Antigen-specific Interactions Research Articles

Prediction of antibody-antigen interaction based on backbone aware with invariant point attention.

Antibodies play a crucial role in disease treatment, leveraging their ability to selectively interact with the specific antigen. However, screening antibody gene sequences for target antigens via biological experiments is extremely time-consuming and labor-intensive. Several computational methods have been developed to predict antibody-antigen interaction while suffering from the lack of characterizing the underlying structure of the antibody. Beneficial from the recent breakthroughs in deep learning for antibody structure prediction, we propose a novel neural network architecture to predict antibody-antigen interaction. We first introduce AbAgIPA: an antibody structure prediction network to obtain the antibody backbone structure, where the structural features of antibodies and antigens are encoded into representation vectors according to the amino acid physicochemical features and Invariant Point Attention (IPA) computation methods. Finally, the antibody-antigen interaction is predicted by global max pooling, feature concatenation, and a fully connected layer. We evaluated our method on antigen diversity and antigen-specific antibody-antigen interaction datasets. Additionally, our model exhibits a commendable level of interpretability, essential for understanding underlying interaction mechanisms. Quantitative experimental results demonstrate that the new neural network architecture significantly outperforms the best sequence-based methods as well as the methods based on residue contact maps and graph convolution networks (GCNs). The source code is freely available on GitHub at https://github.com/gmthu66/AbAgIPA .

Virus-Associated CD8+ T-Cells Are Not Activated Through Antigen-Mediated Interaction Inside Atherosclerotic Lesions.

Viral infections have been associated with the progression of atherosclerosis and CD8+ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8+ T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8+ T-cells in the atherosclerotic lesion. The presence, clonality, tissue enrichment, and phenotype of virus-associated CD8+ T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-β sequencing and single-cell T-cell receptor (α and β) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8+ T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined. Virus-associated CD8+ T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P=0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8+ T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8+ T-cells were phenotypically highly similar to other CD8+ T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8+ T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion. This study suggests that virus-specific CD8+ T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms.

Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens.

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.

Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.

DNMT3A-Mutated Stem and Progenitor Cells Contribute to Altered T Cell Activation in Clonal Hematopoiesis

Clonal hematopoiesis is characterized by an expansion of blood cells derived from a single mutated hematopoietic stem cell (HSC). When this expansion is driven by a leukemia-associated mutation that exceeds a variant allele frequency (VAF) of 2%, it is termed clonal hematopoiesis of indeterminate potential (CHIP). Approximately half of CHIP cases are driven by mutations in the DNA methyltransferase gene DNMT3A. In patients with CHIP, greater clonal expansion is associated with an increased risk of cardiovascular diseases and hematologic malignancies. While DNMT3A impairment has been shown to alter inflammatory signaling in HSCs and myeloid cells, less is known about the impact of DNMT3A-mutated CHIP on T cell biology. Here, we leverage a cohort of human CHIP samples to assess how the expansion of DNMT3A-mutated cells affects gene expression across blood cell types and generate a new mouse model to assess antigen-specific immune cell interactions. We find evidence that the accumulation of DNMT3A-mutated stem and progenitor cells can cause T cell dysfunction. We first identified CHIP in a cohort of 191 sternum bone marrow samples collected during heart surgery. We detected CHIP in 45 samples (23.6%), including 22 samples with mutated DNMT3A (48.9% of CHIP samples). To mitigate confounding factors that might be introduced by comparing CHIP to healthy individuals, we used ten DNMT3A-mutated CHIP samples for this study, splitting them into a low-VAF (<10%, three samples) and high-VAF (>10%, seven samples) cohorts. We performed single-cell RNA sequencing on these 10 samples, generating high-quality data for 95,245 cells. We clustered and annotated 33 cell types according to the expression of canonical marker genes, representing the full spectrum of hematopoietic progenitors to myeloid, erythroid and lymphoid lineages. To evaluate how greater DNMT3A clonal expansion affects immune cell states, we performed differential gene expression analysis in the high-VAF compared to the low-VAF cohort with DESeq2 using a pseudobulk approach followed by gene set enrichment analysis using Hallmark pathways. We found that TGF-beta signaling, known for its immunosuppressive role, is upregulated in hematopoietic stem and progenitor cells (HSPCs), myeloid and T cell populations in the high-VAF compared to the low-VAF cohort. Both interferon alpha and gamma signaling pathways are consistently downregulated in HSPCs and multiple myeloid cell types as well as T cells. TNF-alpha signaling is upregulated in HSPCs and myeloid cells, consistent with recent observations linking TNF-alpha to the survival and proliferation of HSPCs and myeloid cells. Overall, greater expansion of DNMT3A-mutated cells is associated with variable gene expression changes in progenitor and myeloid cell types, whereas gene expression changes in T cells are consistent with diminished activation (Figure 1). To evaluate mechanisms contributing to altered T cell activation in DNMT3A-mutated CHIP, we combined our Dnmt3a-fl-R878H/+ inducible mouse model with the ovalbumin (OVA) trackable antigen system. Endogenously expressed OVA is processed into a peptide that is loaded onto MHCI and recognized by transgenic CD8+ T cells from OT-I mice. We crossed the Dnmt3a-fl-R878H/+ mouse with a mouse expressing OVA under the actin promoter, resulting in Dnmt3a-mutated mice with constitutive OVA expression. To interrogate antigen-specific T cell responses, we co-cultured Dnmt3a-mutated OVA+ lineage negative (Lin-) stem and progenitor cells with CD8+ T cells from OT-I mice. By measuring T cell proliferation using CFSE dilution, we observed an increase in T cell proliferation when CD8+ T cells were co-cultured with Dnmt3a-mutated OVA+ Lin- cells compared to wildtype OVA+ Lin- cells (Figure 2). These results suggest that Lin- Dnmt3a-mutated cells have an increased capacity for antigen-specific T cell activation. In conclusion, our studies demonstrate that greater expansion of DNMT3A-mutated CHIP is associated with reduced expression of activation signatures in T cells. In apparent contrast, Dnmt3a-mutated mouse cells have an increased capacity for antigen-specific T cell stimulation. Since chronic antigenic stimulation causes T cell exhaustion, our findings are consistent with a model in which the expansion of DNMT3A-mutated progenitor and myeloid cells causes chronic stimulation and dysfunction of T cells.

Spatiotemporal co-dependency between macrophages and exhausted CD8+ Tcells in cancer.

T cell exhaustion is a major impediment to antitumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here, we show that the biology of tumor-associated macrophages (TAMs) and exhausted Tcells (Tex) in the TME is extensively linked. We demonstrate that invivo depletion of TAMs reduces exhaustion programs in tumor-infiltrating CD8+ Tcells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ Tcells engage in unique, long-lasting, antigen-specific synaptic interactions that fail to activate Tcells but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.

Follicular dendritic cells restrict interleukin-4 availability in germinal centers and foster memory B cell generation

B cells within germinal centers (GCs) enter cycles of antibody affinity maturation or exit the GC as memory cells or plasma cells. Here, we examined the contribution of interleukin (IL)-4 on B cell fate decisions in the GC. Single-cell RNA-sequencing identified a subset of light zone GC B cells expressing high IL-4 receptor-a (IL4Ra) and CD23 and lacking a Myc-associated signature. These cells could differentiate into pre-memory cells. B cell-specific deletion of IL4Ra or STAT6 favored the pre-memory cell trajectory, and provision of exogenous IL-4 in a wild-type context reduced pre-memory cell frequencies. IL-4 acted during antigen-specific interactions but also influenced bystander cells. Deletion of IL4Ra from follicular dendritic cells (FDCs) increased the availability of IL-4 in the GC, impaired the selection of affinity-matured B cells, and reduced memory cell generation. We propose that GC FDCs establish a niche that limits bystander IL-4 activity, focusing IL-4 action on B cells undergoing selection and enhancing memory cell differentiation.

The Dynamic Entropy of Tumor Immune Infiltrates: The Impact of Recirculation, Antigen-Specific Interactions, and Retention on T Cells in Tumors.

Analysis of tumor infiltration using conventional methods reveals a snapshot view of lymphocyte interactions with the tumor environment. However, lymphocytes have the unique capacity for continued recirculation, exploring varied tissues for the presence of cognate antigens according to inflammatory triggers and chemokine gradients. We discuss the role of the inflammatory and cellular makeup of the tumor environment, as well as antigen expressed by cancer cells or cross-presented by stromal antigen presenting cells, on recirculation kinetics of T cells. We aim to discuss how current cancer therapies may manipulate lymphocyte recirculation versus retention to impact lymphocyte exclusion in the tumor.

Spatiotemporal Co-Dependency between Macrophages and Exhausted CD8 <sup>+</sup> T Cells in Cancer

T cell exhaustion is a major impediment to anti-tumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here we show that the biology of tumor-associated macrophages (TAM) and exhausted T cells (Tex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAM reduces exhaustion programs in tumor-infiltrating CD8+ T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ T cells engage in unique long-lasting antigen-specific synaptic interactions that fail to activate T cells, but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.

Coordinated co-migration of CCR10+ antibody-producing B cells with helper T cells for colonic homeostatic regulation.

In the intestine, IgA antibody-secreting B cells (IgA-ASCs) and helper T cells coordinate to maintain local homeostasis while their dysregulation could lead to development of intestinal inflammatory diseases. However, mechanisms underlying the coordinated localization and function of the B and T cells into the intestine, particularly the colon, are poorly understood. We herein report the first evidence that the gut-homing chemokine receptor CCR10+ IgA-ASCs form conjugates with helper T cells, preferentially regulatory T cells, at their differentiation sites of gut-associated lymphoid organs for their coordinated co-localization into the colon to promote local homeostasis. In CCR10-knockout mice, defective migration of IgA-ASCs also resulted in defective T cell migration and homeostasis, and development of inflammatory symptoms in the colon. Antigen-specific interaction of CCR10+ IgA-ASCs and T cells is crucial for their homeostatic establishment in the colon. On the other hand, in IgA-knockout mice, preferential expansion of CCR10+ IgG1-ASCs with regulatory functions compensated for CCR10+ IgA-ASCs to help maintain colonic homeostasis. The preferential expansion of specific subclasses of CCR10+ IgG-ASCs with regulatory functions was also found in asymptomatic IgA-deficient patients. These findings suggest coordinated cell migration as a novel mechanism underlying localization and function of B and T cells in colonic homeostatic regulation.

Pro-inflammatory Effect of Downregulated CD73 Expression in EAE Astrocytes.

CD73, an ectonucleotidase, participates in the regulation of immune responses by controlling the conversion of extracellular AMP to adenosine. In this study, we investigated whether any type of brain cells, especially neuroglia cells, exhibit altered CD73 expression, localization or activity upon experimental autoimmune uveitis (EAU) induction and whether altered CD73 manipulates the activation of effector T cells that interact with such cell types. First, the amount of cell membrane-exposed CD73 was detected by flow cytometry in various types of brain cells collected from either naïve or EAE mice. Compared to that in astrocytes from naïve control mice, the amount of membrane-bound CD73 was significantly decreased in astrocytes from EAE mice, while no significant differences were detected in other cell types. Thereafter, wild-type and CD73-/- astrocytes were used to study whether CD73 influences the function of inflammatory astrocytes, such as the production of cytokines/chemokines and the activation of effector T cells that interact with astrocytes. The results indicated that the addition of exogenous AMP significantly inhibited cytokine/chemokine production by wild type astrocytes but had no effect on CD73-/- astrocytes and that the effect of AMP was almost completely blocked by the addition of either a CD73 inhibitor (APCP) or an adenosine receptor A1 subtype (ARA1) antagonist (DPCPX). Although the addition of AMP did not affect CD73-/- astrocytes, the addition of adenosine successfully inhibited their cytokine/chemokine production. The antigen-specific interaction of astrocytes with invading CD4 cells caused CD73 downregulation in astrocytes from mice that underwent EAE induction. Collectively, our findings support the conclusion that, upon EAE induction, likely due to an interaction with invading CD4+ cells, astrocytes lose most of their membrane-localized CD73; this inhibits the generation of adenosine in the local microenvironment. As adenosine has anti-inflammatory effects on astrocytes and CNS-infiltrating effector T cells in EAE, the downregulation of CD73 in astrocytes may be considered a pro-inflammatory process for facilitating the pathogenesis of EAE.

Abstract IA11: Developing and improving personalized neoantigen-targeting cancer vaccines

Abstract At the heart of any cancer and host immune cell response is the tumor antigen and host antigen-specific T-cell interaction. The cytotoxic T cell-cognate antigen interaction forms the mechanistic basis for immune-mediated recognition and the killing of malignant cells. While the search for immunogenic tumor antigens has been the subject of decades-long studies, multiple lines of evidence have convincingly demonstrated tumor neoantigens as an important class of immunogenic tumor antigens. Neoantigens arise from amino acid changes encoded by somatic mutations in the tumor cell and have the potential to bind to and be presented by personal HLA molecules. Using next-generation sequencing approaches, we can now systematically identify mutations leading to amino acid changes that can be potentially recognized immunologically through the implementation of neoantigen discovery pipelines. In recent studies, we have tested the feasibility and immunogenicity of a vaccine that targets up to 20 personal neoantigens predicted to be presented by autologous patient tumor. This approach can expand the repertoire of neoantigen-specific T cells, providing a strong rationale for the further development of this approach, alone and in combination with checkpoint therapies. Citation Format: Catherine J. Wu. Developing and improving personalized neoantigen-targeting cancer vaccines [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA11.

Upregulation of bacterial-specific Th1 and Th17 responses that are enriched in CXCR5+ CD4+ T cells in non-small cell lung cancer

The microbial community in the mucosal surfaces is involved in the development of human cancers, including gastric cancer and colorectal cancer. The respiratory tract in the lung also hosts a distinctive microbial community, but the correlation between this community and lung cancer is largely unknown. Here, we examined the Th1 and Th17 responses toward several bacterial antigens, in CD4+ T cells sourced from the peripheral blood (PB), the lung cancer (LC) tissue, and the gastrointestinal (GI) tract of non-small cell lung cancer (NSCLC) patients. Compared to healthy controls, the NSCLC patients presented significantly higher frequencies of Th1 and Th17 cells reacting to Streptococcus salivarius and S. agalactiae, in the PB, LC, and GI tract. Further investigation showed that the upregulation in anti-bacteria response was likely antigen-specific for two reasons. Firstly, the frequencies of Th1 and Th17 cells reacting to Escherichia coli, a typical GI bacterium, were not upregulated in the PB and the LC of NSCLC patients. Secondly, the S. salivarius and S. agalactiae responses could be partially blocked by Tü39, a MHC class II blocking antibody, suggesting that antigen-specific interaction between CD4+ T cells and antigen-presenting cells was required. We also found that S. salivarius and S. agalactiae could potently activate the monocytes to secrete higher levels of interleukin (IL)-6, IL-12, and tumor necrosis factor, which were Th1- and Th17-skewing cytokines. Interestingly, whereas CXCR5+CD4+ T cells represented <20% of total CD4+ T cells, they represented 17%–82% of bacteria-specific Th1 or Th17 cells. Together, these data demonstrated that NSCLC patients presented a significant upregulation of bacterial-specific Th1 and Th17 responses that were enriched in CXCR5+CD4+ T cells.

The role of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 2 (IDO2) in initiation, development, and treatment of autoimmune disorders

Abstract The tryptophan catabolizing enzymes indoleamine 2,3-dioxygenase (IDO) 1 and 2 lie at the interface between metabolism and immunity. Recently, we identified a unique functional role for IDO2 in modulating the initiation and severity of autoimmune arthritis in a murine model of disease. We find that IDO2, but not its better-studied counterpart IDO1, acts as a pro-inflammatory mediator affecting autoantibody production and T helper cell function in the KRN preclinical model of arthritis. Reciprocal adoptive transfer experiments demonstrate that IDO2 acts by a B-cell intrinsic mechanism to regulate inflammation. IDO2 function in B cells was contingent on a cognate, antigen-specific interaction to exert its immunomodulatory effects on arthritis development. Alterations in costimulatory molecules and associated cytokines involved in cross-talk between B and T cells suggest that IDO2 acts at the T:B cell interface to modulate the potency of T cell help needed to promote autoantibody production. Given the paucity of novel therapies for rheumatoid arthritis and related autoimmune disorders, IDO2 should be considered as a potential novel therapeutic target for modulating disease pathways leading to autoimmunity. However, therapeutically targeting IDO2 has been challenging due to the lack of small molecules that specifically inhibit IDO2 without also affecting the closely related IDO1. To this end, we have recently begun development and characterization of approaches that ameliorate disease by specifically targeting IDO2. We find that we are able to recapitulate the reduction in arthritis seen in genetic knockouts with IDO2-targeted therapies in preclinical models of disease.

Abstract B34: Melanoma cells respond to CD8+ T cell attack by upregulation of genes associated with antigen processing and presentation

Abstract T cell-based immunotherapies have brought great progress for cancer patients, but many questions still remain open with regard to mechanisms of action and immune regulation in the tumor microenvironment. Methods: We setup a well-controlled co-culture system to study the dynamic T cell - cancer cell interplay. Low passage MelanA-expressing melanoma cell lines were cultured with MelanA-specific CD8+ T cells and characterized using differential gene expression analysis and flow cytometry. Results: As expected, significant fractions of melanoma cells died in presence of melanoma-specific CD8+ T cells. However, still some melanoma cells persisted during up to three days of co-culture. Characterization of the surviving melanoma cells revealed increased mRNA levels of genes associated with antigen processing and presentation: HLA Class I-encoding genes (HLA-A and HLA-B), HLA Class 2-encoding genes (HLA-DRA and HLA-DRB) and CD74, the invariant chain that stabilizes the complex of HLA Class II α and β chains until it is loaded with a peptide, were strongly increased after co-culture, as well as TAP1 and TAP2 (encoding transporter associated with antigen presentation), responsible for peptide transfer from the cytosol to the endoplasmic reticulum (Neefjes et al., 2011). HLA-Class I and HLA-DR expression was also strongly increased at the protein level as assessed at 48h of co-culture. These gene and protein expression changes were dependent on antigen-specific interaction of CTLs with melanoma cells and were mediated by IFNγ and TNFα, two cytokines secreted by CTLs upon antigen recognition. Discussion: So far, mainly immunosuppressive mechanisms of cancer cells upon exposure to CTLs were described, such as increased expression of IDO1 and the inhibitory receptor ligand PDL1 (Spranger et al., 2013). Here we show that further to these immunosuppressive mechanisms, melanoma cells respond to melanoma-specific CTLs with an IFNγ-driven upregulation of genes involved in antigen presentation. Increased antigen-presentation likely increases target cell recognition of CTLs. These findings may play a role in successful immunotherapy, and might explain why IFNγ blockade in situations of anti-tumor immune attack not only blocks the immunosuppressive response of melanoma cells but also the anti-tumor T cell response (McGray et al., 2013).

CD8+ T Cells Orchestrate pDC-XCR1+ Dendritic Cell Spatial and Functional Cooperativity to Optimize Priming

Adaptive cellular immunity is initiated by antigen-specific interactions between T lymphocytes and dendritic cells (DCs). Plasmacytoid DCs (pDCs) support antiviral immunity by linking innate and adaptive immune responses. Here we examined pDC spatiotemporal dynamics during viral infection to uncover when, where, and how they exert their functions. We found that pDCs accumulated at sites of CD8+ Tcell antigen-driven activation in a CCR5-dependent fashion. Furthermore, activated CD8+ Tcells orchestrated the local recruitment of lymph node-resident XCR1 chemokine receptor-expressing DCs via secretion of the XCL1 chemokine. Functionally, this CD8+ Tcell-mediated reorganization of the local DC network allowed for the interaction and cooperation of pDCs and XCR1+ DCs,thereby optimizing XCR1+ DC maturation and cross-presentation. These data support a model in which CD8+ Tcells upon activation create their own optimal priming microenvironment by recruiting additional DC subsets to the site of initial antigen recognition.

Abstract 4006: Oxaliplatin-induced immune factors and long-term disease control in rectal cancer - a reflection of immunogenic tumor cell death

Abstract Purpose: In contemporary cancer therapy, the ability of the immune system to recognize and destroy tumor cells is increasingly acknowledged. Following cytotoxic damage of tumor cells, the priming of tumor-targeting T lymphocytes via presentation of shed tumor antigens by dendritic cells may ultimately result in systemic anti-tumor activity, a concept referred to as immunogenic cell death (ICD). Recent preclinical studies have highlighted oxaliplatin as an ICD-inducing agent. In a prospective study (NCT00278694) of curatively intended treatment of locally advanced rectal cancer (LARC), oxaliplatin-containing induction neoadjuvant chemotherapy (NACT; two cycles of the Nordic FLOX regimen) was administered prior to chemoradiotherapy (CRT) and surgery. Within the spectrum of possible ICD responses to NACT, we analyzed circulating osteoprotegerin (OPG) and fms-related tyrosine kinase 3 ligand (FLT3LG), both of which are factors implicated in activation of the antigen-specific interaction between dendritic cells and cytotoxic T lymphocytes. Experimental procedures: Immunoassay measurements of OPG and FLT3LG were performed in serum samples collected from study patients at baseline and following the induction NACT. Estimated 5-year progression-free survival (PFS) and overall survival (OS) were assessed using the log-rank test. Results: Fifty-six patients (38 T2-3 cases and 18 T4 cases) with median follow-up time of 65 months were included in the analysis. During follow-up, 18 patients (nine in each of the T2-3 and T4 groups) experienced metastatic progression as a PFS event. For the entire study population, significant increases in serum levels of both OPG and FLT3LG (from median baseline values of 46.1 pg/ml and 66.7 pg/ml to median post-NACT values of 53.6 pg/ml and 140 pg/ml, respectively) were observed. Patients were separated into cases with (n = 37) or without (n = 19) increase in circulating OPG levels and more (n = 30) or less (n = 26) than two-fold increase in circulating FLT3LG levels. When grouped together, patients with the designated increases in both factors (n = 25) or in either of OPG or FLT3LG values (n = 21) during NACT had significantly better PFS rates than the remaining cases (n = 10) (86%, 72%, and 20%, respectively; p &amp;lt; 0.001). Corresponding OS rates were 100%, 88%, and 60% (p &amp;lt; 0.001), suggesting that even patients who experienced metastatic progression had reasonably good OS. Conclusion: Increase in circulating OPG and FLT3LG following oxaliplatin-containing induction chemotherapy was associated with favorable long-term outcome in LARC patients given curatively intended neoadjuvant treatment consisting of CRT and surgery. These immune effectors may have mediated systemic anti-tumor immunity invoked by ICD-inducing oxaliplatin effects. Citation Format: Sebastian Meltzer, Erta Kalanxhi, Svein Dueland, Kjersti Flatmark, Kathrine Røe Redalen, Anne Hansen Ree. Oxaliplatin-induced immune factors and long-term disease control in rectal cancer - a reflection of immunogenic tumor cell death. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4006.

Abstract 4900: Increased IgG antibody responses to neoepitope and native peptides containing high affinity domains for MHCI following combination cancer immunotherapy

Abstract The importance of anti-tumor immunity by CD8+ T cells is well defined, but antigen-specific immune monitoring for CD8+ responses across many potential tumor antigens is expensive and difficult. This difficulty is compounded by increasing evidence that every tumor has unique mutant neoepitopes. We hypothesized that new technologies in peptide printing might allow us to partially overcome this obstacle by instead characterizing antibody responses against a paired array of neoepitope and native peptides. We designed a 15-mer peptide array of mutated and native peptides from the murine mammary carcinoma 4T1 and used it to characterize the serum IgG antibody responses to vaccination with 4T1 autophagosome-enriched vaccine (DRibbles), poly-IC adjuvant, the combination of both, or naïve animals across 4 independent experiments. This array was printed by JPT peptides and included 75 pairs of single-nucleotide polymorphic and native peptides identified by both our own whole exome sequencing and the current 4T1 literature. We also included other sequences of interest to 4T1 immunity such as the retrovirus fragment AH1. MHCI peptide predictions were robust and used the top NetMHCpan v2.8 score from all possible windowed 8,9,10, and 11-mers for H2-Kd,Ld, and Dd surrounding the mutation site in its native protein context. The results of this work identify a linear correlation between higher predicted peptide MHCI binding affinity and the IgG antibody signals increased in combination treated groups versus controls. Linear regression analysis demonstrates that normalized antibody responses to mutated and native 4T1 peptides are more likely to be higher in sera from autophagosome-enriched vaccine plus poly-IC treated animals than naïve animals if these peptides contain higher affinity domains for MHCI (p&amp;lt;.0001). This correlation is not significant in the groups treated with vaccine or adjuvant alone. Animals pre-treated with this combination therapy also benefit from a significant delay in tumor growth upon challenge with 4T1 versus naïve animals (p&amp;lt;.001). These results identify a previously unknown link between predicted MHCI binding affinity and the anti-tumor antibody response following combination immunotherapy - suggesting some antigen-specific interaction between B cells and CD8+ T cells that might be exploited to improve the immune monitoring of cancer therapies. Citation Format: Tyler W. Hulett, Shawn Jensen, Christopher Dubay, Carlo Bifulco, Michael Afentoulis, Ashok Reddy, Larry David, Bernard A. Fox. Increased IgG antibody responses to neoepitope and native peptides containing high affinity domains for MHCI following combination cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4900.

Regulation of bifurcating B cell trajectories by mutual antagonism between transcription factors IRF4 and IRF8

Upon recognition of antigen, B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GCs). Here we identified a double-negative feedback loop between the transcription factors IRF4 and IRF8 that regulated the initial developmental bifurcation of activated B cells as well as the GC response. IRF8 dampened signaling via the B cell antigen receptor (BCR), facilitated antigen-specific interaction with helper T cells, and promoted antibody affinity maturation while antagonizing IRF4-driven differentiation of plasmablasts. Genomic analysis revealed concentration-dependent actions of IRF4 and IRF8 in regulating distinct gene-expression programs. Stochastic modeling suggested that the double-negative feedback was sufficient to initiate bifurcation of the B cell developmental trajectories.

Modes of Antigen Presentation by Lymph Node Stromal Cells and Their Immunological Implications.

Antigen presentation is no longer the exclusive domain of cells of hematopoietic origin. Recent works have demonstrated that lymph node stromal cell (LNSC) populations, such as fibroblastic reticular cells, lymphatic and blood endothelial cells, not only provide a scaffold for lymphocyte interactions but also exhibit active immunomodulatory roles that are critical to mounting and resolving effective immune responses. Importantly, LNSCs possess the ability to present antigens and establish antigen-specific interactions with T cells. One example is the expression of peripheral tissue antigens, which are presented on major histocompatibility complex (MHC)-I molecules with tolerogenic consequences on T cells. Additionally, exogenous antigens, including self and tumor antigens, can be processed and presented on MHC-I complexes, which result in dysfunctional activation of antigen-specific CD8+ T cells. While MHC-I is widely expressed on cells of both hematopoietic and non-hematopoietic origins, antigen presentation via MHC-II is more precisely regulated. Nevertheless, LNSCs are capable of endogenously expressing, or alternatively, acquiring MHC-II molecules. Transfer of antigen between LNSC and dendritic cells in both directions has been recently suggested to promote tolerogenic roles of LNSCs on the CD4+ T cell compartment. Thus, antigen presentation by LNSCs is thought to be a mechanism that promotes the maintenance of peripheral tolerance as well as generates a pool of diverse antigen-experienced T cells for protective immunity. This review aims to integrate the current and emerging literature to highlight the importance of LNSCs in immune responses, and emphasize their role in antigen trafficking, retention, and presentation.