Impaired Antigen Presentation Research Articles

Abstract 627: NLRC5 co-mutations are associated with impaired antigen presentation and immune exclusion in KRAS-mutant lung adenocarcinoma

Abstract Background: We recently reported that co-occurring genetic events constitute major determinants of the molecular diversity of KRAS-mutant lung adenocarcinoma (LUAC) (Skoulidis et al., Cancer Discovery, 2015). However, comprehensive evaluation of the functional impact of KRAS co-mutations on key cancer hallmarks is thus far lacking. Here, we find that inactivating mutations in NLRC5, a major transactivator of MHC class I molecules, are significantly enriched in KRAS-mutant LUAC and examine the impact of NLRC5 loss on the composition of the tumor immune microenvironment. Methods: Our cohorts consist of 513 LUACs from the TCGA (145 KRAS-mutant), 152 chemotherapy-naïve surgically resected LUAC from the PROSPECT cohort, 20 platinum-refractory KRAS-mutant LUAC from the BATTLE-2 clinical trial, as well as a panel of 31 KRAS-mutant NSCLC cell lines. Analysis of immune cell sub-population was performed using automated IF-based enumeration. Antigen presentation score was defined as the geometric mean mRNA expression of HLA-A, HLA-B, HLA-C and β2M. Results: In an unbiased analysis for genes significantly co-mutated with KRAS in LUAC (TCGA cohort) we identified NLRC5 (NLR family, CARD domain containing 5), encoding a recently discovered major transactivator of MHC class I genes (~11% of KRAS-mutant LUAC, odds ratio 2.99, P=0.0197).The spectrum of NLRC5 somatic mutations includes several nonsense and frameshift mutations, as well as missense mutations, many of which are predicted to abrogate normal NLRC5 function. In the TCGA cohort, KRAS/NLRC5 co-mutated tumors exhibited lower antigen presentation score compared to KRAS-mutant NLRC5 wild-type tumors (P=0.0369, t-test). Among KRAS-mutant LUAC from the TCGA, PROSPECT, BATTLE-2 cohorts expression of NLRC5 mRNA correlated tightly with the expression of core antigen presentation pathway components including HLA-A, HLA-B, HLA-C, β2M, TAP1, TAP2, PSMB8 and PSMB9 [in BATTLE-2: HLA-A r=0.7616, P=9.572e-05, HLA-B r=0.834, P=4.884e-06, HLA-C r=0.8029, P=2.036e-05, TAP1 r=0.8189, P=1.009e-05 ]. Similar results were obtained in a panel of 31 KRAS-mutant NSCLC cell line. Thus, both mutational and non-mutational mechanisms can account for NLRC5 inactivation. Finally, in a tumor microarray encompassing surgically resected, chemotherapy naïve LUAC (PROSPECT), NLRC5-low tumors (lower tertile for NLRC5 mRNA expression, N=34), exhibited reduced density of infiltrating CD3+ (P<0.0001, Mann-Whitney U test), CD8+(P<0.0001, Mann-Whitney U test) as well as PD-1+ cells (P<0.0001, Mann-Whitney U test) and PD-L1 Histo-score (P=0.0036, Mann-Whitney U test) compared to NLRC5-high tumors (N=41). Conclusions: Co-mutations in NLRC5, are enriched in KRAS-mutant LUAC and are associated with immune exclusion. KRAS co-mutations can shape the tumor immune micro-environment and may therefore predict for response - or lack thereof- to immunotherapy. Citation Format: Ferdinandos Skoulidis, Taghreed Hirz, Xiang Dong Lee, Jaime Rodriguez Canales, Edwin R. Parra, Pan Tong, Carmen Behrens, Vassiliki A. Papadimitrakopoulou, Jing Wang, Ignacio Wistuba, John V. Heymach. NLRC5 co-mutations are associated with impaired antigen presentation and immune exclusion in KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 627. doi:10.1158/1538-7445.AM2017-627

Abstract 3663: An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice

Abstract Administration of an anti-CD4 depleting antibody induces tumor-specific CD8+ T cell responses in tumor-bearing mice, although the mechanisms underlying this phenomenon remain unclear. CD4+ Foxp3+ regulatory T cells (Treg) impair antigen presentation by DCs through CTLA4-mediated down-regulation of CD80/CD86. In the present study, we investigated the effects of an anti-CD4 depleting monoclonal antibody (mAb) on DC responses in the B16F10 and LLC subcutaneous tumor models, and compared these effects with those of Treg targeting therapy. Administration of anti-CD4 mAb up-regulated CD80/CD86 in tumor-infiltrating DCs, and in migrating-type but not resident-type DC subsets in the tumor-draining lymph node. The upregulation of CD80/CD86 on these DC populations was correlated with increased proliferation of tumor-specific CD8+ T cells in the tumor-draining lymph node. Anti-CD4 mAb-induced CD80/CD86 up-regulation and tumor-specific CD8+ T cell responses were remarkably higher than that induced by an anti-CD25 depleting mAb or an anti-CTLA4 blocking mAb, and were equivalent to that induced by diphtheria-toxin-induced selective depletion of Tregs in Foxp3-DTR mice. Importantly, the activation of CD4+ conventional T cells and B cells in the peripheral lymph nodes that was observed following Treg depletion was not observed following anti-CD4 mAb administration. In conclusion, anti-CD4 depleting antibody therapy is an effective way to evoke anti-tumor CD8+ T cell responses by reversing regulatory T cell-induced suppression of dendritic cells, and to reduce the risk of severe autoimmunity in cancer patients. Citation Format: Satoshi Ueha, Haru Ogiwara, Shoji Yokochi, Yoshiro Ishiwata, Francis Shand, Shohei Hori, Kazuhiro Kakimi, Satoru Ito, Kouji Matsushima. An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3663. doi:10.1158/1538-7445.AM2017-3663

Abstract 422: Lymph node metastasis evolution drives immune evasion and targeted therapy resistance in gastro-esophageal adenocarcinomas (GEAs)

Abstract GEAs are aggressive tumors in which several targeted therapy trials have failed. We assessed intratumor heterogeneity (ITH) and its impact on progression and therapy failure by applying an 81-gene NGS panel and SNP array copy number aberration (CNA) analysis to multiple primary tumor (T) regions and lymph node (LN) metastases from 9 GEAs. Analysis of 39 samples found ITH in all cases. 8 chromosomally instable (CIN) GEAs predominantly evolved through CNAs, with 17-76% of the genome affected by heterogeneous CNAs. A microsatellite instable GEA showed parallel evolution and diversified exclusively through point mutations (58% ITH). This demonstrates ongoing genomic instability rather than punctuated evolution and that specific instability mechanisms impact evolutionary trajectories. LN metastases contributed more to ITH (p<0.01) than any anatomic location within T. Further, subclonal aberrations that activate the Mitogen Activated Protein Kinase-pathway (MAPK-pw), including ERBB3, ERK2, KRAS and NRAS amplifications (amp) and NRAS mutations, were detected in LN metastases from 4/8 CIN GEAs. Subclonal MAPK-activating amp were enriched in LN (p=0.019) compared to T regions that only exhibited a single subclonal MET amp. Convergent evolution of LN subclones across several GEAs suggests that selection pressures differ systematically between LN and T ecosystems. To assess the phenotypes established by MAPK-activating amp evolution, we analyzed 135 published primary CIN subtype GEAs. Cytolytic activity (CYT), estimating tumor immune recognition from RNA expression data, correlated with the mutation load in GEAs with EGFR, ERBB2 or MET amp (p=0.04). In contrast, CYT did not correlate with mutation load in GEAs with KRAS or ERBB3 amp (p=0.22, NRAS/ERK2: insufficient data), indicating that these specific alterations, that also recurrently evolved in LN, may enable immune evasion. Downregulation of TAP and Class I MHC genes (p<0.05) in KRAS or ERBB3 amp GEAs suggested impaired antigen processing and presentation as the mechanisms driving T cell immune evasion. Moreover, ITH of MAPK-activating amp is likely to confer resistance to upstream tyrosine kinase inhibition. We used GEA cell lines with various MAPK-activating amp (ERBB2, MET, NRAS) to investigate downstream MAPK-pw inhibition as a novel strategy to broadly target heterogeneous subclones. Growth control was incomplete with ERK- and MEK-inhibitors but the panRAF/SRC inhibitor CCT196969 was effective in all lines, suggesting that it can effectively intercept subclonal heterogeneity in GEAs. In conclusion, we identified ITH with parallel and convergent evolution in 9/9 metastatic GEAs. Distinct selection pressures in LN foster the evolution of subclonal MAPK-activating amp that decrease immunogenicity and drive evolutionary pre-adaptation to future targeted drugs that can be intercepted by panRAF/SRC inhibitors. Citation Format: Matthew N. Davies, Louise J. Barber, Georgia Spain, Filipa Lopes, Katharina von Loga, Beatrice Griffiths, Andrew Woolston, Donat Alpar, Marta Gomez, Kamil A. Lipinski, Kerry Fenwick, Zakaria Eltahir, Stefano Lise, Emese I. Agoston, Laszlo Harsanyi, Richard Marais, Andrew Wotherspoon, A Szasz, Caroline Springer, Marco Gerlinger. Lymph node metastasis evolution drives immune evasion and targeted therapy resistance in gastro-esophageal adenocarcinomas (GEAs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2017-422

Cytokine mediators of chronic graft-versus-host disease.

Substantial preclinical and clinical research into chronic graft-versus-host disease (cGVHD) has come to fruition in the last five years, generating a clear understanding of a complex cytokine-driven cellular network. cGVHD is mediated by naive T cells differentiating within IL-17-secreting T cell and follicular Th cell paradigms to generate IL-21 and IL-17A, which drive pathogenic germinal center (GC) B cell reactions and monocyte-macrophage differentiation, respectively. cGVHD pathogenesis includes thymic damage, impaired antigen presentation, and a failure in IL-2-dependent Treg homeostasis. Pathogenic GC B cell and macrophage reactions culminate in antibody formation and TGF-β secretion, respectively, leading to fibrosis. This new understanding permits the design of rational cytokine and intracellular signaling pathway-targeted therapeutics, reviewed herein.

Tregitopes and impaired antigen presentation: Drivers of the immunomodulatory effects of IVIg?

IntroductionAlthough intravenous immunoglobulin (IVIg) is commonly used in the clinic to treat various autoimmune and severe inflammatory diseases, the mode of action is not fully elucidated. This work investigates two proposed mechanisms: (1) the potential role of regulatory T‐cell epitopes (Tregitopes) from the constant domain of IgG in the immunosuppressive function of IVIg; and (2) a potential impact of IVIg on the ability of antigen presenting cells (APCs) to present peptides.Methods and ResultsInvestigation of the HLA class II peptide repertoire from IVIg‐loaded dendritic cells (DCs) via MHC‐associated peptide proteomics (MAPPs) revealed that numerous IgG‐derived peptides were strongly presented along the antibody sequence. Surprisingly, Tregitopes 167 and 289 did not show efficient natural presentation although they both bound to HLA class II when directly loaded as “naked” peptides on human DCs. In addition, both Tregitopes could not reproduce the inhibitory effect of IVIg in a human in vitro T‐cell proliferation assay as well as in vivo in mice. MAPPs data demonstrate that presentation of peptides from several antigens remained unchanged even when competed with high doses of IVIg, in both human and mouse.ConclusionThese data suggest that the effects mediated by IVIg are not caused by Tregitopes nor by impaired antigen presentation.

Inhibition of endocytic lipid antigen presentation by common lipophilic environmental pollutants

Environmental pollutants as non-heritable factors are now recognized as triggers for multiple human inflammatory diseases involving T cells. We postulated that lipid antigen presentation mediated by cluster of differentiation 1 (CD1) proteins for T cell activation is susceptible to lipophilic environmental pollutants. To test this notion, we determined whether the common lipophilic pollutants benzo[a]pyrene and diesel exhaust particles impact on the activation of lipid-specific T cells. Our results demonstrated that the expression of CD1a and CD1d proteins, and the activation of CD1a- and CD1d-restricted T cells were sensitively inhibited by benzo[a]pyrene even at the low concentrations detectable in exposed human populations. Similarly, diesel exhaust particles showed a marginal inhibitory effect. Using transcriptomic profiling, we discovered that the gene expression for regulating endocytic and lipid metabolic pathways was perturbed by benzo[a]pyrene. Imaging flow cytometry also showed that CD1a and CD1d proteins were retained in early and late endosomal compartments, respectively, supporting an impaired endocytic lipid antigen presentation for T cell activation upon benzo[a]pyrene exposure. This work conceptually demonstrates that lipid antigen presentation for T cell activation is inhibited by lipophilic pollutants through profound interference with gene expression and endocytic function, likely further disrupting regulatory cytokine secretion and ultimately exacerbating inflammatory diseases.

Lipophilic air pollutants inhibit endocytic lipid antigen presentation

Abstract Air pollutants as non-heritable factors are recognized as triggers for various human inflammatory diseases, in which T cells are central in producing cytokines and determining disease outcomes. We postulated that lipid antigen presentation mediated by cluster of differentiation 1 (CD1) proteins for T cell activation is susceptible to lipophilic air pollutants. To test this notion, we determined whether common lipophilic pollutants benzo[a]pyrene and diesel exhaust particles impact on the activation of lipid-specific T cells. Our results demonstrated that the expression of CD1a and CD1d proteins, and the activation of CD1a- and CD1d-restricted T cells were sensitively inhibited by benzo[a]pyrene even at low concentrations detectable in exposed human populations. Similarly, diesel exhaust particles showed marginal inhibitory effect. Using transcriptomic profiling, we discovered that gene expression for regulating endocytic and lipid metabolic pathways was perturbed by benzo[a]pyrene. Imaging flow cytometry also showed that CD1a and CD1d proteins were respectively retained in early and late endosomal compartments, supporting an impaired endocytic lipid antigen presentation for T cell activation upon benzo[a]pyrene exposure. This work conceptually demonstrates that lipid antigen presentation for T cell activation is inhibited by lipophilic pollutants through profound interference with gene expression and endocytic function, likely further disrupting regulatory cytokine secretion and ultimately exacerbating inflammatory diseases.

AIF1 expression regulates differentiation of dendritic cells from hematopoietic stem cell multipotent progenitor subsets and regulates antigen presentation capacity

Abstract Dendritic cells (DC) are indispensable in orchestrating adaptive immunity, serving as the principal messengers between innate and adaptive arms. DC subsets are derived from CD34+ hematopoietic stem cells (HSC). DC development progress through differentiation from HSC multipotent progenitors towards common myeloid progenitors. However, additional proteins involved in this selective differentiation process remain to be identified. For these studies, use of transcriptomic profiling and RNA interference (RNAi) approaches have now identified that Allograft Inflammatory Factor-1 (AIF1) directly contributes to DC differentiation and immunomodulatory processes. HSC multipotent progenitors, identified as CD117+Lin-CD34+SCA1+, were found to not express AIF1. However, CD117+Lin-CD34+SCA1- and CD117+Lin-CD34-SCA1- subsets, known as the oligopotent progenitors, did express AIF1. Importantly, this was restricted to common myeloid progenitors, as the common lymphoid progenitors, defined as CD117+Lin-SCA1-IL-7Rα+ did not express AIF1. RNAi knockdown of AIF1 in sorted Lin-CD34−CD117+SCA1+ cells led to restrained differentiation in DC upon culturing with GM-CSF. Interestingly, knockdown of AIF1 in committed CD11c+ DC resulted in impaired antigen presentation to cognate CD4+ T cells in vitro, suggesting an important immunostimulatory role in committed myeloid lineages. Current studies are now adoptively transferring HSC knocked out for AIF1 into mice to assess in vivo DC differentiation ability and capacity to migrate and populate the splenic compartment. This is the first report showing the novel role of AIF1 in dendritic cell development and antigen presentation.

449 - Infiltrating Macrophages Show Evidence of Impaired Antigen-Presentation Ability at the Growing Edge of the Tumour in Patients with Colorectal Cancer

449 - Infiltrating Macrophages Show Evidence of Impaired Antigen-Presentation Ability at the Growing Edge of the Tumour in Patients with Colorectal Cancer

Functional Impairment of Murine Dendritic Cell Subsets following Infection with Infective Larval Stage 3 of Brugia malayi.

Filarial parasites cause functional impairment of host dendritic cells (DCs). However, the effects of early infection on individual DC subsets are not known. In this study, we infected BALB/c mice with infective stage 3 larvae of the lymphatic filarial parasite Brugia malayi (Bm-L3) and studied the effect on fluorescence-activated cell sorter (FACS)-sorted DC subsets. While myeloid DCs (mDCs) accumulated by day 3 postinfection (p.i.), lymphoid DCs (LDCs) and CD8+ plasmacytoid DCs (pDCs) peaked at day 7 p.i. in the spleens and mesenteric lymph nodes (mLNs) of infected mice. Increased tumor necrosis factor alpha (TNF-α) but reduced interleukin 12 (IL-12) and Toll-like receptor 4 (TLR4), -6, and -9 and reciprocal secretion of IL-4 and IL-10 were also observed across all DC subsets. Interestingly, Bm-L3 increased the expression of CD80 and CD86 across all DC subsets but decreased that of major histocompatibility complex class II (MHC-II) on mDCs and pDCs, resulting in their impaired antigen uptake and presentation capacities, but maximally attenuated the T-cell proliferation capacity of only mDCs. Furthermore, Bm-L3 increased phosphorylated p38 (p-p38), but not p-ERK, in mDCs and LDCs but downregulated them in pDCs, along with differential modulation of protein tyrosine phosphatases SHP-1, TCPTP, PTEN, and PTP1B across all DC subsets. Taken together, we report hitherto undocumented effects of early Bm-L3 infection on purified host DC subsets that lead to their functional impairment and attenuated host T-cell response.

Immune Cell Intolerance for Excess Cholesterol

Chronic metabolic challenges have severe consequences on physiological systems. In this issue of Immunity, Ito etal. (2016) show that defects in cholesterol metabolism in CD11c+ immune cells result in impaired antigen presentation and ultimately in autoimmune disease.

Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation

Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis.

The T Cell in Myeloma

An active role for the immune system in controlling the malignant plasma cell clone in myeloma has been postulated for many years. The clinical states of monoclonal gammopathy of undetermined significance, plateau phase disease, and smoldering myeloma all suggest that a significant host-tumor interaction is taking place. The fundamental role of the cytotoxic T cell in tumor elimination and control has been exemplified by the dramatic efficacy of adoptive T-cell therapies in many hemopoietic malignancies. However, tumor-host cross-talk results in suppression of the endogenous cytotoxic T-cell response against the malignant plasma cell. Whereas patients with myeloma do not clinically exhibit a T-cell immunodeficiency state, with, for example, increased mycobacterial infections, a number of abnormalities of T-cell function are evident. The major abnormalities of T cells include clonal expansions and associated immunosenescence, alterations of regulatory T cells/T helper 17 cells (Treg/Th17 ratio) and acquired membrane abnormalities, due to trogocytosis, which result in acquired Treg cells. Dendritic cell dysfunction associated with impaired antigen processing and presentation caused by abnormalities of the bone marrow microenvironment plays an additional role. In this perspective, we examine the T-cell abnormalities in myeloma and postulate that, whereas cytotoxic T cells interacting with the tumor are dysfunctional, residual T cells still function adequately against external pathogens and thus protect patients from the infections normally associated with a generalized T-cell immunodeficiency state. The so-called 3 E's of host-tumor interaction (elimination, equilibrium, and escape) are clearly reflected in the immune landscape and clinical behavior of myeloma.

Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner.

In order to explore the potential of HLA-independent T cell therapy for human cytomegalovirus (HCMV) infections, we developed a chimeric antigen receptor (CAR) directed against the HCMV encoded glycoprotein B (gB), which is expressed at high levels on the surface of infected cells. T cells engineered with this anti-gB CAR recognized HCMV-infected cells and released cytokines and cytotoxic granules. Unexpectedly, and in contrast to analogous approaches for HIV, Hepatitis B or Hepatitis C virus, we found that HCMV-infected cells were resistant to killing by the CAR-modified T cells. In order to elucidate whether this phenomenon was restricted to the use of CARs, we extended our experiments to T cell receptor (TCR)-mediated recognition of infected cells. To this end we infected fibroblasts with HCMV-strains deficient in viral inhibitors of antigenic peptide presentation and targeted these HLA-class I expressing peptide-loaded infected cells with peptide-specific cytotoxic T cells (CTLs). Despite strong degranulation and cytokine production by the T cells, we again found significant inhibition of lysis of HCMV-infected cells. Impairment of cell lysis became detectable 1 day after HCMV infection and gradually increased during the following 3 days. We thus postulate that viral anti-apoptotic factors, known to inhibit suicide of infected host cells, have evolved additional functions to directly abrogate T cell cytotoxicity. In line with this hypothesis, CAR-T cell cytotoxicity was strongly inhibited in non-infected fibroblasts by expression of the HCMV-protein UL37x1, and even more so by additional expression of UL36. Our data extend the current knowledge on Betaherpesviral evasion from T cell immunity and show for the first time that, beyond impaired antigen presentation, infected cells are efficiently protected by direct blockade of cytotoxic effector functions through viral proteins.

The dark side of the light: mechanisms of photocarcinogenesis

Ultraviolet radiation (UVR) can have a beneficial biologic impact on skin, but it is also the most significant environmental risk factor for skin cancer development. Photocarcinogenesis comprises a complex interplay between the carcinogenic UVR, skin, and the immune system. UVB is absorbed by the superficial skin layers and is mainly responsible for direct DNA damage, which, if unrepaired, can lead to mutations in key cancer genes. UVA is less carcinogenic, penetrates deeper in the dermis, and mainly causes indirect oxidative damage to cellular DNA, proteins, and lipids, via photosensitized reactions. UVR not only induces mutagenesis, altering proliferation and differentiation of skin cells, but also has several immunosuppressive effects that compromise tumor immunosurveillance by impairing antigen presentation, inducing suppressive cells, and modulating the cytokine environment. This review focuses upon molecular and cellular effects of UVR, regarding its role in skin cancer development.

Relative contribution of medullary thymic epithelial cells and dendritic cells to thymocyte central tolerance as assessed by multi-photon microscopy

Abstract Following positive selection, thymocytes migrate into the medulla where they encounter diverse tissue-restricted antigens (TRAs) that induce self-tolerance. Both medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present TRAs to induce negative selection of autoreactive thymocytes, but their relative contribution remains unresolved. Previous studies using genetic models to impair antigen presentation by mTECs or DCs demonstrated both cell types could induce negative selection. However, because interactions between thymocytes and stromal cells maintain a properly differentiated stromal compartment, these studies may not reflect physiologic interactions or relative contributions of mTECs and DCs when both are intact. Thus, we developed a 2-photon microscopy approach to directly observe interactions between thymocytes and DCs or mTECs during negative selection, while monitoring T cell receptor (TCR) activation. Despite earlier studies indicating DCs are required for negative selection to a soluble TRA, we find that mTECs and DCs contribute equivalently to TCR stimulation of CD4 and CD8 single positive (SP) thymocytes responding to membrane-bound or secreted TRAs. Interactions with mTECs and DCs generally accounted for all TCR activation events; however, a significant fraction of CD4SP activation events were not accounted for in response to a soluble TRA, suggesting presentation by other medullary stromal subsets. Initial analysis of polyclonal thymocytes also indicates equal contributions of mTECs and DCs to medullary TCR activation. In summary, mTECs and DCs contribute equivalently to presentation of soluble and membrane-bound mTEC-expressed TRAs on MHC-I and MHC-II during negative selection.

The Significance and Challenges of Monocyte Impairment: For the Ill Patient and the Surgeon.

Trauma, major elective surgery, and overt sepsis can lead to a cascade of immunological change. A subset of these patients will have a degree of immune suppression that leads to hyporesponsive innate defenses, increasing the risk of infective co-morbidity and death. This article is an overview of monocyte impairment in the high-risk surgical patient. Specifically, our primary focus is on observations made pertaining to monocyte function and pathophysiological mechanisms underpinning this impairment. Clinical factors influencing monocyte function are also discussed. A Pubmed search was conducted to review aspects of monocyte impairment in the surgical patient. Search terms included "monocyte impairment," "immunoparalysis," and "endotoxin tolerance" cross-referenced against terms including "trauma," "major surgery," and "sepsis." Findings revealed a broad variety of monocyte defects reported in surgical patients. They ranged from altered cytokine responses, particularly ex vivo TNF-α production, to impaired antigen presentation such as depressed HLA-DR expression. The latter is the most commonly described marker of secondary infection and death. Studies of underlying mechanisms have commonly utilized a model of endotoxin tolerance with in vitro monocytes, revealing a complex array of dysregulated pathways. For our purposes, endotoxin tolerance and monocyte impairment are sufficiently similar entities to permit further study as a single subject. In the high risk patient, microRNAs (also referred to as miRNA or miR) are emerging as potential biomarkers that may modify such pathways. Creation of a reliable impaired human monocyte model could be important to all such considerations. Impairment of monocyte function continues to be predictive of nosocomial infection, multi-organ failure, and death in some surgical patients. However, the optimal marker that could identify a patient as high risk early enough, and whether it might guide potential therapy, still is yet to be proven.

Abstract B054: Can CLL-B cells treated with novel combination therapies be used to promote anti-CLL immune responses?

Abstract The B cell malignancy Chronic Lymphocytic Leukemia (CLL) is associated with humoral and cellular immunodeficiencies, which result in recurrent opportunistic infections, contributing to patient morbidity and mortality. This profound immunodeficiency arises in part from impaired antigen presenting cell (APC) function, which is likely to impede the development of a robust anti-CLL T cell response. Therefore, we wanted to explore the ability of CLL-B cells treated with novel combination therapies to promote anti-CLL immune responses. We investigated the therapeutic capacity of activated CLL-B cells to be licensed in vitro or in vivo in order to promote tumor antigen presentation and drive an anti-CLL immune response. We are testing the ability of novel combinations of new immunotherapies to activate CLL-B cells in untreated primary patient samples, or after samples from patients on treatment in selected clinical trials. We utilized novel combinations of the TLR agonists and Lenalidomide (Len), to activate both healthy donor B cells, and CLL-B cells. Activation of B cells was determined using flow cytometry to measure cell surface costimulatory protein expression (CD80, CD86, CD83), and secretion of cytokines (IL-6, IL-10, and TNFa) into the supernatant by cytokine bead array. CLL-B cells were profoundly defective in IL-6 and IL-10 secretion after stimulation with novel combination therapies, in comparison to healthy B cells. By contrast, costimulatory protein expression in CLL-B cells was upregulated to a similar level observed in healthy B cells. We are currently investigating if activated CLL-B cells can present the specific NKT ligand, aGalCer, to NKT cells, to expand autologous anti-CLL T cells in vitro. The Bruton's Tyrosine Kinase inhibitor, Ibrutinib, inhibits T and NK cell function in CLL patients, in addition to inhibiting BCR and NF-kB signaling in CLL-B cells. Therefore, we tested the ability of CLL-B cells collected from patients prior to, and after Ibrutinib treatment, to respond to immunomodulatory stimuli. After 48 hours stimulation, the expression of CD80 was impaired in CLL-B cells collected post-Ibrutinib treatment, compared with the pre-treatment CLL-B cells. This warrants further investigation, in particular how pro-inflammatory cytokine secretion is regulated by Ibrutinib treatment, and if downregulation of CLL-B cell activation is associated with clinical outcome. Citation Format: Joanne Davis, Kylie Mason, Chia Sharpe, Rachel Koldej, Brendon Chua, David Jackson, Paul Neeson, Constantine Tam, David Ritchie. Can CLL-B cells treated with novel combination therapies be used to promote anti-CLL immune responses? [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B054.

STAT3 promotes CD1d-mediated lipid antigen presentation by regulating a critical gene in glycosphingolipid biosynthesis.

Cytokines that regulate the immune response signal through the Janus kinase / signal transducer and activation of transcription (JAK/STAT) pathway, but whether this pathway can regulate CD1d-mediated lipid antigen presentation to natural killer T (NKT) cells is unknown. Here, we found that STAT3 promotes antigen presentation by CD1d. Antigen-presenting cells (APCs) in which STAT3 expression was inhibited exhibited markedly reduced endogenous lipid antigen presentation to NKT cells without an impact on exogenous lipid antigen presentation by CD1d. Consistent with this observation, in APCs where STAT3 was knocked down, dramatically decreased levels of UDP glucose ceramide glucosyltransferase (UGCG), an enzyme involved in the first step of glycosphingolipid biosynthesis, were observed. Impaired lipid antigen presentation was reversed by ectopic expression of UGCG in STAT3-silenced CD1d(+) APCs. Hence, by controlling a fundamental step in CD1d-mediated lipid antigen presentation, STAT3 signalling promotes innate immune responses driven by CD1d.

Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.