Antigen-specific Manner Research Articles

T cell antigen discovery via signaling and antigen-presenting bifunctional receptors.

CD8+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, identifying their target antigens remains a challenge. Here, we describe the use of chimeric receptors called Signaling and Antigen-presenting Bifunctional Receptors (SABRs) in a novel cell-based platform for T Cell Receptor (TCR) antigen discovery. SABRs present an extracellular peptide-MHC complex and induce intracellular signaling via a TCR-like signal upon binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery. The antigen discovery platform reported here will provide a scalable and versatile way to develop novel targets for immunotherapy.

Pluripotent Cell-Derived Off-the-Shelf TCR-Less CAR-Targeted Cytotoxic T Cell Therapeutic for the Allogeneic Treatment of B Cell Malignancies

▪The advent of off-the-shelf chimeric antigen receptor (CAR) T cell therapeutics is widely recognized to be a major potential advancement for the treatment of cancer. Several obstacles currently hamper the broad use of CAR T cells, including the inherent variability and cost of manufacturing of autologous cellular populations, the absolute requirement for precise genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity. Here we present pre-clinical data for FT819, a first-of-kind off-the-shelf human induced pluripotent stem cell (hiPSC)-derived CAR T cell product. FT819 is defined by the precise genetic engineering of multiple targeting events at the single cell level to create a clonal master iPSC line. The engineered features include the targeted integration of a novel, modified CD19 CAR into the T cell receptor α (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease (GvHD), and the expression of a high-affinity, non-cleavable form of CD16 (hnCD16) to deliver an adjustable system to address tumor antigen escape.Through a proprietary cellular reprogramming platform, peripheral blood derived T cells are converted to hiPSCs, engineered to contain the modified CD19 CAR targeted into the TRAC locus and hnCD16, and clonally selected to create a master hiPSC line (TRAC-TiPSC, FT819). Molecular characterization of the TRAC-TiPSC master cell line by 5‘ junction, 3‘ junction and internal sequence PCR confirmed homology directed repair and bi-allelic targeting of the CD19 CAR into the TRAC locus. The origin of the clonal master cell bank was confirmed to be a TCRαβ T cell by PCR-mediated detection of TCRδ locus deletion and methyl-seq analysis of the TCRα locus. Flow cytometric analysis demonstrated the maintenance of a uniform population of hiPSCs (>95% SSEA4/TRA-1-81/OCT4/NANOG) and expression of hnCD16 transgene (>95% CD16).Utilizing our stage-specific T cell differentiation protocol, we demonstrate that the TRAC-TiPSCs yield TRAC-iT cells with uniform expression of the CAR (>95%), complete elimination of TCR surface expression and clinically enabling expansion through the manufacturing process (>50,000 fold). To confirm the lack of alloreactivity conferred by the deletion of endogenous TCR expression, mixed lymphocyte reactions were performed using TRAC-iT, primary TCR+ T cells and primary TCR+CAR+ T cells as responders and HLA-mismatched peripheral blood mononuclear cells (PBMCs) as targets. In comparison to primary T cells and primary CAR-T cells, TRAC-iT did not respond and proliferate in response to TCR stimulation or HLA-mismatched PBMCs indicating that the risk of GvHD was alleviated.In vitro functional studies established that TRAC-iT possess a potent cytotoxic T lymphocyte response to CD19 antigen challenge in a similar manner to peripheral blood CAR T cells as demonstrated by expression of markers indicative of degranulation (CD107a/b, Granzyme B), T cell activation (CD69, CD25), and production of INFγ, TNFα and IL2. Importantly, TRAC-iT targeted tumor in an antigen specific manner as verified by lysis of CD19+, but not CD19-, tumor cell lines as seen by in vitro cytolytic assays (50% killing E:T; TRAC-iT = 1:8, primary CAR-T = 1:4).In vivo studies demonstrated that TRAC-iT cells effectively control tumor progression in a mouse model of acute lymphoblastic leukemia Nalm6 (TRAC-iT versus no treatment, p<0.0001). To validate the capability of TRAC-iT to simultaneously target multiple antigens, TRAC-iT was co-cultured with mixtures of CD19+CD20+ and CD19-CD20+ tumor cells in the presence of anti-CD20 monoclonal antibody, Rituxan. In vitro cytolytic assays demonstrate that only TRAC-iT cells can effectively identify and eliminate CD19-CD20+ tumor cells when combined with Rituxan. Importantly, the antibody-dependent cellular-cytotoxicity did not appear to interfere with CAR function as TRAC-iT maintained its directed cytotoxic capacity. Collectively, these preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf product than can be effectively and safely used in the treatment of B cell malignancies in the allogeneic setting. DisclosuresClarke:Fate Therapeutics Inc.: Employment. Chang:Fate Therapeutics Inc.: Employment. Husain:Fate Therapeutics Inc.: Employment. Lai:Fate Therapeutics Inc.: Employment. Peralta:Fate Therapeutics Inc.: Employment. Stokely:Fate Therapeutics Inc.: Employment. Abujarour:Fate Therapeutics Inc.: Employment. Dinella:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Pribadi:Fate Therapeutics Inc.: Employment. Chu:Fate Therapeutics Inc.: Employment. Truong:Fate Therapeutics Inc.: Employment. Sabouri-Ghomi:Fate Therapeutics Inc.: Employment. Meza:Fate Therapeutics Inc.: Employment. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Sadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

A novel and simple method to produce large amounts of recombinant soluble peptide/major histocompatibility complex monomers for analysis of antigen-specific human T cell receptors

Soluble peptide/major histocompatibility complex (p/MHC) tetramers that directly bind to T cell receptors (TCRs) allow the direct quantification, phenotypic characterization and isolation of antigen-specific T cells. Conventionally, soluble p/MHC tetramers have been produced using Escherichia coli, but this method requires refolding of the recombinant proteins. Here, a novel and technically simple method that does not require protein refolding in vitro has been developed for the high-throughput generation of soluble and functional p/MHC-single chain trimer (SCT) monomers and tetramers in a mammalian cell system. The p/MHC-SCT tetramers generated by this method bound to the corresponding antigen-specific TCRs. Moreover, the immobilized p/MHC-SCT monomers effectively activated antigen-specific T cell lines as well as primary T cells in an antigen-specific manner. This technique provides a robust improvement in the technology, such that recombinant soluble p/MHC monomers and tetramers can be produced more readily and which enables their use in analysis of antigen-specific T cells in basic and clinical studies.

Comparative transcriptomic profile of tolerogenic dendritic cells differentiated with vitamin D3, dexamethasone and rapamycin

Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However, the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic, as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim, in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC), dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However, individually, CYP24A1, MUCL1 and MAP7 for vitD3-tolDC; CD163, CCL18, C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC, constituted good candidate biomarkers for each respective cellular product. In addition, a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways, while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes.

Development of a Tetravalent Anti-GPA33/Anti-CD3 Bispecific Antibody for Colorectal Cancers.

Despite progress in the treatment of colorectal cancer, curing metastatic colorectal cancer remains a major unmet medical need worldwide. Here, we describe a T-cell-engaging bispecific antibody (T-BsAb) to redirect polyclonal cytotoxic T cells to eradicate colorectal cancer. A33, a murine antibody specific for GPA33, was humanized to huA33 and reformatted to huA33-BsAb, based on a novel IgG(L)-scFv platform by linking the anti-CD3 huOKT3 scFv to the carboxyl end of the light chain. This T-BsAb was stably expressed in CHO cells and purified as a stable monomer by HPLC, retaining immunoreactivity by FACS through 30 days of incubation at 37°C. In vitro, it induced activation and expansion of unstimulated T cells and elicited potent T-cell-dependent cell-mediated cytotoxicity against colon and gastric cancer cells in an antigen-specific manner. In vivo, huA33-BsAb inhibited the colon and gastric cancer xenografts, in both subcutaneous and intraperitoneal tumor models. More importantly, both microsatellite instable and microsatellite stable colorectal cancer were effectively eliminated by huA33-BsAb. These preclinical results provide further support for the use of IgG(L)-scFv platform to build BsAb, and especially one targeting GPA33 for colorectal cancer. These preclinical results also support further development of huA33-BsAb as a potential immunotherapeutic. Mol Cancer Ther; 17(10); 2164-75. ©2018 AACR.

Comparison of immunogenicity and vaccine efficacy between heat-shock proteins, HSP70 and GrpE, in the DnaK operon of Mycobacterium tuberculosis

Antigens (Ags) in Mycobacterium tuberculosis (Mtb) that are constitutively expressed, overexpressed during growth, essential for survival, and highly conserved may be good vaccine targets if they induce the appropriate anti-Mtb Th1 immune response. In this context, stress response-related antigens of Mtb might serve as attractive targets for vaccine development as they are rapidly expressed and are up-regulated during Mtb infection in vivo. Our group recently demonstrated that GrpE, encoded by rv0351 as a cofactor of heat-shock protein 70 (HSP70) in the DnaK operon, is a novel immune activator that interacts with DCs to generate Th1-biased memory T cells in an antigen-specific manner. In this study, GrpE was evaluated as a subunit vaccine in comparison with the well-known HSP70 against the hyper-virulent Mtb Beijing K-strain. Both HSP70- and GrpE-specific effector/memory T cells expanded to a similar extent as those stimulated with ESAT-6 in the lung and spleen of Mtb-infected mice, but GrpE only produced a similar level of IFN-γ to that produced by ESAT-6 stimulation during the late phase and the early phase of Mtb K infection, indicating that GrpE is highly-well recognised by the host immune system as a T cell antigen. Mice immunised with the GrpE subunit vaccine displayed enhanced antigen-specific IFN-γ and serum IgG2c responses along with antigen-specific effector/memory T cell expansion in the lungs. In addition, GrpE-immunisation markedly induced multifunctional Th1-type CD4+ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the lungs of Mtb K-infected mice, whereas HSP70-immunisation induced mixed Th1/Th2 immune responses. GrpE-immunisation conferred a more significant protective effect than that of HSP70-immunisation in terms of bacterial reduction and improved inflammation, accompanied by the remarkable persistence of GrpE-specific multifunctional CD4+ T cells. These results suggest that GrpE is an excellent vaccine antigen component for the development of a multi-antigenic Mtb subunit vaccine by generating Th1-biased memory T cells with multifunctional capacity, and confers durable protection against the highly virulent Mtb K.

BDCA1+CD14+ Immunosuppressive Cells in Cancer, a Potential Target?

Dendritic cell (DC) vaccines show promising effects in cancer immunotherapy. However, their efficacy is affected by a number of factors, including (1) the quality of the DC vaccine and (2) tumor immune evasion. The recently characterized BDCA1+CD14+ immunosuppressive cells combine both aspects; their presence in DC vaccines may directly hamper vaccine efficacy, whereas, in patients, BDCA1+CD14+ cells may suppress the induced immune response in an antigen-specific manner systemically and at the tumor site. We hypothesize that BDCA1+CD14+ cells are present in a broad spectrum of cancers and demand further investigation to reveal treatment opportunities and/or improvement for DC vaccines. In this review, we summarize the findings on BDCA1+CD14+ cells in solid cancers. In addition, we evaluate the presence of BDCA1+CD14+ cells in leukemic cancers. Preliminary results suggest that the presence of BDCA1+CD14+ cells correlates with clinical features of acute and chronic myeloid leukemia. Future research focusing on the differentiation from monocytes towards BDCA1+CD14+ cells could reveal more about their cell biology and clinical significance. Targeting these cells in cancer patients may improve the outcome of cancer immunotherapy.

Antibody Light Chains Dictate the Specificity of Contact Hypersensitivity Effector Cell Suppression Mediated by Exosomes.

Antibody light chains (LCs), formerly considered a waste product of immunoglobulin synthesis, are currently recognized as important players in the activation of the immune response. However, very little is known about the possible immune regulatory functions of LCs. Recently, we reported that hapten-specific LCs coat miRNA-150-carrying exosomes produced by CD8+ suppressor T cells downregulating the contact hypersensitivity (CHS) reaction in an antigen-specific manner, in mice tolerized by intravenous administration of a high dose of hapten-coupled syngeneic erythrocytes. Thus, the current studies aimed at investigating the role of hapten-specific LCs in antigen-specific, exosome-mediated suppression of CHS effector cells. Suppressor T cell-derived exosomes from tolerized B-cell-deficient µMT−/−, NKT-cell-deficient Jα18−/−, and immunoglobulin-deficient JH−/− mice were nonsuppressive, unless supplemented with LCs of specificity strictly respective to the hapten used for sensitization and CHS elicitation in mice. Thus, these observations demonstrate that B1-cell-derived LCs, coating exosomes in vivo and in vitro, actually ensure the specificity of CHS suppression. Our research findings substantially expand current understanding of the newly discovered, suppressor T cell-dependent tolerance mechanism by uncovering the function of antigen-specific LCs in exosome-mediated, cell–cell communication. This express great translational potential in designing nanocarriers for specific targeting of desired cells.

Armed and Ready: Transcriptional Regulation of Tissue-Resident Memory CD8 T Cells.

A fundamental benefit of immunological memory is the ability to respond in an enhanced manner upon secondary encounter with the same pathogen. Tissue-resident memory CD8 T (TRM) cells contribute to improved protection against reinfection through the generation of immediate effector responses at the site of pathogen entry. Key to the potential of TRM cells to develop rapid recall responses is their location within the epithelia of the skin, lungs, and intestines at prime entry sites of pathogens. TRM cells are among the first immune cells to respond to pathogens that have been previously encountered in an antigen-specific manner. Upon recognition of invading pathogens, TRM cells release IFN-γ and other pro-inflammatory cytokines and chemokines. These effector molecules activate the surrounding epithelial tissue and recruit other immune cells including natural killer (NK) cells, B cells, and circulating memory CD8 T cells to the site of infection. The repertoire of TRM effector functions also includes the direct lysis of infected cells through the release of cytotoxic molecules such as perforin and granzymes. The mechanisms enabling TRM cells to respond in such a rapid manner are gradually being uncovered. In this review, we will address the signals that instruct TRM generation and maintenance as well as the underlying transcriptional network that keeps TRM cells in a deployment-ready modus. Furthermore, we will discuss how TRM cells respond to reinfection of the tissue and how transcription factors may control immediate and proliferative TRM responses.

Antigen-Specificity in the Thymic Development and Peripheral Activity of CD4+FOXP3+ T Regulatory Cells.

CD4+Foxp3+ T regulatory cells (Treg) are essential for the life of the organism, in particular because they protect the host against its own autoaggressive CD4+Foxp3− T lymphocytes (Tconv). Treg distinctively suppress autoaggressive immunity while permitting efficient defense against infectious diseases. This split effect indicates that Treg activity is controlled in an antigen-specific manner. This specificity is achieved first by the formation of the Treg repertoire during their development, and second by their activation in the periphery. This review presents novel information on the antigen-specificity of Treg development in the thymus, and Treg function in the periphery. These aspects have so far remained imprecisely understood due to the lack of knowledge of the actual antigens recognized by Treg during the different steps of their life, so that most previous studies have been performed using artificial antigens. However, recent studies identified some antigens mediating the positive selection of autoreactive Treg in the thymus, and the function of Treg in the periphery in autoimmune and allergic disorders. These investigations emphasized the remarkable specificity of Treg development and function. Indeed, the development of autoreactive Treg in the thymus was found to be mediated by single autoantigens, so that the absence of one antigen led to a dramatic loss of Treg reacting toward that antigen. The specificity of Treg development is important because the constitution of the Treg repertoire, and especially the presence of holes in this repertoire, was found to crucially influence human immunopathology. Indeed, it was found that the development of human immunopathology was permitted by the lack of Treg against the antigens driving the autoimmune or allergic T cell responses rather than by the impairment of Treg activation or function. The specificity of Treg suppression in the periphery is therefore intimately associated with the mechanisms shaping the formation of the Treg repertoire during their development. This novel information refines significantly our understanding of the antigen-specificity of Treg protective function, which is required to envision how these cells distinctively regulate unwanted immune responses as well as for the development of appropriate approaches to optimally harness them therapeutically in autoimmune, malignant, and infectious diseases.

Abstract LB-108: Generation of off-the-shelf TCR-less CAR-targeted cytotoxic T cells from renewable pluripotent cells for cancer immunotherapy

Abstract Genetically engineered T cells are at the vanguard of an emerging wave of scientific breakthrough focused on harnessing the power of the immune system to treat cancer and other immune disorders. Perhaps the most dramatic clinical outcome to date has been demonstrated in clinical trials evaluating autologous chimeric antigen receptor (CAR) therapy for the treatment of refractory B cell ALL where complete responses in the majority of the patients have been reported. While adoptive CAR-T cell treatments hold great promise, pressing challenges remain to ensure multi-parameter genetically engineered T-cell immunotherapies can be successfully derived, cost-effectively and consistently manufactured, and safely and reliably delivered at the scale necessary to support wide patient base commercialization.Human induced pluripotent stem cell (hiPSC)-derived T cells uniquely represent a practical and renewable supply of well-defined engineered CAR-T cells for various therapeutic applications. We have previously described a novel platform to facilitate multi-gene locus-specific engineering of hiPSCs at the single cell level to establish highly characterized master cell banks which can then be repeatedly applied to our stage-specific lymphocyte directed differentiation process to reproducibly and reliably generate engineered cytotoxic T cells.Here we present the first set of pre-clinical data for FT819, a first-of-kind off-the-shelf hiPSC-derived CAR-T cell product. To generate FT819, we successfully combined reprogramming of peripheral blood derived T cells with targeted insertion of a CD19 CAR into the T cell receptor α (TRAC) locus under the transcriptional control of its endogenous regulatory elements to generate a single cell-derived clonal TRAC-targeted CAR expressing master hiPSC line. The clone was characterized to be pluripotent (&amp;gt;95% SSEA4 / TRA181) and consisted of bi-allelic disruption of TRAC locus. During the stage-specific differentiation, the hiPSC line faithfully converted into CD34 positive cells which were then differentiated towards CD8 positive cells with uniform CAR expression (95 +/- 5%) in the absence of TCR expression, eliminating the likelihood of GvHD. In vitro functional studies demonstrated that FT819 elicits an efficient cytotoxic T lymphocyte response to CD19 antigen challenge with production of effector cytokines (IFNg, TNFa, IL2), degranulation (CD107a/b, Perforin, Granzyme B), proliferation (&amp;gt;85% entry into cell cycle) and upregulation of activation markers CD69 and CD25. Importantly, FT819 targets tumor in an antigen specific manner as demonstrated by lysis of CD19+, but not CD19-, Raji and Nalm6 tumor cell lines. In summary, FT819 holds the promise of a safe and efficacious off-the-shelf cytotoxic CAR-T cell product derived in a renewable and highly reproducible process analogous to biopharmaceutical drug products. Citation Format: Raedun L. Clarke, Sjoukje van der Stegen, Tom Lee, Jorge Mansilla-Soto, Chia-wei Chang, Jeffrey Sasaki, Mushtaq Husain, Eigen Peralta, Ian Hardy, Edwin Ortiz, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. Generation of off-the-shelf TCR-less CAR-targeted cytotoxic T cells from renewable pluripotent cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-108.

Abstract 3795: Novel mechanism underlying tumor immune evasion through tumor endothelial cells

Abstract Background: There has been a recent interest in how tumor cells escape from immune surveillance while establishing tumor microenvironments. Tumor endothelial cells (TECs), which constitute the lining of the tumor vessels, develop in the tumor microenvironment and come in contact with circulating immune cells. As such, TECs may control the trafficking or the anti-tumor reaction of an immune cell. We hypothesized that TECs may have an immune-suppressive potential in tumor microenvironments; thus, TECs may contribute to tumor immune-evasion. Objective: We aimed to investigate the immune-suppressive functions of TECs. Materials and Methods: We established a mouse cancer-model using the B16 mouse melanoma cell line and isolated TECs from the tumors. The immunological character of TECs was assessed by performing flow cytometric phenotyping of TECs and in vitro antigen endocytosis assay using exogenous antigen ovalbumin (OVA). Next, to identify the antigen presentation and immune-suppressive functions of TECs, B16 cells transfected with ovalbumin (B16-OVA) were inoculated in mice and the TECs isolated from the B16-OVA tumor were analyzed. An antibody reacting specifically to complex of OVA-derived antigen presented with mouse MHC class I was used to test antigen presentation. Further, to verify antigen-specific suppressive functions, TECs isolated from B16-OVA tumors were co-cultured with OVA-specific CD8+ T cells (OT-I cells) stimulated by OVA-pulsed bone marrow derived dendritic cells (BMDCs) in vitro. In this suppression assay, we performed a blocking assay using an anti-programmed death-ligand 1 (PD-L1) antibody. We also performed an antigen-specific cytotoxicity assay using B16-OVA cells as targets and OVA-specific CD8+ T cells which had been co-cultured with TECs as effectors. Results: TEC expressed MHC class I/II and co-stimulating molecules and PD-L1, suggesting that TECs had antigen-presenting and immune-suppressive properties. We found that TECs endocytosed B16-OVA derived protein and presented tumor-derived OVA peptides with their MHC class I molecule. TECs from B16-OVA tumor showed significantly greater suppression of OT-I CD8+ T cell proliferation compared to those from non-transfected B16 tumor. Similarly, in a cytotoxicity assay, TECs from B16-OVA tumor suppressed cytotoxicity of CD8+ T cells in an antigen specific manner. Strikingly, this suppressive function of TECs for the proliferation and cytotoxicity of OVA-specific T cells was abrogated by blocking PD-L1 using an anti-PD-L1 antibody, suggesting that the PD-1/PD-L1 axis contributes towards the immune-evasion mechanism. Conclusions: TECs have the characteristics of antigen presenting cells and regulate the immune response of tumor antigen specific T cells via the PD-1/PD-L1 pathway. Our data may suggest the novel mechanism of tumor immune-evasion through TECs. Citation Format: Kazuhiro Taguchi, Takashi Onoe, Hideki Ohdan. Novel mechanism underlying tumor immune evasion through tumor endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3795.

Identifying shared patterns in the T cell receptor repertoire specific to IE-1 CMV

Introduction Cytomegalovirus (CMV) continues to be a major problem for organ transplant recipients. T cells specific to the CMV antigens immediate-early (IE-1) and pp65 CMV govern the adaptive immune response to CMV. Molecular studies indicate that CMV-specific responses usually comprise clonally expanded viral-specific T cell receptors (TCR) that can be conserved among subjects with a similar HLA haplotype. However, our understanding of the CMV-specific T cell receptor (TCR) repertoire is incomplete and the characteristics of TCR repertoire conferring protection to CMV are not known. The aim of this study is to identify specificity groups in the IE-1-specific TCR repertoire of CMV seropositive A02:01 subjects.Methods: PBMC from three CMV seropositive, IE-1-ELISPOT+ A02:01 healthy subjects were stimulated with IE-1 peptides. Antigen-reactive T cells were FACS-sorted based on the expression of CD137 for CD8+ T cells and CD154 for CD4+ T cells, or the loss of CFSE expression in T cells that had been prelabeled with CFSE prior to stimulation. We sequenced the IE-1 specific TCRβ chains following TCRb CDR3 segment amplification using a PCR template switch methodology that minimizes bias through the use of unique molecular barcodes. IE-1 TCRb sequences were analyzed using the recently published Grouping of Lymphocyte Interactions Paratope Hotspots (GLIPH) algorithm. Results We obtained a total of 2003 TCRβ sequences from IE-1-specific CD8+ T cells from three CMV seropositive subjects. The IE-1-specific TCR repertoire analysis revealed clonally expanded populations for two of the three subjects, with ~50% of the repertoire comprised of 1-10 dominant clones. In contrast, the corresponding non-IE-1 specific T cells had highly diverse TCR repertoires and a minimal number of expanded clones (<1%). Analysis of all CD8+ IE-1-specific TCRβ sequences by GLIPH identified multiple possible clusters. The best scoring identity cluster grouped 95 TCR sequences across two subjects within either the CD137+, or the CFSE-populations. To note, cluster 1 motiff was found in a published IE-1 TCR sequence in the literature, binding to an HLA A2:01 restricted epitope (VLEETSVML).Cluster 2 grouped 43 TCR sequences across two subjects within either the CD137+, or the CFSE-, populations. Importantly, 58 TCR sequences were found to cluster across the IE-1-specific CD8+ T cell populations in all three donors. Here we demonstrate that is feasible to characterize the CMV-specific TCR repertoire in an antigen-specific manner. This methodology has the potential to cluster TCR sequences in transplant patients controlling CMV replication.

Abstract 4679: Rectal cancer-infiltrating T cells show clonal expansion associated with spatially restricted tolerogenic phenotypes

Abstract T cell infiltration correlates with prognosis and outcome in colorectal cancer regardless of the tumor stage. However, data on clonal expansion, phenotypes, functions, and spatial distribution of tumor-infiltrating T cells (TILs) are limited. We hypothesized that subsets of clonally expanded rectal cancer-associated T cells are specifically recruited into the tumor and show characteristic phenotypes and functions. Paired TILs and T cells from adjacent unaffected mucosa were isolated from five treatment-naïve rectal cancer patients. Although T cell immune phenotypes were heterogeneous, the frequencies of CD38+, PD-1+, and TIM-3+ cells were significantly higher in CD8+ TILs when compared to T cells from unaffected mucosa (p &amp;lt; 0.05). To track clonal expansion and phenotypes at the single cell level, we combined 13-parameter FACS single cell index sorting with next generation T cell receptor (TCR) and phenotype sequencing. Paired TCRαβ sequences were obtained from 1237 TILs (on average 412 TILs per patient) and 1257 T cells infiltrating the unaffected mucosa (on average 419 T cells per patient) from three selected patients. Clonal T cell expansion was not restricted to TILs and occurred predominantly in CD8+ T cells (p &amp;lt; 0.05). Expanded TIL clones were more frequently TIM-3+, PD-1+, and CD38+ (p &amp;lt; 0.05) and occupied characteristic phenotype compartments when compared to T cells from unaffected mucosa with t-stochastic neighbor embedding (t-SNE) visualization. FOXP3 expression was enriched in non-expanded TILs (p &amp;lt; 0.05). Only 11 out of 179 TIL clones were also detectable in unaffected mucosa of the same patients and these overlapping clones were exclusively PD-1- TIM-3-. In conclusion, rectal cancer is infiltrated by oligoclonal T cells. Selected clones were exclusively detected in TILs suggesting their expansion and recruitment driven by tumor-associated cues. Tolerance was induced i) in an antigen-specific manner by the expression of immune checkpoint molecules predominantly on clonally expanded TILs, and ii) in an antigen-independent manner by infiltrating polyclonal FOXP3+ regulatory T cells. Citation Format: Livius Penter, Kerstin Dietze, Felix Aigner, Lars Bullinger, Thomas Blankenstein, Leo Hansmann. Rectal cancer-infiltrating T cells show clonal expansion associated with spatially restricted tolerogenic phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4679.

Tregs orchestrate antigen specific suppression via stripping cognate peptide-MHCII from the DC surface

Abstract Tregs are professional suppressors of the immune response, yet their mechanism of action in vivo remains unclear. We compared the morphology and stoichiometry of the interactions of activated 5CC7 T cells and 5CC7 iTregs with MCC88–103 pulsed splenic DCs by electron and confocal microscopy. Image analyses revealed that Tregs interacting with DC displayed a distinct morphology with finger like membrane projections at the DC binding site and uropods at the rear end within three hours of in vitro culture. In contrast, activated T cells maintained their round morphology. Tregs occupied a greater extent of the DC surface than activated T cells consistent with a higher binding avidity. Dynamic intravital two-photon microscopy of adoptively transferred OTII iTregs and activated OTII cells demonstrated that Treg displayed a greater volume and duration of contact with OVA323–339 pulsed DC compared to that of activated OTII cells. Subsequent to their high avidity interactions with DC, 5CC7 iTregs captured MCC88–103-I-Ek complexes from DC surface reducing the amount of MCC presented on DC. Reduced antigen presentation was not due to global suppression of the DC’s ability to present antigen as 5CC7 and 3A9 iTregs only captured their cognate peptide-MHCII from MCC/HEL double pulsed DC, reducing the DC presentation in an antigen specific manner. When double pulsed DCs were cultured with iTregs specific for one peptide, separated from the iTregs, the DC failed to prime T cells specific for the antigen seen by the iTreg, but primed T cells specific for the second antigen. Altogether, we propose antigen specific depletion of peptide-MHCII complexes as a new mechanism for Treg-mediated suppression.

Induction of neuroantigen-specific CD8+ T cell responses as a treatment for relapsing autoimmune demyelinating disease

Abstract Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). We have shown that CNS-specific CD8+ T cells possess a disease suppressive function in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In humans, this suppressive function of CD8+ T cells is deficient during MS relapses and is restored during quiescence, suggesting that lack of CD8+ T cell mediated regulatory function may contribute to relapses in MS. We thus evaluated the role of CD8+ T cells in the context of the relapsing-remitting model (RR-EAE), using SJL mice. We demonstrate here that PLP178–191- and MBP84–104-specific CD8+ T cells ameliorated disease severity in an antigen-specific manner. Moreover, PLP178–191 CD8+ T cells reduced the number of relapses in PLP178–191-induced disease, even when transferred as a treatment during ongoing disease. We also probed the role of endogenously generated CNS-specific CD8+ T cell responses by using Listeria monocytogenes (LM) encoding CNS antigens to preferentially prime suppressive CD8+ T cells in vivo. Using this system, we show infection with LM expressing PLP175–194 endogenously induced disease suppressive CD8+ T cells that protected and treated PLP178–191 disease. Importantly, LM-PLP175–194 infection during disease remission of PLP139–151-induced EAE could effectively prevent relapses. These studies support a potential immunotherapeutic strategy using LM vaccination to endogenously prime disease regulatory CD8+ T cells.

Gene Therapy induced Tregs: A treatment for relapsing-remitting MS in mice

Abstract Autoimmune disease is caused by a breakdown of tolerance against endogenous protein(s). New therapies are being developed that utilize the natural abilities of regulatory T (Treg) cells to suppress autoimmune effector cells in an antigen-specific manner. Recently, we demonstrated a clinically relevant in vivo gene immunotherapy approach for restoring Ag-specific tolerance using hepatic adeno-associated virus (AAV) gene transfer. This approach was shown to be remarkably successful at preventing and reversing a monophasic, MOG-induced EAE disease. Additionally, we have shown the dynamic ability of this treatment to prevent and/or reverse disease induced with various MOG epitopes, in genetically diverse mouse strains. However, it is unclear if this approach would be effective in the more complicated, PLP induced relapsing-remitting disease model. Thus, we re-engineered our therapeutic vector to contain the full-coding sequence of PLP. This is critically important as the established RR-EAE (SJL) model is characterized by epitope spreading; a phenomenon that may more closely resemble multiple sclerosis. SJL mice received a single peripheral injection of AAV. PLP vector, or control, 2 weeks prior to PLP 139–151 induced EAE. By day 14, control mice developed significant neurological deficits followed by a relapsing-remitting disease course. In contrast, the treated mice failed to exhibit any neurological symptoms of disease throughout the duration of the experiment, ~75 days. The long-term prevention of PLP induced disease, provides the first evidence of an effective Treg based therapy for combating epitope drift in RR-EAE, and potentially a new paradigm for treating the autoimmune disease multiple sclerosis.

Early, in situ re-activation of lung memory T cells drives chronic lung inflammation

Abstract Acute immune responses within the respiratory tract generate memory T lymphocyte populations that are retained within the lung tissue and contribute to chronic inflammatory responses upon subsequent re-exposures to antigen. The generation and maintenance of antigen-specific memory T cell populations within the lung are therefore processes which, if inhibited, could ameliorate the symptoms of chronic inflammatory lung diseases. We sought to develop a model system that consistently generates lung inflammation and antigen-specific memory T cells within the lung. We show that prolonged respiratory exposure to lipopolysaccharide and a protein antigen generates a long-lived memory T cell population in the lung. These cells are retained in this compartment even after a week-long period of induced lymphopenia. Furthermore, lung memory T cells are re-activated in an antigen-specific manner upon intranasal challenge with antigen. The early in situ reactivation of memory T cells in the lung induced the recruitment of innate immune cells into the lung parenchyma and into the airways. Thus, elucidating the pathways involved in the tissue retention of the lung-retained memory T cell population may lead to therapies that can reduce harmful T cell populations within the lung.

Transcriptional profiling of pathogen-specific CD4 T cells reveals key events that regulate Th17 cell priming and differentiation

Abstract CD4 T cells play a central role in adaptive immunity through secretion of specific cytokines which exert effector functions against invading pathogens. They also drive B cell activation and coordinate CD8 T cell responses. Productive CD4 T cell responses are determined not only by the specificity of the T cell receptor, but also by innate immune cues provided by dendritic cells (DCs) which shape the nature of the effector lineage commitment. However, to what extent pathogen-recognizing DCs prime naïve T cells on a global transcriptional level remains elusive, because of the inability to track and identify pathogen-specific T cells during cognate DC-T cell interaction. Here, we analyzed transcriptional profiles of pathogen-specific mouse Th17 cells utilizing an in vitro approach, where naïve CD4 T cells were differentiated by mature DCs in a highly antigen-specific manner. These pathogen-specific Th17 cells exhibit enormous lineage heterogeneity and resemble Th17 cells found in vivo following infections. We further discovered that DC-mediated priming not only generates a distinct transcriptional landscape in pathogen-specific Th17 cells, but also warrants their acquisition of memory gene signature and metabolic switch from glycolysis to oxidative phosphorylation. These results indicate that pathogen-activated dendritic cells play a critical role in CD4 T cell transcription programming that extends beyond determining lineage commitment. By mining this dataset, we have identified a previously unappreciated gene in promoting Th17 lineage commitment. Conceptually, our study underlines the necessity of investigating physiologically differentiated CD4 T cells in order to reveal novel regulators of T cell biology.

Revealing the specificity of regulatory T cells in murine autoimmune diabetes

Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαβ pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9-23 and proinsulin. Consistently, insulin B:9-23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28-/- recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.