Chronic Antigenic Stimulation Research Articles

PF366 CXCR5+PD‐1+ CD8+ T‐CELLS IN HUMAN BLOOD AND LYMPH NODES

Background:Re‐invigorating exhausted T‐cells through immune checkpoint blockade (ICB) has shown promising activity in several malignancies. However, response rates vary between malignancies, and it is unclear which parameters predict response to ICB. Recently, a subset of CXCR5+PD‐1+ CD8+ T‐cells has been identified in mice. This memory‐like population appears to be crucial for the expansion of CD8+ T‐cells during ICB, and is characterized by expression of the transcription factor Tcf1. CXCR5+ CD8+ T‐cells have also been identified in humans, but it's currently unknown whether this distinctive CD8+ T cell subset has a similar function as described in mice, and plays an equally important role during ICB.Aims:To investigate the human CXCR5+PD‐1+ CD8+ T‐cell subset, we studied peripheral blood and lymph node samples derived from healthy donors and patients with a variety of B cell malignancies.Methods:PB and LN samples were analyzed by flow cytometry.Results:The frequency of CXCR5+PD‐1+ CD8+ T‐cells was higher in lymph nodes. CXCR5+PD‐1+ CD8+ T‐cells resemble antigen‐experienced memory‐like cells, with high expression of KLRG1, CD127, granzyme K, and Eomes, and low expression of effector molecules granzyme B and T‐bet. They express high levels of Tcf1, in accordance with the phenotype described in mice. CXCR5+PD‐1+ CD8+ T‐cells were present within the EBV‐specific T‐cell population, especially directed against epitopes expressed during viral latency. In line with mice data, we could not detect CXCR5+PD‐1+ CD8+ T‐cells within the FLU‐specific T‐cell compartment, suggesting that chronic antigen stimulation is required for this population to develop. On a functional level, CXCR5+PD‐1+ CD8+ T cells produce effector cytokines IL‐2, IFNγ and TNFα after stimulation, in line with their memory‐like phenotype. CXCR5+PD‐1+ CD8+ T cells proliferate and differentiate into effector CD8+ T cells, with higher expression of granzyme B and loss of Tcf1.We assessed the role of CXCR5+PD‐1+ CD8+ T cells in ICB by analyzing their phenotype in samples derived from patients with various B cell malignancies with differential clinical response rates to ICB. In chronic lymphocytic leukemia (CLL), a disease with no objective response to ICB, CXCR5+PD‐1+ CD8+ T cells express lower levels of Tcf1 and markers compatible with an advanced differentiation state, and express lower levels of PD‐1. We did not observe these changes in CXCR5+PD‐1+ CD8+ T cells in B cell malignancies with better response rates to ICB, like DLBCL, multiple myeloma, and follicular lymphoma. Currently, we perform functional experiments in the context of checkpoint blockade to verify whether this changed phenotype is related to the disappointing response towards ICB in CLL. To explore the potential role of CXCR5+PD‐1+ CD8+ T cells for ICB therapy further, we are analyzing peripheral blood samples derived from cancer patients who receive ICB therapy while also studying the localization and frequency of CXCR5+PD‐1+ CD8+ T‐cells in relation to tumor cells within a spectrum of malignant tissues by confocal microscopy.Summary/Conclusion:In conclusion, we find that on a phenotypical and functional level, human CXCR5+PD‐1+ CD8+ T‐cells closely resemble this subset in mice. In CLL, in which ICB therapy does not lead to clinical responses, CXCR5+PD‐1+ CD8+ T‐cells have an altered phenotype which might explain the lack of response to ICB. Furthermore, if human CXCR5+PD‐1+ CD8+ T‐cells indeed play a similar important role in the response to ICB therapy as in mice, they could potentially serve as a biomarker for clinical response in human malignancies.

PRIMARY THYMIC MUCOSA‐ASSOCIATED LYMPHOID TISSUE LYMPHOMA: 7 CLINICAL CASES REPORT AND A REVIEW OF THE LITERATURE

Introduction: Mucosa-associated lymphoid tissue (MALT) lymphoma is one kind of non-Hodgkin lymphomas (NHL), accounts for 7% to 8%. MALT lymphoma is an indolent, organophilic lymphoma that can nearly occur at any epithelial tissues and mucosal sites. The predilection sites of MALT lymphoma are gastrointestinal tract, salivary glands, lungs and other organs. Studies have shown that MALT lymphoma is associated with chronic inflammation and antigenic stimulation, including chronic infection of pathogens and autoimmune disease. Primary thymus MALT lymphoma is rare. Only 92 cases have been reported over the past 27 years, and only 15 cases reported from China before. Here we introduce 7 cases of primary thymic MALT lymphoma found in the First Affiliated Hospital of Nanjing Medical University, and takes a review of the literature. Methods: We analyze 7 cases of primary thymic MALT lymphoma in the First Affiliated Hospital of Nanjing Medical University from December 2017 to February 2019. We evaluate the clinical features, laboratory test results, PET-CT scans, pathological specimen stained by hematoxylin–eosin (HE) or immunohistochemistry, etc (Figure 1). The median follow-up of 7 cases is 8 months (range 1 to 15 months). Results: primary thymic MALT lymphoma is a kind of indolent small lymphocytic lymphoma, appears mostly in Asian women and is often accompanied with AID. SS is the most common AID. Both contrast-enhanced CT and other imaging findings before surgery and pathological specimen after surgery can see mass with smooth boundary, containing cystic cavities of different sizes and numbers. We can also observe a wide range of small to medium-sized lymphocytes, with increased plasma cells surrounding small blood vessels. Primary thymic MALT lymphoma is lack of API2-MALT1 fusion gene. All cases only received “watch and wait” approach after surgery. Up to now, none of them had recurrence, and their quality of life was no different from normal people. Keywords: Mucosa-Associated Lymphoid Tissue (MALT); positron emission tomography (PET); thyroid function.

PS1303 CHRONIC TCR STIMULATION PROMOTES PERIPHERAL T‐CELL LYMPHOMAGENESIS AND INDUCES ADDICTION TO SYK AND NK RECEPTOR SIGNALING

Background:Peripheral T cell lymphomas (PTCL) represent about 10% of non‐Hodgkin lymphomas and are associated with poor prognosis. T‐cell receptor (TCR) signaling is the major growth‐regulatory machinery of normal T cells. Although its involvement in T‐cell lymphomagenesis has not been extensively investigated, various genes coding components of the TCR signaling are found altered in human PTCL and several reports indicates a predominant role of chronic antigenic stimulation in some PTCL.Aims:This study was conducted to investigate the role of TCR chronic stimulation in T‐cell lymphomagenesis and to identify crucial survival factors for lymphoma cells.Methods:To mimic chronic TCR stimulation, we took advantage of a phenomenon called homeostatic proliferation, which is known to be driven by TCR signaling generated by self‐MHC/TCR interactions. To this end, purified mature T cells from p53‐deficient mice were transferred into CD3epsilon‐deficient mice.Peripheral T‐cell lymphomas generated were analyzed using different approaches; flow cytometry, transcriptomic analysis, kinome, western blot, immunohistochemistry and by various in vitro or in vivo functional experiments.Human PTCL cases from Grenoble, Lyon and Tenomic or CeVi collections were analyzed using flow cytometry and immunochemistry.Results:Sixty percent of mice died from PTCL originating from conventional alpha‐beta T cells with an effector‐memory (CD44hi, CD62Llo, CD122hi) and exhausted (PD1hi) phenotype. Transcriptomic analysis reveals a chronic TCR activation signature, showing the role of TCR in lymphomagenesis.To study signals involved in lymphoma survival, we performed a kinome analysis showing a constitutive activation of the Syk kinase. Syk expression and activation was confirmed by western blot and constitutive activation of downstream targets of Syk such as PLCg1&2 and the PI3K‐Akt‐mTOR pathway was demonstrated. However, using in vivo experiments, we demonstrated that TCR signaling is not required for PTCL survival, suggesting that Syk may be involved in other signaling pathways. Interestingly, transcriptomic analyses of both mouse and human PTCLs showed an upregulation of a subset of genes involved in Natural Killer (NK) cells biology, such as NK receptors (NKRs) and signaling effectors of NKR, compared to normal T‐cells. This suggests that PTCL acquire “innate‐like” features during chronic TCR stimulation that would be implicated in cell survival and we next show that activatory NKRs are functional and signalize through SYK in murine PTCL. We also demonstrate that activatory NKRs participate to murine PTCL survival as in vivo NKR blockade delays PTCL evolution as well as Syk inhibition.We studied NKRs and Syk expression in a large subset of human PTCL and confirmed their various expression by many PTCL entities.Summary/Conclusion:Using a murine model, we show that chronic TCR stimulation is crucial for T‐cell lymphomagenesis and is associated with an ”innate like“ reprogramming. We characterized potential novel mechanisms of PTCL survival involving NKR and Syk signaling in murine PTCL that could lead to new therapeutic strategy development as a large proportion of human PTCL shares some common features with this murine PTCL generated in this experimental model.

T cell metabolism in chronic viral infection.

T cells are a fundamental component of the adaptive immune response in the context of both acute and chronic viral infection. Tight control over the metabolic processes within T cells provides an additional level of immune regulation that is interlinked with nutrient sensing and the continued balancing of co-stimulatory and co-inhibitory signals. Underpinning T cell responsiveness for viral control are a number of phenotypic and functional adaptations ensuring adequate nutrient uptake and their utilization. T cells responding to persistent viral infections often exhibit a profile associated with immune cell exhaustion and a dysregulated metabolic profile, driven by a combination of chronic antigenic stimulation and signals from the local microenvironment. Understanding alterations in these metabolic processes provides an important basis for immunotherapeutic strategies to treat persistent infections.

Nur77 Links Chronic Antigen Stimulation to B Cell Tolerance by Restricting the Survival of Self-Reactive B Cells in the Periphery.

Nur77 (Nr4a1) belongs to a small family of orphan nuclear receptors that are rapidly induced by BCR stimulation, yet little is known about its function in B cells. We have previously characterized a reporter of Nr4a1 transcription, Nur77-eGFP, in which GFP expression faithfully detects Ag encounter by B cells in vitro and in vivo. In this study, we report that Nur77 expression correlates with the degree of self-reactivity, counterselection, and anergy among individual B cell clones from two distinct BCR transgenic mouse models but is dispensable for all of these tolerance mechanisms. However, we identify a role for Nur77 in restraining survival of self-reactive B cells in the periphery under conditions of competition for a limited supply of the survival factor BAFF. We find that Nur77 deficiency results in the progressive accumulation of self-reactive B cells in the mature repertoire with age and is sufficient to break B cell tolerance in VH3H9 H chain transgenic mice. We thus propose that Nur77 is upregulated in self-reactive B cells in response to chronic Ag stimulation and selectively restricts the survival of these cells, gradually pruning self-reactivity from the mature repertoire to impose a novel layer of peripheral B cell tolerance.

Lymphomas complicating primary Sjögren's syndrome: from autoimmunity to lymphoma.

Lymphoma development is the most serious complication of SS and the main factor impacting on mortality rate in patients with this condition. Lymphomas in SS are most commonly extranodal non-Hodgkin B-cell lymphomas of the mucosa-associated lymphoid tissue and frequently arise in salivary glands that are the target of a chronic inflammatory autoimmune process. Extensive work on lymphomagenesis in SS has established that the progression towards B-cell lymphoma is a multistep process related to local chronic antigenic stimulation of B cells. These neoplastic B cells in SS frequently derived from autoreactive clones, most commonly RF-producing B cells, which undergo uncontrolled proliferation and malignant escape. In this review, we highlight the most important recent findings that have enhanced our understanding of lymphoma development in SS, with particular reference to the close link between autoimmunity and lymphomagenesis. We also discuss how the identification of key factors involved in B-cell malignancies may impact on our ability to identify at early stages patients at increased risk of lymphoma with potential significant repercussions for the clinical management of SS patients. Finally, we identified the most promising areas of current and further research with the potential to provide novel basic and translational discoveries in the field. The questions of finding new biomarkers, developing a validated score for predicting lymphoma occurrence and assessing if a better control of disease activity will decrease the risk of lymphoma in primary SS will be the enthralling questions of the next few years.

Serological Assessment of 18 Pathogens and Risk of AIDS-Associated Non-Hodgkin Lymphoma.

HIV infection is associated with increased susceptibility to common pathogens, which may trigger chronic antigenic stimulation and hyperactivation of B cells, events known to precede the development of AIDS-associated non-Hodgkin lymphoma (AIDS-NHL). To explore whether cumulative exposure to infectious agents contributes to AIDS-NHL risk, we tested sera from 199 AIDS-NHL patients (pre-NHL, average lead time 3.9 years) and 199 matched HIV-infected controls from the Multicenter AIDS Cohort Study, for anti-IgG responses to 18 pathogens using multiplex serology. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using conditional logistic regression models. We found no association between cumulative exposure to infectious agents and AIDS-NHL risk (OR 1.01, 95% CI: 0.91 to 1.12). However, seropositivity for trichodysplasia spinulosa polyomavirus (TSPyV), defined as presence of antibodies to TSPyV capsid protein VP1, was significantly associated with a 1.6-fold increase in AIDS-NHL risk (OR 1.62, 95% CI: 1.02 to 2.57). High Epstein-Barr virus (EBV) anti-VCA p18 antibody levels closer to the time of AIDS-NHL diagnosis (<4 years) were associated with a 2.6-fold increase in AIDS-NHL risk (OR 2.59, 95% CI: 1.17 to 5.74). In addition, high EBV anti-EBNA-1 and anti-ZEBRA antibody levels were associated with 2.1-fold (OR 0.47, 95% CI: 0.26 to 0.85) and 1.6-fold (OR 0.57, 95% CI: 0.35 to 0.93) decreased risk of AIDS-NHL, respectively. Our results do not support the hypothesis that cumulative exposure to infectious agents contributes to AIDS-NHL development. However, the observed associations with respect to TSPyV seropositivity and EBV antigen antibody levels offer additional insights into the pathogenesis of AIDS-NHL.

NR4A transcription factors limit CAR T cell function in solid tumours.

T cells expressing chimeric antigen receptors (CAR) targeting human CD19 (huCD19) have exhibited impressive clinical efficacy against B cell malignancies1,2. CAR-T cells have been less effective against solid tumors3–5, in part because they enter a hyporesponsive (“exhausted” or “dysfunctional”) state6–9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR-T cells in solid tumors, we transferred huCD19-reactive CAR-T cells into huCD19+ tumor-bearing mice. CD8+ CAR+ tumor-infiltrating lymphocytes (TILs) and endogenous TILs expressing inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors (TFs) Nr4a1 (Nur77), Nr4a2 (Nurr1) and Nr4a3 (Nor1) by the initiating TF NFAT (nuclear factor of activated T cells)10–12. CD8+ T cells from humans with cancer or chronic viral infections13,14,15 expressed high levels of Nr4a TFs and displayed enrichment of Nr4a binding motifs in accessible chromatin regions. CAR-T cells lacking all three Nr4a TFs (Nr4aTKO) promoted tumor regression and prolonged the survival of tumor-bearing mice. Nr4aTKO CAR-TILs displayed phenotypes and gene expression profiles characteristic of CD8+ effector T cells, and chromatin regions uniquely accessible in Nr4aTKO CAR-TILs compared to WT were enriched for binding motifs for NFκB and AP-1, TFs involved in T cell activation. Our data identify Nr4a TFs as major players in the cell-intrinsic program of T cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy.

Abstract A178: Nr4a transcription factors limit CAR T-cell function in solid tumors

Abstract In cancer immunotherapy, CD19-targeted CAR T-cells have exhibited impressive clinical efficacy against B cell leukemias and lymphomas; however, they have been less effective against solid tumors. This is in part because CAR T-cells enter a hyporesponsive (“exhausted” or “dysfunctional”) state that is triggered by chronic antigen stimulation and characterized by upregulation of several inhibitory receptors and loss of effector function. To identify transcriptional regulators and other candidates contributing to the diminished function of CAR T-cells in solid tumors, we developed a CAR T-cell model in which recipient mice bearing murine melanoma tumors expressing the human CD19 antigen were adoptively transferred with CD19-targeted CAR T-cells. Genome-wide analyses of these mouse tumor-infiltrating lymphocytes (TILs) showed that endogenous CD8+ TILs and CAR T TILs selected for expression of high levels of PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of the Nr4a nuclear receptor family of transcription factors by the transcription factor NFAT. We demonstrate that in both CAR T TILs and endogenous CD8+ TILs, the Nr4a proteins Nr4a1 (Nur77), Nr4a2 (Nurr1), and Nr4a3 (Nor1) are prominent effectors of the transcriptional program downstream of NFAT: they promote the expression of inhibitory receptors and genes associated with early stages of the exhausted/ dysfunctional state, and limit effector function. Most importantly, treatment of tumor-bearing mice with CAR T-cells lacking all three Nr4a transcription factors (Nr4aTKO) resulted in tumor regression and prolonged survival. Nr4aTKO tumor-infiltrating CAR T-cells displayed a gene expression profile characteristic of effector function, including increased expression of granzymes, cytokines and the transcription factor T-bet. Many of these gene expression changes were associated with altered regulatory element accessibility near effector genes. Our data identify Nr4a transcription factors as major players in the cell-intrinsic program of T-cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy. Citation Format: Joyce Chen, James P. Scott-Browne, Isaac F. Lopez-Moyado, Laura J. Hempleman, Hyungseok Seo, Takashi Sekiya, Akihiko Yoshimura, Anjana Rao. Nr4a transcription factors limit CAR T-cell function in solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A178.

Abstract A215: Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction

Abstract Tumor-specific CD8 T-cells in cancers enter a state of dysfunction characterized by the expression of inhibitory receptors and failure to produce effector cytokines and cytotoxic molecules. Here we identify the nuclear factor, Thymocyte selection-associated HMG box protein, TOX, as a master regulator of tumor-specific T-cell dysfunction. TOX is uniquely expressed in dysfunctional CD8 T-cells from mouse and human tumors but absent in functional T-cells. TOX expression is driven by continuous TCR stimulation and NFAT activity. Forced expression of TOX in functional effector T-cells was sufficient to induce a transcriptional program of dysfunction through the concerted expression of genes encoding numerous inhibitory receptors and dysfunction-associated transcription factors. Notably, TOX-deficient tumor-infiltrating T-cells did not upregulate inhibitory receptors such as PD1, LAG3, CD38, or CD39 and maintained high TCF1 expression. Surprisingly, despite their normal, “non-exhausted” phenotype, TOX-deficient T-cells failed to make effector cytokines, suggesting that loss of effector function in tumor-specific T-cells is uncoupled from inhibitory receptor expression. Furthermore, TOX-deficient T-cells failed to persist in tumors, ultimately undergoing activation-induced cell death. We propose that the TOX-induced transcriptional program of hyporesponsiveness is a physiologic negative feedback mechanism that prevents overstimulation; thus TOX is absolutely required for T-cell survival in the setting of chronic antigen stimulation as in cancers. Citation Format: Andrew C. Scott, Steven Camara, Peter Lauer, Alexandra Synder, Dmitriy Zamarin, Tyler Walther, Olivier Levy, Michael Glickman, Jonathan Kaye, Mary Philip, Andrea Schietinger. Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A215.

Fas/FasL signaling is critical for the survival of exhausted antigen-specific CD8+ T cells during tumor immune response

Antigen (Ag)-specific activated CD8+ T cells are critical for tumor elimination but become exhausted, and thus, dysfunctional during immune response against the tumor due to chronic antigen stimulation. The signaling of immune checkpoint receptors is known to be a critical component in this exhaustion; however, the fate of these exhausted CD8+ T cells remains unclear. Therefore, to elucidate this, we followed the fate of Ag-specific CD8+ T cells by directly visualizing them using MHC class I tetramers coupled with ovoalubumin257–264 in C57BL/6 mice inoculated with EG.7. We found that the number of generated Ag-specific activated CD8+ T cells decreased via apoptosis during a prolonged tumor immune response. However, the number of Ag-specific CD8+ T cells was significantly higher in Fas ligand (FasL)-dysfunctional gld mice than in control mice, resulting in suppressed tumor growth. In contrast, the enforced expression of Bcl-2 failed to rescue apoptosis of the exhausted CD8+ T cells following EG.7 inoculation. These results suggest that Fas/FasL signaling is critical for the survival of exhausted CD8+ T cells during the tumor immune response.

BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.

Origin and Pathogenesis of B Cell Lymphomas.

Immunoglobulin (IG) gene remodeling by V(D)J recombination plays a central role in the generation of normal B cells, and somatic hypermutation and class switching of IG genes are key processes during antigen-driven B cell differentiation. However, errors of these processes are involved in the development of B cell lymphomas. IG locus-associated translocations of proto-oncogenes are a hallmark of many B cell malignancies. Additional transforming events include inactivating mutations in various tumor suppressor genes and also latent infection of B cells with viruses, such as Epstein-Barr virus. Many B cell lymphomas require B cell antigen receptor expression, and in several instances, chronic antigenic stimulation plays a role in lymphoma development and/or sustaining tumor growth. Often, survival and proliferation signals provided by other cells in the microenvironment are a further critical factor in lymphoma development and pathophysiology. Many B cell malignancies derive from germinal center B cells, most likely because of the high proliferation rate of these cells and the high activity of mutagenic processes.

Hyper-N-glycosylated SAMD14 and neurabin-I as driver autoantigens of primary central nervous system lymphoma

AbstractTo address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper-N-glycosylated (SAMD14 at ASN339 and neurabin-I at ASN1277), explaining their autoimmunogenicity. SAMD14 and neurabin-I induced BCR pathway activation and proliferation of aggressive lymphoma cell lines transfected with SAMD14- and neurabin-I-reactive BCRs. Moreover, the BCR binding epitope of neurabin-I conjugated to truncated Pseudomonas exotoxin-killed lymphoma cells expressing the respective BCRs. These results support the role of chronic antigenic stimulation by posttranslationally modified central nervous system (CNS) driver autoantigens in the pathogenesis of PCNSL, serve as an explanation for their CNS tropism, and provide the basis for a novel specific treatment approach.

Regulation of TIM-3 expression in a human T cell line by tumor-conditioned media and cyclic AMP-dependent signaling

T cell immunoglobulin and mucin domain-3 (TIM-3) expression increases in exhausted T cells, which inhibits T cell function. TIM-3 expression is supposedly up-regulated in tumor-bearing individuals via chronic antigenic stimulation of T cells. Considering the immunosuppressive nature of the tumor microenvironment, we investigated whether tumor-secreted molecules might enhance TIM-3 expression in Jurkat T cells. We observed that TIM-3 expression was increased by the activation of prostaglandin (PG) E2 and cyclic AMP (cAMP) signaling pathways. Adenylate cyclase activation led to protein kinase A (PKA)-dependent upregulation of the TIM-3 minimal promoter region and of upstream conserved non-coding sequences. TIM-3 expression in Jurkat T cells was increased by the exposure to breast tumor cell-conditioned media partially through the interaction between PGE2 and its receptor, EP4. Our results propose that tumor-secreted molecules such as PGE2, which activates PKA and EPAC, may regulate TIM-3 expression in T cells.

P53 Isoform D133p53a: A Novel Transcriptional Enhancer of T-Cell Effector Function to Improve T-Cell Based Cancer Immunotherapy

Background:Adoptive transfer of genetically modified T lymphocytes with tumor antigen-specific receptor has proven efficacy in cancer immunotherapy. However, in many patients the overall benefit is still limited due to various tumor escape mechanisms. Cell damage and metabolic/hypoxic stress in the tumor microenvironment (TME) can lead to a dysfunctional anti-tumor T cell response called T cell senescence. The tumor suppressor TP53 is a master molecule in the regulation of cell cycle and senescence. Few studies have demonstrated the critical role of p53 isoforms in the regulation of cellular senescence mainly in tumor cells. However, their role in tumor infiltrating lymphocytes (TILs) remains largely unexplored.Aims:Strategies to prevent T cell senescence in the TME could improve T cell function and thus anti-tumor response. To better understand the role of D133p53 isoform in regulating the cell cycle and senescence we studied the cellular and metabolic/energetic phenotype as well as the effector function of the D133p53-modified tumor-antigen (TA) specific human T cells. We further aimed at identifying the mechanism that may regulate this phenotype.Methods:T cells form healthy donors were retrovirally co-transduced with a TA-specific T cell receptor (TCR) together with the D133p53 isoform or an empty control vector. Modified T cells were characterized for the expression of key activating/inhibitory molecules, homing markers and for their proliferation capacity by flow cytometry. Additionally, we determined the metabolic and energetic phenotype of the cells with an Agilent Seahorse XFp Analyzer. The effector functions i.e. cytokine secretion and antigen-specific killing capacity were assessed by Luminex immunoassay and long-term tumor colony-forming assay, respectively. In an attempt to identify molecules/pathway contributing to this phenotype we performed quantitative proteomic-based analysis.Results:Our analyses of human T cells simultaneously engineered with D133p53a-isoform and a TA-specific TCR revealed reduced cell surface expression of T-cell inhibitory molecules (i.e. PD-1 or TIGIT), senescence markers (CD57, CD160) and increased expression of the homing receptor CD62L upon TA stimulation. First comparative analyses between D133p53a-modified and control T-cells revealed changes in the cell's metabolic and energetic program similar to quiescent/naïve T cells. D133p53a-T cells exhibited lower ATP production, oxygen consumption as well as lower glucose utilization. Upon antigen-specific stimulation, however, they increased their metabolic activity up to the levels of control cells. Importantly, while control T cells exhibited replicative senescence after chronic antigen stimulation, D133p53a-expressing T cells remained highly proliferative, showed superior cytokine secretion and enhanced tumor-specific killing capacity. Comparative proteomic analysis revealed significant differences in more than 100 proteins. Detailed pathway and network analysis as well as validation of the most significantly changing proteins is currently performed.Conclusion:By providing insights in the regulation of T cell metabolic changes and underlying mechanisms that limit immunosenescence, genetic modification with p53 isoforms could be a promising strategy to circumvent tumor-mediated T cell dysfunction and represents a novel approach with high potential for cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Circulating B Cell Subsets from Untreated Diffuse Large B Cell Lymphoma (DLBCL) Patients Resemble Those of Patients with Autoimmune Disease

IntroductionDiffuse large B cell lymphoma (DLBCL) is a clinically and genetically heterogeneous malignancy that can arise from B cell-mediated autoimmune (AI) diseases such as systemic lupus erythematosus (SLE). Profiling B cell subsets by multicolor flow cytometry can delineate SLE pts from healthy controls and correlates with differences in SLE outcomes (Tipton CM, Nat Immunol 2015). Emerging data suggest that expansions of IgD-CD27- (“double-negative,” DN) memory B cells in SLE are comprised of a novel subset representing pre-plasma cell effector B cells (Sanz MS under review), with at least 15% of DN B cells expressing autoantibodies. Given the etiologic link between autoimmunity and DLBCL, we hypothesized that B cell profiling in DLBCL patients (pts) with and without concomitant AI disease could identify variations in B cell repertoire similar to those observed in SLE patients and reveal links to distinct subtype variations based on biology and etiology.MethodsPts with DLBCL were prospectively identified and consented to participate. All pts were followed longitudinally for clinical data annotation and sample re-collection. Peripheral blood mononuclear cells were isolated from whole blood using density-gradient centrifugation, stained with a 13-color cocktail that included fluorochrome-conjugated mouse monoclonal antibodies to CD3, CD11c, CD19, CD20, CD21, CD24, CD27, CD38, IgD, and Ig light chain kappa and lambda, and then analyzed via flow cytometry with gating analysis using FlowJo. After excluding dead cells, doublets, and non-lymphocytes, we gated on CD19+ B cells and assessed subpopulations based first on expression of IgD and CD27 to distinguish mature-naïve B cells (IgD+CD27-) from switched (IgD-CD27+), unswitched (IgD+CD27+), and DN (IgD-CD27-) memory B cells. Additional cell surface markers (e.g., CD24, CD38) defined other subsets such as antibody-secreting cells and transitional B cells. We examined pre-treatment B cell profile to characterize baseline variations in DLBCL. To examine changes in B cell profile relative to treatment, DLBCL pt samples were evaluated: after completion of standard first-line chemoimmunotherapy; in remission ≥ 1 year from treatment; and at relapse. The Kruskal-Wallis test was used to compare B cell subset distributions between samples.ResultsPeripheral blood was collected from a total of 59 DLBCL pts, with 8 pts contributing samples at >1 time-point. B cell profiling of pre-treatment samples (n=12) revealed two distinct phenotypes according to CD27 and IgD expression: 7 pts' profiles resembled those of healthy controls (42%), and 5 exhibited diminished unswitched memory B cells (<10% of CD19+ cells), similar to SLE pts (Figures 1 and 2). Within the latter group, 4 pts also exhibited DN expansion (>10% of CD19+ cells), a phenotype similar to SLE pts with poor outcomes. Plasmablasts, transitional B cells, switched memory and naïve B cells in untreated DLBCL were not significantly expanded. As expected, CD19+ B cells were uniformly depleted following chemoimmunotherapy, constituting <1% lymphocytes until about 9 months post-treatment. Prior to B cell reconstitution, this small B cell population consisted predominantly of DN cells (Figure 3).ConclusionsIntriguingly, a subset of untreated DLBCL pts exhibit diminished unswitched memory B cells and DN expansion characteristic of SLE pts. It is unclear whether the DN expansion we detected in non-AI-associated DLBCL represents an immune response to tumor or a state of subclinical immune dysregulation that predisposes to DLBCL development, perhaps via chronic antigen stimulation as postulated in AI-associated lymphomas. Longitudinal studies are ongoing to evaluate associations between abnormal B cell profiles, clinical factors, and survival outcomes [Display omitted] DisclosuresFlowers:BeiGene: Research Funding; TG Therapeutics: Research Funding; Gilead: Research Funding; Abbvie: Research Funding; Pharmacyclics/ Janssen: Consultancy; Bayer: Consultancy; Acerta: Research Funding; Karyopharm: Consultancy; Genentech/Roche: Consultancy; National Cancer Institute: Research Funding; Celgene: Research Funding; Millennium/Takeda: Research Funding; Spectrum: Consultancy; Genentech/Roche: Research Funding; Janssen Pharmaceutical: Research Funding; V Foundation: Research Funding; OptumRx: Consultancy; Burroughs Wellcome Fund: Research Funding; Pharmacyclics: Research Funding; Abbvie: Consultancy, Research Funding; Eastern Cooperative Oncology Group: Research Funding; Gilead: Consultancy; Denovo Biopharma: Consultancy.

Association between Monoclonal Gammopathy and Rheumatoid Arthritis: A National Inpatient Sample (NIS) Study

Introduction: Monoclonal gammopathy (MG) is a spectrum of conditions including monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). MM is the second most common hematologic malignancy. Despite advances in the treatment of MM it remains an incurable disease that accounts for > 80 000 deaths annually worldwide. Many etiologies for myelomagenesis have been postulated. One theory suggests that chronic antigen stimulation from rheumatologic conditions can increase the risk of MG. Numerous studies have reported an increased risk of MG and other lymphoproliferative disorders in patients with systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS). However, the association between rheumatoid arthritis (RA) and MG is controversial as conflicting results have been reported. Case control studies have described an increased risk of RA but this finding was not corroborated in a subsequent meta-analysis. We used the national inpatient sample (NIS) database to explore the association of RA with MGUS and MM.Methods: The NIS is a validated database derived from discharge billing information which provides a sample of all non-federal hospital discharges in the Unites States. We identified adult patients with discharge diagnosis codes for MGUS (ICD-9 273.1), MM (ICD-9 203.0), RA (ICD-9 714.0), SLE (ICD-9 710.0) and SS (ICD-9 710.2) from 2014. Demographic data such as age, sex and race were also obtained. Multiple logistic regressions were used to analyze data for association of RA with MGUS and MM after adjusting for age, gender, race and other rheumatic diseases. Statistical analysis was performed with STATA 14.Results: A total of 7,071,762 discharges were analyzed. There were 20,066 hospitalized adults with discharge diagnosis codes for MM and 6,433 patients with a discharge diagnosis of MGUS. The prevalence of RA in patients with MGUS is 5.1% (p<0.001) and the prevalence of RA in patients with MM is 1.6 % (p=0.92). By logistic regression MGUS was associated with RA (OR 2.04, CI 1.80 - 2.32, p<0.0001) when adjusted for age, gender, race, SLE and SS. However, logistic regression did not show a significant association between MM and RA when adjusted for the same variables (OR 1.05, CI 0.95-1.15, p=0.23). MGUS was significantly associated with SS (OR 4.4, CI 3.29-5.90, p<0.001) and SLE (OR 1.9, CI 1.44-2.43, p<0.001) when adjusted for variables.Discussion: Our review of the NIS database found a significant association between rheumatologic disorders (RA, SLE and SS) and MGUS. These results are in concordance with previous retrospective studies. However, we did not find a significant relationship between RA and MM. There is known to be a high prevalence of MGUS in the general population, the clinical significance of which is being debated. The association between RA and MGUS that our study found may be due to a selection bias as patients with rheumatic conditions such as RA are tested more frequently for MGUS than the general population. DisclosuresNo relevant conflicts of interest to declare.

Highly competent, non-exhausted CD8+ T cells continue to tightly control pathogen load throughout chronic Trypanosoma cruzi infection.

Trypanosoma cruzi infection is characterized by chronic parasitism of non-lymphoid tissues and is rarely eliminated despite potent adaptive immune responses. This failure to cure has frequently been attributed to a loss or impairment of anti-T. cruzi T cell responses over time, analogous to the T cell dysfunction described for other persistent infections. In this study, we have evaluated the role of CD8+ T cells during chronic T. cruzi infection (>100 dpi), with a focus on sites of pathogen persistence. Consistent with repetitive antigen exposure during chronic infection, parasite-specific CD8+ T cells from multiple organs expressed high levels of KLRG1, but exhibit a preferential accumulation of CD69+ cells in skeletal muscle, indicating recent antigen encounter in a niche for T. cruzi persistence. A significant proportion of CD8+ T cells in the muscle also produced IFNγ, TNFα and granzyme B in situ, an indication of their detection of and functional response to T. cruzi in vivo. CD8+ T cell function was crucial for the control of parasite burden during chronic infection as exacerbation of parasite load was observed upon depletion of this population. Attempts to improve T cell function by blocking PD-1 or IL-10, potential negative regulators of T cells, failed to increase IFNγ and TNFα production or to enhance T. cruzi clearance. These results highlight the capacity of the CD8+ T cell population to retain essential in vivo function despite chronic antigen stimulation and support a model in which CD8+ T cell dysfunction plays a negligible role in the ability of Trypanosoma cruzi to persist in mice.

BRONCHIAL-ASSOCIATED LYMPHOID TISSUE LYMPHOMA (BALTOMA) PRESENTING AS A NONRESOLVING PNEUMONIA

SESSION TITLE: Lung Pathology 2 SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/09/2018 01:15 PM - 02:15 PM INTRODUCTION: Pulmonary mucosa-associated lymphoid tissue lymphoma (also known as bronchial associated lymphoid tissue lymphoma or BALToma) is a rare type of extranodal marginal zone B-cell lymphoma. Marginal zone lymphomas in other parts of the body have been associated with chronic antigen stimulation, such as in autoimmune disease. Pathogenesis from chronic infection has been hypothesized but a causative organism or antigen has not been identified for the lungs. We present a soldier with initial infectious symptoms who had persistent dense pulmonary consolidation that was found to be a BALToma. CASE PRESENTATION: 48 year old male underwent chest x-ray for cough and dyspnea on exertion that developed shortly after returning from deployment to Afghanistan. He was found to have a right middle lobe consolidation on chest radiograph that was redemonstrated along with multiple bilateral ground glass opacities on confirmatory chest CT. His symptoms resolved after treatment with oral azithromycin and follow-up chest radiograph 3 weeks later showed improvement in the right middle lobe consolidation. Chest imaging two years later while asymptomatic revealed persistent, slightly enlarged right middle lobe consolidation and persistent bilateral ground glass lesions. PET-CT of the chest, abdomen, and pelvis showed hypermetabolism (SUV-m of 3.7) limited to the pulmonary mass without any other hypermetabolic foci. There was a single pathologic-sized right paratracheal lymph node measuring up to 1.5 cm that showed normal FGD activity. Bronchoscopy with bronchial brushing, fine needle aspiration, and transbronchial forceps biopsy of the mass revealed a dense infiltrate composed of small B-cells that tested positive for the MALT1 translocation (t(11;18)), consistent with a BALToma. No infectious organism was isolated. DISCUSSION: Bronchial associated lymphoid tissue lymphoma is a slow growing malignant pulmonary tumor. Clinical manifestations are usually nonspecific and the majority of patients are asymptomatic at diagnosis. Biopsy sample of a BALToma are characterized by presence of a nodular lymphoid infiltrate expanding the marginal zone of reactive lymphoid follicles with tumor cells infiltrating the bronchiolar or the alveolar epithelium. The diagnosis is confirmed via immunohistochemical analysis that excludes lung localization of other lymphomas such as mantle cell and follicular lymphoma. PCR or FISH probes for the MALT1 gene can be supportive. Diagnosis may be missed due to the radiographic similarity to community acquired pneumonia, but appropriate diagnosis is important since this process typically responds well to therapy. CONCLUSIONS: BALToma should be considered in the differential of asymptomatic patients with imagining suggest of persistent nonresolving pneumonia. Reference #1: Borie R, Wislez M, Antoine M, et al. Pulmonary mucosa-associated lymphoid tissue lymphoma revisited. Eur Respir J. 2016 Apr;47(4):1244-60 DISCLOSURES: No relevant relationships by Benjamin Rosen, source=Web Response No relevant relationships by Krystle Salazar, source=Web Response No relevant relationships by John Sherner, source=Web Response