Citations

Abstract Description Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy derived from transformed B cells of germinal center (GC) or post-GC origin. DLBCL incidence is elevated in immunodeficient persons with reduced T cells, such as among HIV+ patients. Yet a causal link between T cell deficiency and DLBCL is incompletely understood. Using a mouse model of spontaneous DLBCL, we show that suboptimal T cell function, but neither zero nor full T cell function, can be a driver of B cell lymphomagenesis. In CD3δ-/- mice, which exhibit reduced number, signaling, and function of T cells, we observed progressive disruption of splenic architecture and eventual DLBCL development with ∼100% incidence. In contrast, lymphoma did not develop in wild-type mice, or CD3e-/-z-/- mice with complete T cell absence. This showed that poor-but-positive suboptimal T cells were required for GC B cell transformation and outgrowth. Mechanistically, CD3δ-/- mice exhibited altered GC dynamics at steady state and in response to immunization. Compared with wild-type mice, CD3δ-/- mice had a diminished T follicular regulatory (TFR) compartment and exhibited defective T follicular helper (TFH) cell differentiation when antigen challenged, indicating compromised regulation of the GC reaction. Indeed, defective GC B cell entry and differentiation was also observed. Our data thus suggest that suboptimal function of T helper subsets can lead to dysegulated GC reactions and B cell lymphomagenesis. Funding Sources Supported by AAI Intersect Fellowship Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Abstract Previously we and others observed that the signaling module of the TCR, the CD3 complex, undergoes a conformational change at the level of its cytoplasmic tails when T cells are stimulated. At that time, we interpreted this data as proof that antigen recognition modifies the conformation of CD3 as part of the receptor triggering mechanism. In absence of specific structural information, the induced conformation has been referred to as "open CD3". Here we present new evidence that thymocytes display detectable amounts of "open CD3" even in the absence of antigenic stimulation. However, treatment of thymocytes with stimulatory anti-CD3 antibodies significantly increases the amount of "open CD3". We conclude that ligand binding causes many CD3 molecules to adopt the "open CD3" conformation. We propose that CD3 is expressed in an equilibrium between two distinguishable conformations, which may reflect "on" and "off" states that are perturbed by TCR engagement. The accumulation of a certain level of "open CD3" might trigger T cell activation. Research support: Diana Gil, Travis Kruger and Javier de la Cruz were supported with start-up funds from the Mayo Foundation.

Abstract Upon positive selection of conventional αβ T cells, CD4+ CD8+ double-positive (DP) cells transition to single-positive (SP) and reduce their responsiveness to weak peptide/MHC ligands (affinity threshold conversion, ATC). We report that ATC actually begins prior to the completion of positive selection in the DP stage, and this checkpoint is where the TCRα/CD3δ signaling axis controls positive selection. We dissected ATC in the OT1 TCR transgenic system in conjunction with several different genes whose null mutations block thymic development at DP stage: β2m, CD3δ, and/or TCRα-Connecting Peptide Motif (αCPM). In the absence of MHC class I (β2m0), CD3δ or αCPM mutants were blocked at pre-selection DP stage and displayed immature, high-sensitivity to weak TCR ligands. In contrast, when MHC class I was present (β2m+), CD3δ or αCPM mutants were blocked at DP stage but underwent partial ATC; they began positive selection but did not complete it. Upon exogenous TCR engagement of partial-ATC thymocytes, the proximal signaling protein interaction network showed overall weakened activity, notably decreasing SOS1 participation linked to the downstream Ras/Raf/ERK selection pathway. Provision of high affinity peptide/MHC in FTOC rescued completion of positive selection for partial-ATC mutant thymocytes. Finally, comparing nontransgenic, polyclonal B6 vs. CD3δ0 DP thymocytes, although most cells showed equal maturation state, a subset of cells in B6 progressed in maturation relative to CD3δ0. We propose the existence of an intermediate checkpoint inside DP stage, in which ATC already begins to reduce responsiveness to selection ligands, yet further TCR signaling is required to complete positive selection.

Abstract TCR mediated antigen recognition results in a signaling cascade leading to T cell activation. Antigen binding to its cognate TCR drives receptor aggregation and conformational changes that trigger signaling from the receptor. Conformational change in the CD3 complex (CD3Δc) is required for robust signaling and productive T cell responses. In contrast, weakly recognized antigens do not induce CD3Δc and the resulting T cell response is poor. Thus, T cell responses to weak antigens may be improved by induction of CD3Δc. We previously described an anti-mouse CD3ɛ monovalent Fab (Mono-7D6-Fab) that induced CD3Δc and synergized with weak antigens to enhance the T cell response, a process we termed co-potentiation. However, Mono-7D6-Fab did not induce classical T cell signaling in the absence of antigenic ligation. Mono-7D6-Fab effectively co-potentiated T cells against murine B16-F10 melanoma cells and reduced tumor burden in vivo in a T cell dependent manner. We exploited a model of human cytomegalovirus (CMV) to generate proof of concept that co-potentiation may enhance T cell antigen recognition in humans. Anti-human CD3ɛ monovalent Fab (Mono-OKT3-Fab) improved the CD8 T cell response to the CMV pp65495–503 peptide as measured by increased T cell proliferation, cytokine production, and the ability to kill target cells. Co-potentiation was dependent on antigen specificity, as 1) the effects were dampened when antigen recognition was impaired and 2) sequencing of the TCRs revealed a restricted number of expanded TCR clones. Our results suggest that Mono-OKT3-Fab may have therapeutic clinical applications to achieve a more robust antigen-specific T cell response in the context of diverse human disease, from cancer to chronic infection.

Abstract The αβ T cell antigen Receptor (TCR) is a multi-protein complex expressed together with CD3 subunits (γδεζ). The conventional model has it as monovalent with a stochiometry of (αβγδε2ζ2); however, some previously published work reported observation of bivalent complexes with two αβ heterodimers. To reevaluate the valency of the TCR, we have subjected T cell digitonin-solubilized complexes to immunoprecipitation detected by flow cytometry (IP-FCM), size exclusion chromatography, and other methods for detecting complex size and subunit composition. Our results indicate that precisely bivalent TCRs are found among surface-expressed complexes independent of cross-linking by strong antigenic ligands. Their detection can be masked in the presence of Coomassie reagent from Blue-native PAGE conditions, or if antibodies used in detection assays are themselves bivalent. Therefore, bivalent TCR/CD3 complexes can be found under the biochemical conditions that have classically defined the minimally expressed TCR/CD3 unit (digitonin solubilization). Further investigation is required to determine the functional relevance of these bivalent TCR complexes, although greater antigenic binding and signaling is predicted over that of monovalent TCRs.

Abstract TCR interactions with agonist pMHCs induce a conformational change in the tails of the associated CD3 complex (CD3Δc) that contributes to efficient TCR/CD3 triggering and productive T cell responses. In contrast, TCR engagement by antagonistic pMHCs does not induce either CD3Δc or full TCR/CD3 signaling in mature T cells. Here, we have used a monovalent Fab fragment (Mono-Fab) specific for the murine CD3 complex to provide induction of CD3Δc to mature T cells engaging pMHC ligands. This Mono-Fab does not interfere with TCR capacity to bind pMHC and fails to elicit signaling in the absence of TCR/MHC interactions. Using OT-I Tg T cells and a collection of altered peptide ligands derived of the agonist peptide OVA, we show that addition of CD3Δc to OT-I T cells using Mono-Fab enables T cell responses to antagonist ligands that fail to induce CD3Δc on their own. Moreover, using a mouse model of metastatic melanoma, we show that Mono-Fab promotes in vivo T cell dependent anti-melanoma responses that are antigen-specific. This Mono-Fab can also be combined with established therapies targeting signals 2 and 3 involved in T cell activation for a synergistic effect. Our data suggests that in-trans CD3Δc provision decreases the activation threshold of T cells. We propose that targeting the CD3 complex for the induction of CD3Δc could be exploited to achieve efficient TCR/CD3 triggering by poorly immunogenic antigens in the context of vaccination against cancer and infectious diseases.

Abstract Mature T cells recognize and respond to antigens through the T cell antigen receptor (TCR). We have established that upon TCR antigenic engagement, the TCR/CD3 complex undergoes a conformational change (CD3dc) of its CD3 epsilon tails to expose a proline-rich sequence and allow downstream signaling to occur. CD3dc has been shown to be required for full TCR/CD3 signaling and function of mature T cells. Pre-TCR assembly with the CD3 complex serves as a developmental checkpoint that ensures the expression of a function variant TCRβ chain in thymocytes. Interestingly, stimulation of the preTCR complex has been found to be ligand-independent; assembly is sufficient. Knowing this, we first determined the stoichiometry of the preTCR/CD3 complex and asked the question, how does assembly impact its ability to undergo conformational change? To answer this question, we employed various novel biochemical methods developed in our laboratory including a flow-cytommetry based immunoprecipitation (IP-FCM) as well as a CD3-pull down assay (CD3-PD). We aim to study the conformation of CD3 in the preTCR/CD3 complex and establish whether or not the assembly of this receptor is enough to induce the active CD3 conformation that supports functional CD3 signaling in mature cells. With our data, we are further able to characterize previously unstudied requirements for CD3 conformational change in the preTCR/CD3 complex with the potential of using this knowledge to manipulate CD3dc in the future.

During normal T cell development in mouse and human, a low-frequency population of immature CD4 − CD8 − double-negative (DN) thymocytes expresses early, mature αβ T cell antigen receptor (TCR). We report that these early αβ TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αβ T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αβ TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Abstract Malignant melanoma is the most aggressive form of skin cancer, accounting for 10,000 deaths annually. Largely resistant to conventional cytotoxic chemotherapy and radiation, immune checkpoint blockade (ICB) therapies have revolutionized patient care, accounting for partial or complete responses in up to 70% of patients. An important aspect of cancer elimination is activation of the cytotoxic T lymphocyte (CTL) response. Recent data suggest that ICB mediated broadening of the peripheral blood CTL repertoire correlates with good clinical outcomes. Thus, recent studies in cancer immunotherapy have focused on targeting tumor neoantigens (tNeoAg) with cancer vaccines for use in combination with ICB. Computational algorithm-driven predictions of immunogenic tNeoAgs yield vast numbers of potential peptide targets, only a fraction of which may be immunogenic in patients. This likely contributed to the modest success of cancer vaccines targeting predicted, high-avidity tNeoAgs, emphasizing the importance of identifying true tumor rejection antigens, including sub-dominant antigens, outside the scope of predictive models. In addition to promoting T cell-mediated tumor rejection, ICB often comes at the expense of treatment-induced immune-related adverse effects (irAE) that frequently require discontinuation of treatment. Modulation of ICB towards antitumor immunity and away from autoimmunity may dramatically improve the therapeutic index of modern cancer therapy. This appears feasible considering the clinical observation that antitumor efficacy does not correlate with type/severity of irAE, suggesting that the mechanisms of both processes, though likely related (CTL mediated), may not be identical. We aim to identify and separate non-cross-reacting antigenic targets mediating tumor rejection from those mediating irAEs to enable therapeutic interventions that maximize the efficacy of ICB, expanding tumor-specific CTLs with vaccines while minimizing irAEs through desensitization. We have access to samples from responding/nonresponding melanoma patients subjected to ICB with varying degrees of irAEs. We have designed an experimental approach that combines established mass spectrometry and sequencing techniques to identify peptides and matching TCR clones with a novel strategy that targets the TCR-associated CD3 complex to allow inclusion of subdominant antigens in our studies. T-cell “co-potentiation” is achieved when anti-CD3 monovalent Fabs induce a conformational change in the CD3 complex that sustains the T-cell response to weak antigenic stimulation. We have successfully used anti-human CD3 Fabs to co-potentiate in vitro the activation and subsequent response to weak peptide-HLA/TCR interactions of human T cells found in PBMCs isolated from healthy donors. Our preliminary data suggest T-cell co-potentiation may allow identification of ICB-induced CTL clones specific for subdominant tNeoAgs and irAE targets in patients with melanoma undergoing active immunotherapy. Citation Format: Laura Elsbernd, Alfreda Nelson, Hien Huynh, Michele Hoffmann, Christopher Parks, Wendy Nevala, Shari Sutor, Larry Pease, Adam Schrum, Diana Gil, Svetomir N. Markovic. Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B78.

Abstract Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.