OT-I Cells Research Articles

Identification of a core miRNA signature unique to exhausted cytotoxic CD8+ T cells

Abstract Cytotoxic CD8+ T lymphocytes (CTLs) play a major role in protection against chronic viral infections and tumors. Chronic antigen stimulation in both conditions leads to CTL exhaustion and failure of CTLs to efficiently eradicate tumors and virally infected cells. miRNAs are small, non-coding RNA molecules that participate in post-transcriptional gene regulation. This study aimed to determine the core miRNA signature of CTL exhaustion in mouse models of both tumors and chronic infection. Exhausted anti-tumor CTLs were isolated from tumors of mice that received 10^6 mouse mesothelioma cells (AE17sOVA) followed by an adoptive transfer of 10^4 CD45.1+ OT-I cells. Effector anti-viral CTLs were isolated from mice adoptively transferred with 10^4 CD45.1+ OT-I cells or P14 cells and infected with WSN-OVA or LCMV Armstrong respectively. For exhausted anti-viral CTLs, mice were adoptively transferred with 10^4 CD45.1+ P14 cells and infected with LCMV-Cl13. Single cell suspensions were prepared from the different tissues, live CTLs were FACS sorted, RNA isolated and small RNA-seq was performed. Principle component analysis (PCA) of miRNA expression revealed that exhausted anti-tumor CTLs and anti-LCMV Cl13 CTLs cluster apart from effector anti-viral CTLs (WSN-OVA and LCMV Armstrong). Differential expression analysis identified 73 miRNAs commonly shared between the exhausted day anti-tumor CTL and anti-viral CTL. Using RNA sequencing of two different models of exhaustion, distinct RNA profiles common in exhausted CTLs were found. This core miRNA signature can now be used to manipulate the expression of specific miRNA to potentially prevent or revert CTL exhaustion and improve the CTL response against tumors and/or chronic infections.

A road map of memory CD8+ T cell functional imprinting and fitness

Abstract Previous studies established that TCR signaling strength determines the size of CD8+ T cell expansion and memory pool. Here we hypothesized that TCR signaling strength further modulates the functional features of memory CD8+ T cells. We immunized mice transferred with naïve OT-I cells, which recognize the Ovalbumin (Ova)-derived epitope SIINFEKL, with Listeria monocytogenes expressing different variants, exposing OT-I cells to various TCR signaling strength. Transcriptomic analysis at early times after priming revealed that compared to OT-I cells primed with strong TCR signals, those primed with weak TCR signals upregulated genes involved in cell cycle (IL-2) and type I IFN response, suggesting that TCR signaling strength could determine how CD8+ T cells respond to cytokines at the time of priming. We postulated that disrupting cytokine signaling in T cells primed with weak TCR signals could alter memory T cell fate. As proposed, OT-I memory cells primed with weak TCR signals and lacking IL-2 or type I IFN cytokine signaling (Ifnar−/−, Il2ramut/mut) failed to re-expand upon secondary challenge compared to WT counterparts. Moreover, transcriptomic and epigenetic analyses of WT or Il2ramut/mut OT-I memory cells primed with strong versus weak TCR signals revealed that while gene expression remained similar, chromatin accessibility was significantly different. TCR and IL-2 signaling together regulate an epigenetic landscape reflecting a broad functional fitness of memory CD8+ T cells, whereas TCR signaling selectively imprinted an epigenetic landscape related to metabolism. These results suggest that epitope selection for effective CD8+ T cell vaccine design is pivotal to modulate the functional programming of memory T cells.

Dysregulated TGF-β Signaling Leads to Cell-Intrinsic Changes in T cell Development that Promote Allergic Inflammation

Abstract Variants in genes in the Transforming Growth Factor Beta (TGFb) signaling pathway are associated with allergic diseases suggesting that this cytokine may be a central player in allergic disease. The role of TGFb in preserving T cell mediated self-tolerance is well-appreciated but how disruption in this pathway promotes allergic inflammation is not clear. TGFb signals through its receptor TGFβRI and II, leading to phosphorylation and nuclear translocation of Smad proteins 2 and 3. We previously demonstrated that patients with Loeys-Dietz Syndrome (LDS), an autosomal dominant disorder caused by mutations in TGFBR1 and TGFBR2 are highly predisposed to developing allergic disease. Both LDS patients and mice harboring a knock-in allele (Tgfbr1mut) of an LDS mutation known to cause severe disease in humans exhibit increased levels of total and food-specific IgE, consistent with their allergic tendencies. These serologic changes were associated with increased numbers of T follicular helper (Tfh) cells which promote memory B cell and plasma cell formation, and decreased T follicular regulatory (Tfr) cells, which suppress humoral immune responses, in LDS patients and mice. Using a mixed bone marrow chimera model we demonstrated that these alterations in T cell development are cell-intrinsic. Furthermore, OVA specific OTII cells harboring the Tgfbr1mut were more likely to accumulate in the peyer’s patch, differentiate into Tfh cells, and less likely to upregulate Foxp3 in response to orally ingested OVA. Tgfbr1mut OTII cells also produced more IL-4 and less IL-10 in response to OVA. These findings suggest that TGF-β signaling plays an important role in T cell development and function that, when disrupted, promotes allergic inflammation.

Immunogenicity and the CXCL12-coat control T cell accumulation in murine pancreatic cancer

Abstract Improving the efficacy of immunotherapies in pancreatic cancer can increase patient survival rate. Microsatellite-stable pancreatic adenocarcinoma (PDA) exclude T cells and resist immunotherapy. This may occur by cancer cells coating themselves with CXCL12, a chemokine produced by cancer associated fibroblasts (CAFs). However, PDA with microsatellite instability (MSI-H) is infiltrated with T cells and responsive to PD-1 checkpoint inhibition. We found that human MSI-H pancreatic cancer cells display the CXCL12-coat, suggesting additional factors control pancreatic cancer immune accessibility. We hypothesized that expression of neoantigens by MSI-H PDA is an independent variable that overcomes T cell exclusion mediated by the CXCL12-coat. To test this, we established a mouse hepatic PDA metastases model in which PDA cells express doxycycline-induced ovalbumin (OVA), and adoptively transferred OVA-specific OT-I CD8+ T cells report intra-tumoral accumulation of T cells. Inducing OVA expression in established metastases increases intra-tumoral accumulation and activation of OT-I cells, despite the presence of the CXCL12-coat. We are currently investigating whether high- and low-affinity antigen-expressing cancers are subject to T cell mediated elimination in the presence and absence of the CXCL12-coat, respectively. The CXCL12-coat on PDA cells and the expression of neoantigens can independently control immune response. As the formation of the CXCL12-coat is facilitated by the Transglutaminase-2 (TGM2) and can be inhibited by TGM2 inhibitors (Z Wang et al., bioRxiv, 2019), these findings reveal potential combination therapies to overcome pancreatic cancer immunotherapy resistance.

Cancer immunotherapy using a polysaccharide from Codium fragile in a murine model

ABSTRACT Natural polysaccharides have shown immune modulatory effects with low toxicity in both animal and human models. A previous study has shown that the polysaccharide from Codium fragile (CFP) promotes natural killer (NK) cell activation in mice. Since NK cell activation is mediated by dendritic cells (DCs), we examined the effect of CFP on DC activation and evaluated the subsequent induction of anti-cancer immunity in a murine model. Treatment with CFP induced activation of bone marrow-derived dendritic cells (BMDCs). Moreover, subcutaneous injection of CFP promoted the activation of spleen and lymph node DCs in vivo. CFP also induced activation of DCs in tumor-bearing mice, and combination treatment with CFP and ovalbumin (OVA) promoted OVA-specific T cell activation, which consequently promoted infiltration of IFN-γ-and TNF-α-producing OT-1 and OT-II cells into the tumors. Moreover, combination treatment using CFP and cancer self-antigen efficiently inhibited B16 tumor growth in the mouse model. Treatment with CFP also enhanced anti-PD-L1 antibody mediated anti-cancer immunity in the CT-26 carcinoma-bearing BALB/c mice. Taken together these data suggest that CFP may function as an adjuvant in the treatment of cancer by enhancing immune activation. Abbreviations CFP: Codium fragile polysaccharide; NK: natural killer; IFN: interferon; TNF: tumor necrosis factor; IL: interleukin; tdLN: tumor draining lymph node; BMDC: bone marrow-derived dendritic cell; OVA: ovalbumin; Ab: antibody; Ag: antigen; DC: dendritic cell; CTL: cytotoxic T lymphocyte; APC: antigen-presenting cell; pDC: plasmacytoid dendritic cell; mDC: myeloid dendritic cell; MHC: major histocompatibility complex; CR3: complement receptor type 3; TLR: Toll-like receptor; LPS: lipopolysaccharide; SP: sulfated polysaccharide; TRP2: tyrosinase-related protein 2; SR-A: scavenger receptor-A

Stochastic Expansions Maintain the Clonal Stability of CD8+ T Cell Populations Undergoing Memory Inflation Driven by Murine Cytomegalovirus

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV.

Restraining neuroinflammation during antigen delivery as an immunosuppressive approach to prevent and treat contact dermatitis

Abstract Neuroinflammation promotes the initiation and sustains chronic inflammatory disorders. Substance-P, released by sensory neurons, is the prototype neuropeptide that signals via the neurokinin 1 receptor (NK1R) to enhance cellular immunity. We observed that NK1RKO or SPKO mice do not develop contact dermatitis (CD). Hence, we hypothesized that limiting neuroinflammation during Ag entrance induces an immune-suppressive environment to prevent T cell priming and eliminate memory T cells that cause CD. We co-delivered OVA or 2,4-dinitrocholorobencene (DNCB) and two different NK1R antagonists during sensitization of a DTH reaction induced to C57/BL6 mice reconstituted or not with OTI and OTII cells. For efficient skin delivery, we generated microneedle arrays loaded with OVA or DNCB and NK1R antagonists. We demonstrate that our approach prevents the innate and adaptive immunity accounting for the initiation of CD and mitigates pre-existing pathogenic memory T cells that cause local or systemic CD relapses. Mechanistic studies demonstrate that NK1R antagonists suppress the release of pro-inflammatory cytokines in the skin, promote the death of activated CD4 Th1 and CD8 T-cells, and generate Tregs in the skin draining lymph nodes. Our data demonstrates that controlling neuroinflammation during Ag entrance prevents the generation of pathogenic effector and memory T cells accounting for the initiation and relapses of chronic inflammatory skin disorders like CD. NIH R01 AR068249 and AR071277 to ATL and LDF.

NLRP3 inflammasome-activating arginine-based liposomes promote antigen presentations in dendritic cells.

Purpose: The NLRP3 inflammasome activation has been proposed as a common mechanism for some adjuvants to boost the immune system, and cationic liposomes were reported to potentially activate the NLRP3 inflammasome. Herein, we questioned whether the NLRP3 inflammasome-activating cationic liposomes could promote antigen presentation and be applied as an immune adjuvant. In addition, we aimed to investigate the structure effect of lipid on triggering these immune responses.Materials and methods: A series of structurally similar lipids, consisting of arginine (Arg) head group and varied lengths of alkyl chains or spacers in between were used to prepare cationic liposomes. Lipopolysaccharide-primed human or murine macrophages or phorbol 12-myristate 13-acetate-primed THP-1 cells were treated with these liposomes, and interleukin (IL)-1β secretion was measured to quantify the NLRP3 inflammasome activation. Lysosome rupture was examined in THP-1 cells by the fluorescence loss of acridine orange, a lysosome dye. Further, chicken ovalbumin (OVA) was loaded on the liposome surface and applied to murine bone marrow-derived dendritic cells (BMDCs), which activate OT-I and OT-II lymphocytes upon major histocompatibility complex (MHC) class I- and class II-mediated antigen presentation, respectively. OT-I and OT-II cell division and IL-2 secretion were measured to evaluate the antigen presentation efficiency. The expressions of MHC molecules and co-stimulatory molecules ie, CD80, CD86, and CD40 on BMDCs were investigated by flow cytometry.Results: All the liposomes showed size distributions of 80–200 nm and zeta potentials of around 50 mV. A3C14 liposomes, consisting of Arg-C3-Glu2C14 lipids induced the most potent lysosome rupture and NLRP3 inflammasome activation. OVA-A3C14 also exhibited the most potent MHC class I- and class II-mediated antigen presentation in BMDCs without interfering MHC and co-stimulatory molecules.Conclusion: The hydrophobic moieties of arginine-based liposomes are crucial in stimulating innate immune cells. A3C14 liposomes were non-immunogenic but strongly activated innate immune cells and promoted antigen presentation, and therefore can be applied as immune adjuvants.

Antigen presentation by CD301b+ dermal dendritic cells dictates CD4+ T cell fate

Abstract Different subsets of dendritic cells (DCs) induce different types of effector CD4T cells. Naïve mouse skin is populated with three major DC subsets, including epidermal Langerhans cells, CD103+ dermal DCs and CD301b+ dermal DCs, all of which migrate to the draining lymph nodes (dLNs). We showed previously that CD301b+DCs are required for the differentiation of antigen specific T helper type 2 (Th2) cells upon immunization with ovalbumin (OVA) and papain in the footpad. However, the mechanism by which CD301b+DCs induce Th2 differentiation is unclear. Here, we examined the role of antigen presentation by CD301b+DCs in the priming and Th2 differentiation of antigen-specific CD4T cells. As in CD301b+DC-depleted mice, mice lacking MHC class II (MHCII) specifically in CD301b+ cells (CD301bΔMHCII mice) showed significantly reduced IL-4 production from OVA-specific OT-II CD4T cells 7 days after immunization, suggesting that cognate interaction between CD301b+DCs and CD4T cells is required for the development of Th2 cells. In addition, although the overall cell cycle progression of OT-II cells was unaffected, their entry into the cell cycle was significantly delayed. In both CD301b+DC-depleted and CD301bΔMHCII mice, this reduction of OT-II cells was associated with impaired retention of OT-II cells in the dLN at much earlier timing. Furthermore, CD301b+DCs were required for retaining and activating wild-type polyclonal CD4T cells in the dLNs in an MHCII-dependent manner. These results indicate that CD301b+DCs are required for effective priming of CD4T cells and Th2 differentiation through their cognate interaction with antigen-specific CD4T cells.

Robust enhancement of tumor-dictated CD8 T cell trafficking and activation by QS21 adjuvanted vaccine

Abstract Adjuvants enhance immune response to antigens and increase vaccine efficacy. We investigated the relative potency of adjuvants with diverse mechanism of action: a natural killer T cell activating ligand (alpha-galactosylcermide, αGalCer), a toll-like receptor 9 ligand (CpG B), a liposomal nanoparticle (R-DOTAP) and a plant saponin from Quillajira saponaria (QS21). We used OVA TCR transgenic model and also compared systemic vs mucosal routes of delivery for inducing antigen-specific CD8 T cell responses in naïve and syngeneic tumor-bearing mice. Wild type C57Bl/6 mice were vaccinated with OVA admixed with the adjuvants via subcutaneous (SC) or intranasal (IN) route 1d after adoptive transfer of the OT-I cells. Single cell suspensions of tissues 7d post-vaccination were analyzed using flow cytometry for OT-1 cells (CD45.1+Vα2+). Vaccination by either SC or IN route using QS21 adjuvant showed the most potent trafficking and activation of CD8+ T cells to both systemic and at mucosal sites, with the highest response observed in the lungs. Vaccination using the αGalCer by the IN route was relatively more effective than when delivered by the SC route, but the responses with αGalCer were lower compared to that with the QS21 adjuvant. However, vaccination of mice bearing B16F10-OVA resulted in preferential trafficking of OT-I cells to the tumors with much reduced numbers at the other tissues. Once again, these responses were robustly higher with QS21 vaccine delivered by either SC or IN route and the OT-1+ T cells from the tumors exhibited higher levels of functional and proliferating properties based on IFN-γ and Ki67 staining. Our results demonstrate that QS21 is a potent adjuvant for prophylactic as well as therapeutic vaccination applications.

Spatial distribution of antigen-specific CD8+ T cells in virally infected skin

Abstract CD8+ T cells play a critical role in the elimination of viral infections. After epicutaneous infection with vaccinia virus (VACV), virus replicates in skin keratinocytes and antigen drains to the lymph node, priming antigen-specific CD8+ T cells. Once activated, CD8+ T cell migration into the skin occurs independent of cognate antigen recognition. In laboratory mice, which are immunologically naive prior to viral infection, only antigen-specific CD8+ T cells are activated and can migrate into the skin. However, as humans are exposed to many more pathogens than laboratory mice, activated CD8+ cells present in the circulation can be recruited to the site of infection, regardless of their antigen specificity. To model this phenomenon in mice, we adoptively transferred OT-I CD8+ T cells, infected in the footpad with non-replicating virus to activate these T cells, and then infected epicutaneously with recombinant VACV expressing or lacking SIINFEKL (the cognate antigen for OT-I cells). This system allows the robust recruitment of both antigen-specific and non-specific antiviral CD8+ T cells into the skin. Using a combination of flow cytometry and confocal and intravital microscopy, we are currently investigating the spatial distribution of antigen-specific and non-specific CD8+ T cells in the skin. Additionally, we are quantifying cytokine production by these CD8+ T cells under various conditions, including after infection with viruses expressing distinct antigens, in close proximity. These studies should provide additional clarity on the role of antigen in CD8+ T cell spatial distribution in infected peripheral tissues.

Differential effect of GITR on lung effector T cell subpopulations during influenza infection

Abstract Influenza remains an important global threat. Tissue resident memory T cells (Trm) are critical in protection against influenza, and it is important to identify key mechanisms regulating their formation and persistence. GITR, an NF-κB activating TNFR family member, is required on CD8 T cells for maximal responses against influenza. Our recent work (Mucosal Immunology, doi:10.1038/s41385-018-0105-5) provided evidence that GITRL on monocyte-derived inflammatory APCs provides crucial signals through GITR on T cells in the lung tissue (termed Signal 4), allowing effector T cell accumulation and optimal Trm formation during influenza infection in mice. During influenza infection, responding lung T cells are heterogeneous, some will give rise to terminally differentiated effector cells, while others will become Trm. It remains unknown how GITR affects these subpopulations. To address this issue, we characterized these CD8 T cell subsets using markers Ly6C, KLRG1, T-bet, and the chemokine receptor CX3CR1. By staining with Ly6C and CX3CR1, we identified three different CD8 T cell subsets with differential expression of Ly6C and CX3CR1 (Ly6ChiCX3CR1 hi, Ly6ChiCX3CR1 lo, Ly6CloCX3CR1 lo). Ly6ChiCX3CR1 hi CD8 T cell subset likely represents the most terminally differentiated effector cells as they have the highest expression of KLRG1 and T-bet. Using the adoptive transfer of transgenic OT-I cells, we discovered that there is a larger loss of the Ly6CloCX3CR1 loCD8 T cell in the absence of GITR when compared to the other two subsets. We are currently investigating which of the three subsets give rise to the lung Trm population after influenza infection and the timing and nature of the GITR-dependent signals involved. Funded by CIHR:FDN143250

638 SPNS2 enables T cell egress from lymph nodes during an immune response

The signaling lipid sphingosine 1-phosphate (S1P), which is abundant in lymph, guides T cell exit from lymph nodes (LN) into lymph by signaling through T cell S1P receptor 1 (S1PR1). By trapping pathogenic T cells within LN and preventing them from reaching their target tissues, drugs targeting S1PR1 have been successful in treating autoimmune diseases such as multiple sclerosis, and are being tested in dermatomyositis, psoriasis, graft versus host disease, and subacute cutaneous lupus erythematosus. Simultaneous inhibition of S1PR1 within the cardiovascular system, however, results in adverse effects on the heart and vasculature. At steady state, the major facilitator superfamily transporter SPNS2 is required to supply lymph S1P, which drives lymphocyte egress, but does not play a major role in maintaining the levels of blood S1P required for cardiovascular function. Inhibition of SPNS2 was therefore proposed as a strategy to trap autoreactive T cells in LN while avoiding cardiovascular side effects. However, it was unknown whether SPNS2 remained necessary to supply lymph S1P during an immune response, or whether other compensatory transporters were up-regulated during inflammation. Here, using an OT-II TCR transgenic CD4 T cell model of dermal inflammation, we demonstrate that in SPNS2-deficient mice, there is a 10-fold decrease in T cell accumulation in inflamed skin compared with littermate controls. This is not due to differential T cell proliferation, as draining LN contain similar numbers of OT-II cells. Consistent with a LN egress defect, there is a decrease in activated OT-II cells in the lymph and blood of SPNS2-deficient mice compared to controls. We then developed a method to measure S1P gradients within lymphatics of inflamed LN and demonstrated that SPNS2 supplies the lymph S1P that guides T cells out of LN with an ongoing immune response. This work adds to our understanding of lymphocyte trafficking in inflammation and suggests a new, more targeted way to treat inflammatory diseases.

Cognate Interaction With CD4+ T Cells Instructs Tumor-Associated Macrophages to Acquire M1-Like Phenotype.

The immunosuppressive tumor microenvironment (TME) established by tumor cells, stromal cells and inhibitory immune cells counteracts the function of tumor reactive T cells. Tumor associated macrophages (TAMs) showing functional plasticity contribute to this process as so called M2-like macrophages can suppress the function of effector T cells and promote their differentiation into regulatory T cells (Tregs). Furthermore, tumor antigen specific CD4+ T effector cells can essentially sustain anti-tumoral immune responses as shown for various tumor entities, thus suggesting that cognate interaction between tumor antigen-specific CD4+ Th1 cells and TAMs might shift the intra-tumoral M1/M2 ratio toward M1. This study demonstrates repolarization of M2-like PECs upon MHC II-restricted interaction with tumor specific CD4+ Th1 cells in vitro as shown by extensive gene and protein expression analyses. Moreover, adoptive transfer of OVA-specific OT-II cells into C57BL/6 mice bearing OVA expressing IAb−/− tumors resulted in increased accumulation of M1-like TAMs with enhanced M1 associated gene and protein expression profiles. Thus, this paper highlights a so far underestimated function of the CD4+ Th1/TAM axis in re-conditioning the immunosuppressive tumor microenvironment.

Abstract A067: Cognate interaction with CD4+ T-cells instructs M2-like macrophages to acquire M1-like phenotype

Abstract Current tumor immunotherapy approaches are based on application of checkpoint inhibitors, such as monoclonal antibodies against PD-1/PD-L1 and CTLA-4 or adoptive T-cell therapy using ex vivo expanded TILs or genetically modified autologous T-cells expressing recombinant T-cell receptors (TCRs) or chimeric antigen receptors (CARs). However, the success of these therapeutic strategies is often limited by various inhibitory immune cell types accumulating in the tumor. In particular, tumor-associated macrophages (TAMs) contribute to the immune suppressive tumor micro-milieu, since so-called M2-like macrophages suppress the function of T effector cells and promote the differentiation of regulatory T-cells (Treg) through secretion of inhibitory cytokines such as TGF-β and IL10. Tumor antigen-specific CD4+ T effector cells, on the other hand, can essentially sustain antitumoral immune responses as shown for various tumor entities. In fact, using peritoneal exudate cells (PECs) as source for macrophages we demonstrate that MHC II restricted interaction between ovalbumin (OVA) specific CD4+ T-cells and M2-like macrophages drives M1 polarization. This was confirmed by detailed gene and protein expression analyses as well as functional assays testing phagocytic and pinocytic activities of repolarized macrophages. Moreover, in a set of preclinical experiments, adoptive transfer of CD4+, OVA-specific OT-II cells into C57BL/6 mice bearing OVA expressing IAb-/- tumors resulted in increased intratumoral number of M1-like TAMs as determined by gene expression analysis and flow cytometry. Furthermore, we observed a significant survival benefit of mice treated with a combination of OVA-specific CD4+ (OT-II) and CD8+ (OT-I) cells after transplantation of B16F10-Ova tumors and a complete response in some mice that rejected the tumor cells also upon a later re-challenge. While the antitumoral effect of this adoptive transfer experiment has been already described, we now offer a possible explanation for the supportive effect of specific CD4+ cells on co-transferred CD8+ cells. Taken together, the instructive impact of CD4+ T-cells on M2-like macrophages described in this presentation points towards a so far underestimated function of the CD4+ T-cell / TAM axis in reconditioning the immunosuppressive tumor micro-milieu. Citation Format: David Eisel, Wolfram Osen, Krishna Das, Franziska Marie-Claire Hoerhold, Rainer König, Stefan B. Eichmüller. Cognate interaction with CD4+ T-cells instructs M2-like macrophages to acquire M1-like phenotype [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 A067.

Abstract A173: The extracellular ATP receptor P2RX7 is required for CD8+ T-cells to maintain and respond to chronic virus and melanoma tumors

Abstract T-cell exhaustion is an acquired state of T-cell dysfunction induced by sustained antigen stimulation (such as in chronic viral infections and cancer). Recently, it was described that exhausted CD8+ T-cells are heterogeneous, with some being preferentially rescued by checkpoint blockade therapy. These cells typically express the chemokine receptor CXCR5 and depend on the transcription factor TCF1 for their development. However, little is known about the extracellular signals required for the generation and function of this subset. Our group has been working with the “danger signal” extracellular ATP (eATP), which sense cellular damage and is recognized by purinergic receptors in mammals. Notably, we recently discovered that the eATP purinergic receptor P2RX7 is crucial for the generation and maintenance of long-lived memory CD8+ T-cells in a cell-intrinsic way, by controlling the mitochondrial function and viability of these cells during acute viral infections. Here, we show that P2RX7 is also required, in a cell-intrinsic way, for CD8+ T-cells to survive and maintain in the presence of chronic antigen. In response to chronic lymphocytic choriomeningitis virus (LCMV Clone 13) infection, we tracked WT vs P2RX7KO LCMV-specific CD8+ T-cells, using specific tetramer staining in germline knock-out vs WT mice, or co-adoptive transfer of antigen-specific (P14) cells into CD4-depleted recipient mice. In response to LCMV-Clone 13, P2RX7KO CD8+ T-cells initially expand but fail to maintain after the first week of infection. This is accompanied by almost no expansion of the CXCR5+ subset – which express higher surface P2RX7 levels. The few CXCR5+ cells that form in the absence of P2RX7 are aberrant in their phenotype, with higher expression of PD1 and Tim3 and lower expression of TCF1. During chronic LCMV, P2RX7KO CD8+ T-cells have increased cell death as early as one week after infection, but no proliferation defects. Correlating with this, both mitochondrial mass and membrane potential (a readout of mitochondrial functionality) of P2RX7KO CD8+ T-cells are severely impaired. Consequently, germline P2RX7KO mice have defective control of viral titers in comparison to WT counterparts. Despite having higher PD1 expression, anti-PD1 therapy with do not induce expansion of adoptively transferred P2RX7KO P14 cells in comparison to WT P14 cells, in pair with the absence of the CXCR5+ subset. Importantly, pharmacologic P2RX7 blockade after establishment of chronic infection leads to a decrease in WT LCMV-specific CD8+ T-cell numbers, indicating P2RX7-mediated eATP sensing is constantly required for CD8+ T-cells to survive during chronic infection. Finally, we used adoptive transfer of in vitro activated WT vs P2RX7KO OT-I cells (in which we observed similar initial activation) and observed that CD8+ T-cells require cell-intrinsic P2RX7 to infiltrate B16-OVA melanoma tumors. Additionally, P2RX7KO mice have impaired B16 melanoma growth control. These results show that the danger signal eATP can aid in CD8+ T-cell long-term viability and function in the presence of chronic antigen. This will open the possibility of using this signaling pathway to design new alternatives to boost cancer immunotherapies, for example by harnessing the P2RX7/eATP pathway in tumor-specific CD8+ T-cells in adoptive transfer-based immunotherapies, which can be combined with existing checkpoint blockade therapies. Citation Format: Henrique Borges da Silva, Stephen C. Jameson. The extracellular ATP receptor P2RX7 is required for CD8+ T-cells to maintain and respond to chronic virus and melanoma 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 A173.

First Evidence of Dysfunctional Antigen-Specific T Cell Responses in Experimental CLL As a Model for Studies of Autologous T Cell-Based Therapies

Background - Tumor immunosuppression is a major cause for treatment failure and disease relapse, both in solid tumors and in leukemia. New T cell mediated therapeutic modalities (such as CAR T cells, bispecific antibodies and immune checkpoint blockade) can be highly effective, but depend on autologous T cell functionality. In chronic lymphocytic leukemia (CLL), ex vivo patient studies have shown that T cells are skewed toward a terminally differentiated, dysfunctional phenotype, which may contribute to the disappointing results of T cell therapies in this disease. Recently, it has been shown that for CAR T cell efficacy in CLL, the presence of memory subsets both prior to CAR T cell generation and in the infusion product, is instrumental for CAR T cell persistence and complete responses (Fraietta et al., 2018). However, it is currently unknown how the presence of CLL impacts T cell skewing and whether it affects acute antigen-specific immune responses in vivo. As dynamic immune studies are limited in the human setting, we investigated whether acute antigen-mediated T cell responses are affected by CLL using the TCL1 adoptive transfer mouse model. Earlier T cell studies in this model revealed similar changes as seen in the human disease, both with respect to gene expression profiles as well as phenotypic and functional characteristics.Methods - C57BL/6J (CD45.2+) mice were injected i.p. with 20x106 TCL1 transgenic splenocytes (CD45.2+) or PBS. During development of CLL mice were monitored for T cell subset differentiation. When CLL mice reached over 70% CLL in blood, 50.000 OT-I cells (CD45.1+), which specifically recognize ovalbumin) were adoptively transferred (i.v.) into all mice, directly followed by infection with 100.000 PFU mCMV-OVA. This model allowed us to study the endogenous (CD45.2+) T cell compartment during CLL development, as well as the acute response of naïve, antigen-specific (CD45.1+) T cells that had not been influenced by CLL prior to antigen exposure. Seven days after infection the mice were sacrificed and splenocytes were analyzed by flow cytometry either directly or after re-stimulation with OVA-peptide to assess cytokine production.Results - Using this adoptive transfer model of CLL, we found that CLL mice showed a decrease in naïve CD8 T cells, an increase of antigen-experienced cells, and a reversed CD4/CD8 ratio compared to control mice, as reported earlier (McClanahan et al., 2015). At day 7 after injection of naïve OT-I cells and infection with mCMV-OVA, percentages of OT-I cells were similar in CLL and control mice, and all OT-I cells showed an effector phenotype (CD44+CD62L-). However, in this effector OT-I pool CLL mice showed an increased frequency of KLRG1+CD127- short-lived effector cells (SLEC), while KLRG1-CD127+ memory precursor effector cells (MPEC) were decreased compared to control mice. This was associated with an enhanced expression of the effector-associated transcription factor T-bet, and reduced expression of the memory-associated transcription factor Bcl-6 in total OT-I cells and within the OT-I SLEC and MPEC populations of CLL mice. Since poor responses to CAR T cell therapy are associated with effector phenotypes and higher rates of glycolysis (Fraietta et al., 2018), and Bcl-6 directly represses glycolysis (Oestreich et al., 2014), we analyzed glucose uptake by OT-I cells. In line with reduced expression of Bcl-6, we found increased levels of glucose uptake in CLL derived OT-I cells. Despite the skewing towards a more short-lived effector phenotype of the CLL derived OT-I cells, in vitro re-stimulation with OVA peptide resulted in decreased production of IFN-γ and decreased degranulation as measured by CD107a (LAMP-1) expression compared to control derived OT-I cells.Conclusion - Our findings show that in this mouse model CLL development skews the T cell compartment towards more antigen-experienced cells. In addition to this, our results suggest that there is a CLL-antigen independent effect on acute antigen-specific immune responses. This CLL mediated effect drives T cells towards an effector phenotype, that is functionally impaired. This study provides clues for better understanding T cell responses in CLL, and may lead to new strategies to improve T cell mediated therapies in CLL, which are currently being exploited. DisclosuresEldering:Celgene: Research Funding. Kater:Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

Immunogenic cell death of dendritic cells following modified vaccinia virus Ankara infection enhances CD8+ T cell proliferation.

"Immunogenic cell death" (ICD) is associated with the emission of so-called damage-associated molecular patterns (DAMPs) which trigger the immune response against dead-cell associated antigens. The secretion of the DAMP, adenosine triphosphate (ATP) has been shown to be autophagy-dependent. Here, we demonstrate that Modified Vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, induces both cell death and autophagy in murine bone marrow-derived dendritic cells (BMDCs), which in turn confer the (cross-)priming of OVA-specific cytotoxic Tcells (OT-I cells). Additionally, we show that MVA infection leads to increased extracellular ATP (eATP) as well as intracellular ATP (iATP) levels, with the latter being influenced by the autophagy. Furthermore, we show that the increased eATP supports the proliferation of OT-I cells and inhibition of the P2RX7 receptors results in an abrogation of the proliferation. These data reveal novel mechanisms on how MVA enhances adaptive immunity in vaccine strategies.

Lack of Sprouty 1 and 2 enhances survival of effector CD8+ T cells and yields more protective memory cells

Identifying novel pathways that promote robust function and longevity of cytotoxic T cells has promising potential for immunotherapeutic strategies to combat cancer and chronic infections. We show that sprouty 1 and 2 (Spry1/2) molecules regulate the survival and function of memory CD8+ T cells. Spry1/2 double-knockout (DKO) ovalbumin (OVA)-specific CD8+ T cells (OT-I cells) mounted more vigorous autoimmune diabetes than WT OT-I cells when transferred to mice expressing OVA in their pancreatic β-islets. To determine the consequence of Spry1/2 deletion on effector and memory CD8+ T cell development and function, we used systemic infection with lymphocytic choriomeningitis virus (LCMV) Armstrong. Spry1/2 DKO LCMV gp33-specific P14 CD8+ T cells survive contraction better than WT cells and generate significantly more polyfunctional memory T cells. The larger number of Spry1/2 DKO memory T cells displayed enhanced infiltration into infected tissue, demonstrating that absence of Spry1/2 can result in increased recall capacity. Upon adoptive transfer into naive hosts, Spry1/2 DKO memory T cells controlled Listeria monocytogenes infection better than WT cells. The enhanced formation of more functional Spry1/2 DKO memory T cells was associated with significantly reduced mTORC1 activity and glucose uptake. Reduced p-AKT, p-FoxO1/3a, and T-bet expression was also consistent with enhanced survival and memory accrual. Collectively, loss of Spry1/2 enhances the survival of effector CD8+ T cells and results in the formation of more protective memory cells. Deleting Spry1/2 in antigen-specific CD8+ T cells may have therapeutic potential for enhancing the survival and functionality of effector and memory CD8+ T cells in vivo.

Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells.

Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-γ (IFN-γ) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-κB p65 activation and IL-12 and IFN-γ production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-γ production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.