Antigen-independent Activation Research Articles

Antigen-independent activation is critical for the durable antitumor effect of GUCY2C-targeted CAR-T cells

BackgroundChimeric antigen receptor (CAR)-T cells face many obstacles in solid tumor therapy, including heterogeneous antigen expression and inefficient T cell persistence. Guanylyl cyclase C (GUCY2C) has been identified as a...

Cognate Antigen-Independent Differentiation and Activation of Resident Memory T cells in Chronic Kidney Disease

Resident memory T cells (TRMs) are a memory subset of T cells which are retained locally within barrier and epithelial tissues such as the kidney and have also been noted for their role in perpetuating chronic inflammation. While T cells have gained recognition for their ability to modulate kidney inflammation in various models of kidney injury TRMs are still poorly understood and their contributions to sterile models of chronic kidney disease (CKD) are unknown. We hypothesized that kidney TRMs are activated and modulate kidney inflammation in the setting of CKD. We utilized an aristolochic acid (AA) model of murine CKD to investigate the presence of kidney TRMs via flow cytometry and characterized their transcriptional profile via single cell RNA sequencing (scRNAseq). We also measured kidney cytokine levels with an MSD cytokine plate. Six weeks after AA-induced CKD, kidney CD8+ T cells were significantly increased compared to control mice (29.6 ± 1.6% vs. 4.7 ± 0.6% of CD45+, respectively; p<0.0001), as were CD4+ T cells (34.4 ± 1.4% vs. 8.2 ± 0.7% of CD45+, respectively; p<0.0001). 53.6 ± 2.5% of CD8+ T cells expressed CD103, a TRM marker, in AA mice compared to 4.8 ± 1.2% in control mice, and 36.0 ± 1.7% vs 1.6 ± 0.3% of CD4+ T cells. Kidney protein lysates of AA mice had significantly higher levels of IFNγ than control kidneys (18.9 ± 1.6 vs. 2.9 ± 0.7 pg/mg, respectively; p=0.0003) as well as TNF (98.6 ± 8.9 pg/mg vs. 6.9 ± 0.8 pg/mg, respectively; p=0.0002) and IL-6 (247.6 ± 39.1 pg/mg vs. 75.4 ± 22.5 pg/mg, respectively; p=0.0226). scRNAseq of immune cells revealed T cells bearing TRM markers CD103 and CD49a which lacked expression of tissue egress molecules S1pr1, Sell, Ccr7, and Klf2. scRNAseq analysis of this TRM population also revealed downregulation of Eomes and Tbx21 transcription factors and upregulation of Runx3 transcription factor, all of which correspond with previously described TRM transcriptional profiles. We next evaluated signaling pathways involved with the survival and transcriptional profile of TRMs, namely IL-15 and TGF-β. While Il2rb and Il2rg (encoding IL-15 receptor subunits) were upregulated in TRMs, IL-15 protein lysate levels were not elevated in AA kidneys compared to controls (2,600 ± 145 pg/mg protein vs. 2,281 ± 96 pg/mg protein, respectively; p=0.0831). Tgfbr1 and Tgfbr2 (encoding TGF-β receptor subunits) were also upregulated in TRMs, but TGF-β protein lysate levels were not significantly elevated in AA kidneys compared to controls (2,158 ± 159 pg/mg protein vs. 1,924 ± 202 pg/mg protein, respectively; p=0.3886). Gene ontology pathway analysis showed enrichment of pathways involved in T cell receptor signaling. Because of this we assessed the kidney immune profile of transgenic OT-1 mice, which can only recognize specific ovalbumin residues in the context of MHC class 1 H-2Kb. Six weeks after AA-induced CKD, injured OT-1 mice still had a significant increase in the proportion of CD8+ T cells that were CD103+ compared to vehicle control OT-1 mice (47.3 ± 2.9% vs. 9.6 ± 1.6% of CD45+, respectively; p<0.0001) and CD4+ T cells that were CD103+ (26.7 ± 3.1% vs. 6.4 ± 2.4% of CD45+, respectively; p=0.0040). Here we demonstrate the differentiation and activation of TRMs during AA-induced CKD independent of cognate antigen interaction and highlight their activated profile, as well as an increase in kidney IFNγ, TNF, and IL-6, all proinflammatory cytokines typical of TRMs. Future studies will aim to determine the contributions of TRMs to CKD progression. F32 DK136187, T32 DK007545, R01 DK 118932, U54 DK137301. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeting NKG2D ligands in glioblastoma with a bispecific T-cell engager is augmented with conventional therapy and enhances oncolytic virotherapy of glioma stem-like cells

BackgroundGlioblastoma (GBM) almost invariably becomes resistant towards conventional treatment of radiotherapy and temozolomide (TMZ) chemotherapy, partly due to subpopulations of intrinsically resistant glioma stem-like cells (GSC). The oncolytic herpes simplex...

ZFP36 disruption is insufficient to enhance the function of mesothelin-targeting human CAR-T cells

Loss of inflammatory effector function, such as cytokine production and proliferation, is a fundamental driver of failure in T cell therapies against solid tumors. Here, we used CRISPR/Cas9 to genetically disrupt ZFP36, an RNA binding protein that regulates the stability of mRNAs involved in T cell inflammatory function, such as the cytokines IL2 and IFNγ, in human T cells engineered with a clinical-stage mesothelin-targeting CAR to determine whether its disruption could enhance antitumor responses. ZFP36 disruption slightly increased antigen-independent activation and cytokine responses but did not enhance overall performance in vitro or in vivo in a xenograft tumor model with NSG mice. While ZFP36 disruption does not reduce the function of CAR-T cells, these results suggest that singular disruption of ZFP36 is not sufficient to improve their function and may benefit from a multiplexed approach.

P2X7-Mediated Antigen-Independent Activation of CD8+ T Cells Promotes Salt-Sensitive Hypertension.

CD8+ T cells (CD8Ts) have been implicated in hypertension. However, the specific mechanisms are not fully understood. In this study, we explore the contribution of the P2X7 (purinergic receptor P2X7) receptor to CD8T activation and subsequent promotion of sodium retention in the kidney. We used mouse models of hypertension. Wild type were used as genetic controls, OT1 and Rag2/OT1 mice were utilized to determine antigen dependency, and P2X7-knockout mice were studied to define the role of P2X7 in activating CD8Ts and promoting hypertension. Blood pressure was monitored continuously and kidneys were obtained at different experimental end points. Freshly isolated CD8Ts from mice for activation assays and ATP stimulation. CD8T activation-induced promotion of sodium retention was explored in cocultures of CD8Ts and mouse DCTs. We found that OT1 and Rag2/OT1 mice, which are nonresponsive to common antigens, still developed hypertension and CD8T-activation in response to deoxycorticosterone acetate/salt treatment, similar to wild-type mice. Further studies identified the P2X7 receptor on CD8Ts as a possible mediator of this antigen-independent activation of CD8Ts in hypertension. Knockout of the P2X7 receptor prevented calcium influx and cytokine production in CD8Ts. This finding was associated with reduced CD8T-DCT stimulation, reversal of excessive salt retention in DCTs, and attenuated development of salt-sensitive hypertension. Our findings suggest a novel mechanism by which CD8Ts are activated in hypertension to exacerbate salt retention and infer that the P2X7 receptor on CD8Ts may represent a new therapeutic target to attenuate T-cell-mediated immunopathology in hypertension.

Complementarity-determining region clustering may cause CAR-T cell dysfunction

Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as cancer treatment, however, designing an optimal CAR remains challenging. A single-chain variable fragment (scFv) is generally used as CAR targeting moiety, wherein the complementarity-determining regions (CDRs) define its specificity. We report here that the CDR loops can cause CAR clustering, leading to antigen-independent tonic signalling and subsequent CAR-T cell dysfunction. We show via CARs incorporating scFvs with identical framework and varying CDR sequences that CARs may cluster on the T cell surface, which leads to antigen-independent CAR-T cell activation, characterized by increased cell size and interferon (IFN)-γ secretion. This results in CAR-T cell exhaustion, activation-induced cell death and reduced responsiveness to target-antigen-expressing tumour cells. CDR mutagenesis confirms that the CAR-clustering is mediated by CDR-loops. In summary, antigen-independent tonic signalling can be induced by CDR-mediated CAR clustering, which could not be predicted from the scFv sequences, but could be tested for by evaluating the activity of unstimulated CAR-T cells.

After virus exposure, early bystander naïve CD8 T cell activation relies on NAD+ salvage metabolism.

CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.

Crosslinking-mediated activation of the FcεRI: Does it need antigen for success?

Mast cells (MCs), hematopoietic cells of the myeloid lineage, are well-known for their pro-inflammatory nature contributing to the development of various allergic and autoimmune diseases. One of the characteristic receptors on MCs, the high-affinity receptor for IgE (FcεRI), is activated in its IgE-bound state via binding and crosslinking by polyvalent antigen. This results in its phosphorylation by the SRC family kinase LYN, initiating differential signaling pathways, eventually triggering immunological effector functions, such as degranulation and cytokine production. Few publications have reported on FcεRI-dependent but antigen-independent MC activation by antibody-mediated crosslinking of membrane molecules (e.g., transmembrane proteins and glycosphingolipids) that are both localized in membrane rafts and in close vicinity to the FcεRI. In this Viewpoint we will briefly introduce FcεRI-mediated MC stimulation, cite examples of FcεRI-proximal molecules, the crosslinking of which can cause FcεRI-dependent MC activation, and discuss the potential of certain viruses as well as auto-antibodies to act as indirect FcεRI-crosslinking agents. In latter cases, antigen-independent FcεRI-mediated pro-inflammatory MC activation could contribute to the development of detrimental cytokine storms.

Abstract P024: Using innate immune ligands to activate adaptive immune cells for glioblastoma therapy

Abstract Glioblastoma (GBM) is one of the most aggressive and immunosuppressive brain tumors in adults. Despite conventional therapy of surgical resection, radiotherapy and temozolomide (TMZ) chemotherapy, tumors invariably recur and become resistant. The Natural Killer Group 2 Member D (NKG2D) receptor plays a key role in the innate immune control of cancers. The ligands for this receptor (NKG2DLs) are minimally expressed or absent on healthy cells. Stressed or malignant cells upregulate NKG2DLs, resulting in immune activation. We have developed a strategy targeting upregulated NKG2DLs on GBM cells with bi-specific T cell engagers (BiTEs). NKG2D BiTE contains a recombinant NKG2D receptor linked to an anti-CD3 domain. Simultaneous binding of the NKG2D BiTE to NKG2DLs on GBM cells and CD3 on T cells causes antigen-independent activation, pro-inflammatory cytokine release, and tumor cell death. Pre-treating GBM cells with radiation and TMZ causes further NKG2DL upregulation, increased NKG2D BiTE-mediated T cell activation, and sensitises GBM cells to T cell lysis. Meanwhile, healthy non-cancer cells exposed to the same pre-treatment do not trigger T cell activation with NKG2D BiTE. To deliver the BiTE directly into the tumor, the NKG2D BiTE has been encoded into the genome of an oncolytic herpes simplex virus 1 (oHSV-1). This oHSV is capable of selective infection, replication and cell lysis within GBM cells, but not healthy cells. Alongside the direct oncolytic capacity of the oHSV-1, it can also act as a gene therapy vector to express the NKG2D BiTE in situ to activate endogenous tumor infiltrating lymphocytes and direct cytotoxicity towards the remaining cancer cells. Combining conventional radio/chemotherapy with NKG2D BiTE immunotherapy presents an opportunity for synergy by enhancing NKG2DL expression on GBM cells and directing T cell lysis specifically towards these tumor cells, whilst avoiding responses towards non-cancerous cells. Meanwhile, local delivery of the NKG2D BiTE using an oncolytic virus endows tumor-specific expression directly within the tumor. Citation Format: Richard T. Baugh, Hena Khalique, Janet Lei-Rossmann, Len Seymour. Using innate immune ligands to activate adaptive immune cells for glioblastoma therapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P024.

Histologic Patterns and Clues to Autoinflammatory Diseases in Children: What a Cutaneous Biopsy Can Tell Us.

Autoinflammation is defined by aberrant, antigen-independent activation of the innate immune signaling pathways. This leads to increased, pro-inflammatory cytokine expression and subsequent inflammation. In contrast, autoimmune and allergic diseases are antigen-directed immune responses from activation of the adaptive immune system. The innate and adaptive immune signaling pathways are closely interconnected. The group of ‘complex multigenic diseases’ are a result of mutual dysregulation of both the autoinflammatory and autoimmune physiologic components. In contrast, monogenic autoinflammatory syndromes (MAIS) result from single mutations and are exclusively autoinflammatory in their pathogenesis. Studying the clinical and histopathological findings for the various MAIS explains the phenotypical correlates of their specific mutations. This review aims to group the histopathologic clues for autoinflammation into three recognizable patterns. The presence of these histologic patterns in a pediatric patient with recurrent fevers and systemic inflammation should raise suspicion of an autoinflammatory component in MAIS, or, more frequently, in a complex multigenic disease. The three major histopathological patterns seen in autoinflammation are as follows: (i) the ‘neutrophilic’ pattern, seen in urticarial neutrophilic dermatosis, pustular psoriasis, aseptic neutrophilic folliculitis, and Sweet’s syndrome; (ii) the ‘vasculitic’ pattern seen in small vessel-vasculitis (including hypersensitivity/leukocytoclastic vasculitis, thrombosing microangiopathy and lymphocytic vasculitis), and intermediate-sized vessel vasculitis, mimicking polyarteritis nodosa; and (iii) the ‘granulomatous’ pattern. Beyond these three patterns, there are additional histopathologic clues, which are detailed below. It is important for a dermatopathologist to recognize the patterns of autoinflammation, so that a diagnosis of MAIS or complex multigenic diseases may be obtained. Finally, careful histopathologic analyses could contribute to a better understanding of the various clinical manifestations of autoinflammation.

The Invariant NKT Cell Response Has Differential Signaling Requirements during Antigen-Dependent and Antigen-Independent Activation.

Invariant NKT (iNKT) cells are an innate-like population characterized by their recognition of glycolipid Ags and rapid cytokine production upon activation. Unlike conventional T cells, which require TCR ligation, iNKT cells can also be stimulated independently of their TCR. This feature allows iNKT cells to respond even in the absence of glycolipid Ags, for example, during viral infections. Although the TCR-dependent and -independent activation of iNKT cells have been relatively well established, the exact contributions of IL-12, IL-18, and TLRs remain unclear for these two activation pathways. To definitively investigate how these components affect the direct and indirect stimulation of iNKT cells, we used mice deficient for either MyD88 or the IL-12Rβ2 in the T cell lineage. Using these tools, we demonstrate that IL-12, IL-18, and TLRs are completely dispensable for the TCR activation pathway when a strong agonist is used. In contrast, during murine CMV infection, when the TCR is not engaged, IL-12 signaling is essential, and TLR signaling is expendable. Importantly, to our knowledge, we discovered an intrinsic requirement for IL-18 signaling by splenic iNKT cells but not liver iNKT cells, suggesting that there might be diversity, even within the NKT1 population.

Programmed cell death protein 1 activation preferentially inhibits CD28.CAR–T cells

Targeted adoptive immunotherapy with engineered T cells is a promising treatment for refractory hematologic malignancies. However, many patients achieving early complete remissions ultimately relapse. Immunosuppressive ligands are expressed on tumor and supportive cells in the tumor microenvironment (TME). When activated, T cells express associated “checkpoint” receptors. Binding of co-inhibitory ligands and receptors may directly contribute to T-cell functional exhaustion. It is not known whether all T cells engineered to express chimeric antigen receptors (CARs) are subject to checkpoint-mediated regulation. It is also unknown whether distinct CAR signaling moieties modulate T-cell responsiveness to these inhibitory pathways. We have, therefore, directly compared functional co-inhibition in engineered T cells identically targeted to the tumor-associated antigen CD123, but distinct in their mode of T-cell activation: via the endogenous T-cell receptor (ENG), or downstream of CD28 or 41BB-containing CARs. In all cases, we have observed antigen-independent T-cell activation associated with upregulation of the co-inhibitory receptors programmed cell death protein 1 (PD-1, CD279), Tim-3 and Lag-3. Notably, CD28.CAR T cells were uniquely susceptible to PD-1/PD-L1 mediated checkpoint inhibition. Together, our data indicate that PD-1/PD-L1 checkpoint blocking agents may be considered clinically when CD28.CAR T cells do not perform optimally in human trials.

Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rβ2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells.

Balanced secretion of anti-CEA \xd7 anti-CD3 diabody chains using the 2A self-cleaving peptide maximizes diabody assembly and tumor-specific cytotoxicity

Adoptive transfer of genetically engineered human cells secreting bispecific T-cell engagers has shown encouraging therapeutic effects in preclinical models of cancer. However, reducing the toxicity and improving the effectiveness of this emerging immunotherapeutic strategy will be critical to its successful application. We have demonstrated that for gene-based bispecific antibody strategies, two-chain diabodies have a better safety profile than single-chain tandem scFvs (single-chain variable fragments), because their reduced tendency to form aggregates reduces the risk of inducing antigen-independent T-cell activation. Here, we demonstrate that the incorporation of a 2A self-processing peptide derived from foot-and-mouth disease virus conveying co-translational cleavage into a two-chain anti-CD3 × anti-CEA diabody gene enables near-equimolar expression of diabody chains 1 and 2, and thus increases the final amount of assembled diabody. This was found to maximize diabody-mediated T-cell activation and cytotoxicity against carcinoembryonic antigen-positive tumor cells.

Integrated Analysis of IGHV Gene Status, Cell-of-Origin Signature and Genomic Features in Diffuse Large B-Cell Lymphoma

The molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) has been highlighted by gene expression profiling (GEP), dividing DLBCL into the following three main molecular subtypes with different clinical outcomes and responses to immunochemotherapy: the germinal center B-cell-like (GCB) subtype, the activated B-cell-like (ABC) subtype and the primary mediastinal B-cell lymphoma (PMBL) subtype. Despite frequent translocations involving the IGH (heavy chain) locus, B-cell receptor (BCR) expression is retained in almost all DLBCL, indicating that BCR signaling is crucial for the survival of malignant cells. Furthermore, DLBCL are characterized by recurrent somatic mutations that are supposed to be oncogenic drivers. Here, with the aim to more accurately define the DLBCL pathogenic subgroups, we report a detailed immunogenetic analysis of IGH rearrangements in a well-defined GEP DLBCL cohort and correlate these features with the somatic mutation status of recurrently mutated genes involved in lymphomagenesis. Methods: 204 DLBCL enrolled in the prospective LYSA LNH03 trial program were classified into molecular subtypes by GEP using Affymetrix arrays [82 ABC (40.2%), 77 GCB (37.7%), 29 (14.2%) unclassified and 16 PMBL (7.8%)]. The amplification of complete VDJ rearrangements was realized using BIOMED-2 protocols. Somatic hypermutation (SHM) characteristics were studied (mutation rate, glycosylation N-X-S/T sites, RGYW hotspots, composition of CDR3) and correlated with the molecular subtypes. To obtain a more comprehensive molecular portrait of the DLBCL cases, GEP and IGH VDJ analyses were correlated with the mutational status of a panel of 34 recurrently mutated genes in DLBCL such as MYD88, EZH2, CD79B, TNFAIP3, CARD11 and GNA13 (Dubois et al. Clinical Cancer Research 2016). Results: A total of 153/204 (75%) clonal VDJ rearrangements were successfully determined. Failures to detect clonotypic sequences were predominantly seen in PMBL (56%) and unclassified (31%) cases. The ABC subtype display a significantly lower SHM rate than the 3 other subtypes, especially in contrast to the PMBL subgroup that was characterized by a higher SHM rate (p<0.0001 for each). While IGHV gene usage generally reflected that of normal B cells, a marked increase in IGHV4-34 in ABC cases (30%) was confirmed and those cases tended to have less SHM. Acquisition of new N-glycosylation (N-Gly) sites, a hallmark of follicular lymphoma, was more prevalent in the GCB cases (48%) than in ABC (22%) (p=0.012) but was also observed in 5/7 (71%) PMBL cases with functional IGH rearrangement. This acquisition is correlated with a more frequent t(14;18)(q24;q32) in GCB subtype. As reported in FL, this suggests that lectin binding signals via surface Ig that contain N-Gly sites may provide an extrinsic antigen-independent signal in some DLBCL, and more specifically in t(14;18) DLBCL (Linley et al. Blood 2015 ). Finally, we correlated these immunogenetic features with the mutational status of some drivers genes frequently mutated in DLBCL. Within ABC subtype patients, MYD88 or TNFAIP3 mutated cases displayed a higher SHM rate (13.4% vs 8.4%, p <0.0001; 15.44% vs 11.13%, p=0.03), without any IGH family distribution bias. 9/56 (16%) GCB DLBCL harbored an EZH2 mutation. All expressed an IGHV3 rearrangement (100% vs 52% for the GCB-EZH2 wt, p=0.014). By contrast to MYD88 status, a similar IG SHM rate was observed in mutated and wt patients regarding EZH2, CARD11 or GNA13 genes. Conclusion: The IG characterization and the driver gene mutation analysis according to the cell of origin of the tumor allowed us to refine DLBCL subgroups. Our data suggest that there is a strong interaction between extrinsic mechanisms, including lectin-mediated antigen-independent activation or, in the IGHV4-34 cases, via N-acetyl lactosamine ligands, and intrinsic mechanisms to activate the BCR/NFKB pathway. These data could help us classify the different subgroups of DLBCL more accurately and, in particular, to identify new molecular features for BCR target therapy. DisclosuresHaioun:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Salles:Mundipharma: Honoraria; Janssen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding.

Online Haemodiafiltration Improves Inflammatory State in Dialysis Patients: A Longitudinal Study.

BackgroundPatients undergoing conventional hemodialysis (C-HD) present a greater immuno-inflammatory state probably related to uremia, sympathetic nervous system (SNS) activation and /or membrane bioincompatibility, which could improve with a technique-switching to online hemodiafiltration (OL-HD). The antigen-independent pathway activation of this modified immunologic state turns dendritic cells (DC) into an accurate cell model to study these patients. The aim of this study is to further evaluate the immune-inflammatory state of patients in C-HD assessed by DC maturation.Methods31 patients were submitted to C-HD and after 4 months switched to the OL-HD technique. Monocytes-derived DCs from HD patients were cultured in the presence of IL-4/GM-CSF. DC-maturation was evaluated by assessing the maturation phenotype by flow cytometry (FACs). DCs-functional capacity to elicit T-cell alloresponse was studied by mixed leucocyte reaction. Cytokine release was assessed by FACs and SNS was evaluated measuring renalase levels by ELISA.ResultsAn up-regulation of maturation markers was observed in C-HD DCs which induced two fold more T cells proliferation than OL-HD DCs. Also, C-HD-mDCs presented with over-production of pro-inflammatory cytokines (IL-6, IL-1β, IL-8, IL-10 and TNF-α) compared with OL-HD-mDC (P<0·05). Results were correlated with clinical data. When SNS was evaluated, hypotension events and blood pressure were significantly lower and renalase levels were significantly higher after conversion to OL-HD. Diabetes mellitus type 2 patients also found beneficial reduction of mDC when converted to OL-HD compared to non-diabetics.ConclusionsOL-HD could interfere with immuno-inflammatory state in HD patients with an improvement of renalase levels as potential key mediators in the mechanistic pathway of down-regulation of DC maturation.

Analysis of the Innate Response to Adjuvants: Characterization of the Draining Lymph Node by Fluorescence-Activated Cell Sorting.

A clear index for a response to adjuvants is a change in the cellular composition of lymph nodes draining the site of adjuvant injection (Didierlaurent et al., J Immunol 183:6186-6197, 2009; Caproni et al., J Immunol 188:3088-98, 2012; Desbien et al., Eur J Immunol 1-11, 2014). During the steady state, lymph nodes (LNs) are composed of a fixed ratio of innate and adaptive cells awaiting activation signals from tissue draining lymph. Upon exposure to innate stimulants, lymph nodes undergo dramatic changes. The most apparent change to the lymph node is an increase in size. Antigen-independent activation of naïve T cells and B cells, as a consequence of type I interferon signaling, results in upregulation of CD69 (Sun and Zhang, J. Exp. Med 188:2335-2342, 1998), causing increased retention of cells in the lymph node and transient lymphopenia in the blood (Shiow et al., Nature 440:540-544, 2006). In addition tissue-resident dendritic cells, macrophages, as well as circulating inflammatory monocytes will migrate into draining LNs and display maturation markers associated with activation. Such features can provide powerful discrimination of adjuvant potencies.

Innate Effector-Memory T-Cell Activation Regulates Post-Thrombotic Vein Wall Inflammation and Thrombus Resolution.

Immune cells play an important role during the generation and resolution of thrombosis. T cells are powerful regulators of immune and nonimmune cell function, however, their role in sterile inflammation in venous thrombosis has not been systematically examined. This study investigated the recruitment, activation, and inflammatory activity of T cells in deep vein thrombosis and its consequences for venous thrombus resolution. CD4+ and CD8+ T cells infiltrate the thrombus and vein wall rapidly on deep vein thrombosis induction and remain in the tissue throughout the thrombus resolution. In the vein wall, recruited T cells largely consist of effector-memory T (TEM) cells. Using T-cell receptor transgenic reporter mice, we demonstrate that deep vein thrombosis-recruited TEM receive an immediate antigen-independent activation and produce IFN-γ (interferon) in situ. Mapping inflammatory conditions in the thrombotic vein, we identify a set of deep vein thrombosis upregulated cytokines and chemokines that synergize to induce antigen-independent IFN-γ production in CD4+ and CD8+ TEM cells. Reducing the number of TEM cells through a depletion recovery procedure, we show that intravenous TEM activation determines neutrophil and monocyte recruitment and delays thrombus neovascularization and resolution. Examining T-cell recruitment in human venous stasis, we show that superficial varicose veins preferentially contain activated memory T cells. TEM orchestrate the inflammatory response in venous thrombosis affecting thrombus resolution.

646. Development of a Novel Chimeric Antigen Receptor as a Therapy Against Solid Tumors

The use of chimeric antigen receptor (CAR) T cells as a therapy for cancer is gaining traction as they potentially target tumors with high specificity and minimal off-target toxicities. Current CAR designs consist of an antigen-specific single chain antibody fragment (scFv) fused to the transmembrane and signaling domains of receptors capable of activating T cells. In CAR T cell therapy, patients’ T cells are transduced with cDNA encoding the CAR allowing CAR expressing T cells to target tumor cells. Clinical trials using anti-CD19 CAR T cells to target B cell malignancy have demonstrated the potential of this therapy with numerous patients achieving partial or full remission. In the treatment of solid tumors, however, CAR T cell therapy has not proven as effective. Evidence supporting T cell persistence is sparse, suggesting that currently used CARs may not provide optimal signals for memory cell generation. In addition, some patients have experienced life-threating side effects including cytokine release syndrome and on-target off-tumor activity, indicating a need for better modulation of CAR T cell activity. In line with these clinical observations, data from our laboratory and others demonstrate that currently used CARs can activate T cells in the absence of antigen, which promotes toxicity and T cell exhaustion while preventing differentiation into memory T cells. We propose that this antigen-independent activation is a result of high expression of CAR molecules that lack the regulatory context of the endogenous T cell receptor (TCR) complex. To more effectively mimic the temporal regulation of endogenous TCR signaling pathways, we developed a novel TCR-based CAR in which a scFv is fused to the constant region of TCRβ, thereby becoming incorporated into the native complex. We hypothesize that incorporation of our CAR into the TCR complex will mimic endogenous T cell activation and regulation by including contributions from all CD3 members (CD3Δ, CD3e, CD3γ, and CD3ζ). Furthermore, we propose that endogenous regulation of CAR signaling will prevent the constitutive T cell activation observed with currently used CAR constructs and allow for the formation of memory T cells. We have demonstrated that surface expression of our TCR CAR is modulated by endogenous CD3 expression levels and mirrors normal TCR surface expression. In contrast to both first and second generation CARs, T cells expressing our novel CAR lack constitutive activation, are specific for tumor cells expressing high levels of antigen, and retain higher proliferative potential in culture. Moreover, TCR CAR T cells are capable of signaling through the TCR complex and activating T cells in an antigen specific manner. Currently, we are examining the effector potential of our TCR CAR T cells compared to first and second generation CARs. Additionally, we are developing TCR CARs capable of signaling not only through the TCR complex but also through co-stimulatory receptors. In summary, we are designing the next generation of CARs by incorporating the tumor recognition component of a scFv into the endogenous TCR complex.

MIF Is Necessary for Late-Stage Melanoma Patient MDSC Immune Suppression and Differentiation.

Highly aggressive cancers "entrain" innate and adaptive immune cells to suppress antitumor lymphocyte responses. Circulating myeloid-derived suppressor cells (MDSC) constitute the bulk of monocytic immunosuppressive activity in late-stage melanoma patients. Previous studies revealed that monocyte-derived macrophage migration inhibitory factor (MIF) is necessary for the immunosuppressive function of tumor-associated macrophages and MDSCs in mouse models of melanoma. In the current study, we sought to determine whether MIF contributes to human melanoma MDSC induction and T-cell immunosuppression using melanoma patient-derived MDSCs and an ex vivo coculture model of human melanoma-induced MDSC. We now report that circulating MDSCs isolated from late-stage melanoma patients are reliant upon MIF for suppression of antigen-independent T-cell activation and that MIF is necessary for maximal reactive oxygen species generation in these cells. Moreover, inhibition of MIF results in a functional reversion from immunosuppressive MDSC to an immunostimulatory dendritic cell (DC)-like phenotype that is at least partly due to reductions in MDSC prostaglandin E(2) (PGE(2)). These findings indicate that monocyte-derived MIF is centrally involved in human monocytic MDSC induction/immunosuppressive function and that therapeutic targeting of MIF may provide a novel means of inducing antitumor DC responses in late-stage melanoma patients.