helper (Th)1, -2, and -17 cells in the salivary glands vs. the associated lymph nodes. After inoculation with tcMCMV, lymphocytes from the submandibular gland preferentially express the transcription factor T-cell-specific T-box transcription factor (T-bet), which controls the expression of the hallmark Th1 cytokine, IFN-γ. Lymphocytes from the periglandular lymph nodes (PGLNs) express both T-bet and GATA-binding protein 3 (GATA3), which promotes the secretion of IL-4, -5, and -10 from Th2 cells. In contrast, after inoculation with replication-deficient adenoviruses, lymphocytes from the submandibular gland express T-bet, GATA3, and RAR-related orphan receptor γ, thymus-specific isoform (RORγt) (required for differentiation of Th17 cells) and forkhead box P3 (Foxp3) (required for the differentiation of regulatory T cells). Lymphocytes from the PGLNs were not activated. The differential induction of Th responses in the salivary gland vs. the PGLNs after inoculation with attenuated virus vs. a nominal protein antigen supports the use of the salivary as an alternative mucosal route for administering vaccines.-Liu, G., Zhang, F., Wang, R., London, S. D., London, L. Salivary gland immunization via Wharton's duct activates differential T-cell responses within the salivary gland immune system.

Trained Immunity-Based Vaccines: A New Paradigm for the Development of Broad-Spectrum Anti-infectious Formulations.

Challenge with specific microbial stimuli induces long lasting epigenetic changes in innate immune cells that result in their enhanced response to a second challenge by the same or unrelated microbial insult, a process referred to as trained immunity. This opens a new avenue in vaccinology to develop Trained Immunity-based Vaccines (TIbV), defined as vaccine formulations that induce training in innate immune cells. Unlike conventional vaccines, which are aimed to elicit only specific responses to vaccine-related antigens, TIbV aim to stimulate broader responses. As trained immunity is generally triggered by pattern recognition receptors (PRRs), TIbV should be formulated with microbial structures containing suitable PRR-ligands. The TIbV concept we describe here may be used for the development of vaccines focused to promote host resistance against a wide spectrum of pathogens. Under the umbrella of trained immunity, a broad protection can be achieved by: (i) increasing the nonspecific effector response of innate immune cells (e.g., monocyte/macrophages) to pathogens, (ii) harnessing the activation state of dendritic cells to enhance adaptive T cell responses to both specific and nonrelated (bystander) antigens. This capacity of TIbV to promote responses beyond their nominal antigens may be particularly useful when conventional vaccines are not available or when multiple coinfections and/or recurrent infections arise in susceptible individuals. As the set of PRR-ligands chosen is essential not only for stimulating trained immunity but also to drive adaptive immunity, the precise design of TIbV will improve with the knowledge on the functional relationship among the different PRRs. While the TIbV concept is emerging, a number of the current anti-infectious vaccines, immunostimulants, and even vaccine adjuvants may already fall in the TIbV category. This may apply to increase immunogenicity of novel vaccine design approaches based on small molecules, like those achieved by reverse vaccinology.

Activation of host p53 enhances anti-tumor immunity by driving differentiation of immunogenic myeloid DCs in tumors, while simultaneously protecting against collateral autoimmunity against shared self-tumor antigens.

Abstract During immunotherapy, we show that large numbers of immunogenic CD103+ DCs can arise in murine tumors via direct differentiation myeloid-lineage (Ly6c+ monocytic) precursors. These “dual-phenotype” Ly6c+CD103+ DCs differentiate from a precursor population of monocytic myeloid-derived suppressor cells (MDSCs) in tumors. Differentiation was controlled by inflammation-induced activation of the transcription factor p53 in MDSCs. Targeted deletion of myeloid p53 selectively ablated the Ly6c+CD103+ DC population, which resulted in abrogation of all response to multiple forms of tumor immunotherapy. Pharmacologically elevating the level of p53 expression in host cells, by administering the p53-agonist (MDM2-inhibitor) drug nutlin-3a, caused a sustained increase in Ly6c+CD103+ DCs in tumors during immunotherapy, and markedly enhanced anti-tumor immunity. Conversely, however, when a nominal tumor antigen (OVA) was also shared by a critical host tissue (Rip-mOVA mice), elevation of host p53 using nutlin-3a was potent immunosuppressive and protective against lethal autoimmunity outside of the tumor, while still driving enhanced immunity – against the same antigen – within the tumor itself. Thus, p53-agonist drugs may represent a novel class immunotherapy agents, with high discrimination between tumor-associated antigens and collateral autoimmunity.

Autoreactive and immunization-induced follicular helper T cells have opposite glucose and glutamine requirements

Abstract Both glycolysis and mitochondrial oxidative metabolism are elevated in CD4+ T cells from lupus-prone mice, and metabolic inhibitors targeting both of these pathways normalized lupus T cell functions in vitro and reverted disease in mice. Here, we showed that inhibiting glucose metabolism with 2-deoxy-D-glucose (2DG) results in a drastic reduction of follicular helper T (TFH) cell frequency in lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice. However, treatment of TC mice with 2DG had little effect on the production of antibodies or the expansion of TFH cells following immunization with a nominal antigen of T-cell-dependent (TD) humoral responses, and no effect on the frequency of influenza virus-specific TFH cells induced by immunization with PR8 influenza virus. Thus, TFH cells supporting the production of autoantibodies are different from TFH cells providing protective humoral immunity against pathogens. The specific metabolic signature from autoimmune TFH cell is significantly different compared with exogenous antigen-specific TFH cells. Further results obtained with gene expression analysis and treatment with metabolic inhibitors indicated that autoreactive TFH cells depend on glucose metabolism, while exogenous antigen-specific TFH cells are more glutamine-demanding. Overall, our results predict that targeting TFH cellular metabolism provides an effective and safe therapeutic approach for systemic autoimmunity by eliminating autoreactive TFH cells.

Toward a New Kind of Vaccine: A Logical Extension of the Symmetrical Immune Network Theory.

BackgroundThe symmetrical immune network theory, developed in 1975, is based on the existence of specific T cell factors and hypothesizes that normal IgG immune responses comprise the production of 2 kinds of antibodies, namely antigen-specific antibodies and anti-idiotypic antibodies.ObjectiveThe aim of this study was to confirm the existence of specific T cells factors and to show that immunization of C3H mice with BL/6 skin or using nominal antigen for immunization (Tetanus Toxoid) induced production of antigen-specific (anti-BL/6 or antitetanus) antibodies plus anti-idiotypic antibodies (C3H anti-anti-C3H). Subsequently, we investigated the role of combinations of antigen-specific and anti-idiotype antibodies in a variety of animal models of clinical diseases.MethodsAntigen-specific antibodies were produced by conventional immunization of mice (eg, with tetanus toxoid or by skin allografting). Subsequent anti-idiotypic antibodies were derived by exhaustive absorption of antigen-specific antibody, with confirmation of anti-idiotypic specificity by binding to relevant target antigen-specific antibodies in an enzyme-linked immunosorbent assay (ELISA). Antigen-specific plus anti-idiotypic antibodies were then used to modulate skin allograft survival, dextran sulfate sodium (DSS)-induced colitis, ovalbumin (OVA)-induced IgE production, and breast cancer growth in mice.ResultsInfusions of anti-BL/6 antibodies together with BL/6 anti-anti-BL/6 antibodies specifically suppressed (>85%) an immune response to BL/6 lymphocytes in C3H mice. The two kinds of antibodies with complementary specificity are hypothesized to stimulate 2 populations of T lymphocytes. Coselection of these 2 populations leads to a new stable steady state of the system with diminished reactivity to BL/6 tissue. A combination of anti-C3H and C3H anti‑anti-C3H IgG antibodies down-regulated inflammation in a mouse model of inflammatory bowel disease (>75%) and attenuated anti-IgE production and sensitization to produce IL4 cytokines (>70%) in an OVA-allergy model. Combination of C3H anti‑BL/6 and BL/6 anti-anti-BL/6 antibodies decreased tumor growth and metastases (>705) in an EMT6 transplantable breast cancer model.ConclusionsUse of a combination of antigen-specific and anti-idiotypic antibodies has potential as a new class of vaccines.

Metabolic inhibitors elimiante the expansion of autoimmune follicular helper T cells but not that induced by T-dependent-immunization in lupus mice

Abstract The expansion of follicular helper CD4 T (Tfh) cells is tightly associated with disease severity in lupus patients, following multiple studies in various mouse models showing that they are necessary for the production of class-switched high-affinity pathogenic autoAbs. We have recently shown that both glycolysis and mitochondrial oxidative metabolism are elevated in CD4 T cells from lupus prone B6.Sle1.Sle2.Sle3 (TC) mice as compared to B6 controls, and that a treatment combining 2-deoxy-D-glucose (2-DG), which inhibits glucose metabolism, and metformin, which inhibits oxygen consumption, normalized lupus T cell functions in vitro and reverted disease. We have expanded these results to B6.lpr, BXSB.Yaa and NZB/WF1 models of lupus. Remarkably, the number and the frequency of Tfh cells was normalized in these 4 models of lupus treated with a combination of 2DG and metformin, corresponding to a drastic reduction of autoantibodies. Treatment of normal or lupus-prone mice with the same inhibitors had however little effect on the production of TD antibodies or the expansion of Tfh cells following immunization with a nominal antigen. This suggests that Tfh cells supporting the production of autoAbs are quantitatively or qualitatively different from Tfh cells providing protective humoral immunity against pathogens. Moreover, it suggests that autoimmune Tfh cells have higher metabolic requirements than the metabolically quiescent Tfh cells that have been recently described in TD-responses. Studies to characterize the specific metabolic requirements of autoimmune Tfh cells are ongoing. Overall, our results predict that targeting Tfh cellular metabolism provides an effective and safe therapeutic approach for systemic autoimmunity.

Characterisation of an epigenetically altered CD4+ CD28+ Kir+ T cell subset in autoimmune rheumatic diseases by multiparameter flow cytometry

ObjectivesAntigen-specific CD4+ T cells epigenetically modified with DNA methylation inhibitors overexpress genes normally suppressed by this mechanism, including CD11a, CD70, CD40L and the KIR gene family. The altered cells become...

Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma.

Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB) cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id) IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab) ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH) and light chains (VL) were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO) cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18)-zeta) was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC) was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2 overexpressed in NB.

Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine.

Dietary antigens are normally rendered nonimmunogenic through a poorly understood "oral tolerance" mechanism that involves immunosuppressive regulatory T (Treg) cells, especially Treg cells induced from conventional T cells in the periphery (pTreg cells). Although orally introducing nominal protein antigens is known to induce such pTreg cells, whether a typical diet induces a population of pTreg cells under normal conditions thus far has been unknown. By using germ-free mice raised and bred on an elemental diet devoid of dietary antigens, we demonstrated that under normal conditions, the vast majority of the small intestinal pTreg cells are induced by dietary antigens from solid foods. Moreover, these pTreg cells have a limited life span, are distinguishable from microbiota-induced pTreg cells, and repress underlying strong immunity to ingested protein antigens.

Abstract IA18: Adoptive T-cell transfer for metastatic melanoma: What are the relevant antigens?

Abstract The transfer of cultured tumor-reactive T-cells into patients with metastatic melanoma can cause tumor regressions when other immunotherapies have failed. A single transfer of T-cells can mediate complete and durable regressions in some patients. The two main sources of T-cells for transfer have been from the native T-cells infiltrating the tumor (tumor infiltrating lymphocytes; TIL) or from peripheral blood lymphocytes retrovirally engineered with a tumor-reactive T-cell receptor (TCR). In the latter situation, a variety of tumor-associated antigens have been targeted with cloned receptors and although responses are seen, in some cases unacceptable toxicity has occurred due to on-target, off-tumor immune recognition. Although melanoma TIL often contain T-cells that also target the same nominal antigens, their transfer is rarely associated with such undesirable toxicities. In addition, melanoma TIL frequently contain T-cells which recognize the autochthonous tumor, while TIL from other tumors infrequently show this reactivity. Recent data have implicated the presence of abundant tumor-specific mutations in melanoma as the explanation for these various observations. These appear to be significant antigenic targets in therapeutic TIL that cause no collateral damage, and their high frequency as shown by tumor exomic sequencing of melanomas may explain that tumor's high degree of natural immunogenicity. These lessons learned from studying T-cell transfer for melanoma can now be applied to other cancers and a recent example has demonstrated the potential and promise of this approach. Citation Format: James C. Yang. Adoptive T-cell transfer for metastatic melanoma: What are the relevant antigens? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA18.

Immunological long-term follow-up of neuroblastoma stage IV patients after anti-GD2 CH14.18 antibody treatment.

3029 Background: Neuroblastoma stage IV is associated with low cure rates. Treatment with the GD2 antibody ch14.18 could improve the long-term overall survival. Apart from GD2 other tumor antigens (TA) are expressed in neuroblastoma and studies implicated that T-cell responses to TA are associated with a better outcome. For the present study we analyzed the immune response in long term neuroblastoma survivors treated with anti GD2 ch14.18. Methods: Blood and serum of 17 neuroblastoma long-term survivors after GD2 therapy with > 5 years follow up (patients group) and 17 age and gender matched healthy donors (control group) were analyzed. Serum was analyzed for antibodies binding to neuroblastoma cell lines and antibodies to a nominal antigen GD2. T cells were stimulated with overlapping peptides of the tumor antigens (TA) NY-ESO-1, WT-1, Survivin, MAGE-A1 and two mimotope peptides to GD2 for 10 days and analyzed for IFN-γ secretion in flow cytometry. Results: T-cell responses to at least one TA could be found in 82% of healthy and in 93% of patients with no significant difference in the number of TA responders between the two groups to any of these TA (Fisher´s exact test). In contrast, T-cell responses to at least one of the GD2 mimotopes were found in 53% of neuroblastoma survivors but in 0% of healthy donors. Moreover, even priming with GD2 mimotope loaded dendritic cells did not induce a T-cell response in healthy donor cells. No differences between patient and control group could be detected in binding of antibodies to neuroblastoma cell lines and no GD2-specific antibodies were detected in patients and controls. Conclusions: Survivors of neuroblastoma treated with anti-GD2 ch14.18 demonstrated robust T-cell responses to neuroblastoma associated TA that was comparable to healthy donors. This could enable patients to attack minimal residual disease efficiently. Moreover more than half of the patients had T-cell responses to GD2 which could be an effect of the GD2 antibody therapy. Our results show that involvement of the cellular immune system might have an impact on the better outcome of this treatment in stage IV neuroblastoma patients.

Chemotherapy enhances cross-presentation of nuclear tumor antigens.

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8+ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens.

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27−IgD+ (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells.