Pages, Diana Gil

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 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.

Abstract We recently described the concept of T cell co-potentiation as means to target antigen recognition by T cells to achieve antitumor immunotherapy in mouse models. Normally, T cell antigen receptor (TCR) engagement by a strong immunogenic antigen induces a CD3 conformational change that exposes a proline-rich sequence (PRS) in the cytosolic domain of CD3 epsilon (CD3e) to dock cytosolic proteins and contribute to the assembly of the signaling cascade that activates T cell immune function. While induction of CD3 conformational change is required by T cells to achieve productive TCR/CD3 signaling, this molecular event fails to occur when the TCR interacts with poorly immunogenic antigens, like most tumor-associated antigens (TAAs). We found that CD3 conformational change can be exogenously induced on T cells using monovalent Fab fragments (Mono-Fabs) specific for the CD3 complex. Anti-CD3 Mono-Fabs expose the CD3e PRS, but fail to crosslink the TCR/CD3, turning them incompetent to trigger T cell activation in the absence of additional stimulation. However, if TCR has bound a weak antigen, one that fails to induce CD3 conformational change intrinsically, then anti-CD3 Mono-Fabs enable TCR/CD3 signaling, and as a result co-potentiate T cell responses to weak antigenic stimulation. Using a mouse model for melanoma (B6 mice and B16-F10 melanoma cell line), we found that treatment of mice with anti-CD3e Mono-Fabs improved antitumor T cell responses against melanoma in an antigen-dependent manner, and dramatically improved the outcome of complementary T cell immunotherapies. Based on these observations, we hypothesize that T cell co-potentiation using anti-human CD3 Mono-Fabs can be exploited as a novel immunotherapy for cancer patients. We have recently identified one anti-human CD3 Mono-Fab that is capable to induce CD3 conformational change in human T cells and allows the co-potentiation of T cell response to weak antigenic stimulation. Here we present data from human T cells and humanized mouse models using this Mono-Fab that support the potential therapeutic value of exploiting T cell co-potentiation to treat cancer patients. Citation Format: Alfreda D. Nelson, Laura Elsbernd, Adam G. Schrum, Diana Gil Pages. Testing T cell co-potentiation as an antitumor therapeutic strategy in humanized mouse models [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 962.