TCR-Identical CD8+ T-Cells Can Have Distinct Killing Efficacy Determined By Events Beyond the T-Cell Receptor
Authors: Glanville Julie M , Moody Anne-Marie , Peng Yanchun , Taylor Stephan , Ladell Kristin , Gillespie Geraldine , de Bree Godelieve , Baban Dilair , Stewart-Jones Guillaume , Cullen Claire , Mead Adam J , Repapi Emmanuela , Rowland-Jones Sarah , Simmons Alison , Price David A , Dong Tao , McMichael Andrew 
Published: April 2021

Abstract
Introduction: Cytotoxic T lymphocytes (CTL) that encounter antigen proliferate from a single naïve T-cell to form a population with diverse effector function and differentiation states, that control and prevent infection and cancer. Antigen-specific CTL are extremely heterogeneous in terms of clonotype, differentiation state and efficacy. Factors that define CTL of superior efficacy are well explored. TCR signal strength is known to modulate the breadth and magnitude of CTL effector functions, and epigenetic marks modulate intrinsic states such as exhaustion that limit activation. It is not clear if or how TCR signal strength modulates contact-dependent killing, and TCR affinity, TCR avidity, or factors downstream of the TCR could all contribute. There is a critical period of contact when a TCR engages cognate peptide- MHC class I molecules on a target cell that determines if the cell will be killed or survive. This is important to explore as not all T-cell therapy products to treat cancer are equally efficacious despite lentiviral transduction with the same chimeric-antigen-receptor. Lack of CTL efficacy is also encountered in chronic viral infections. Indeed, antigen-specific CTL from HIV controllers suppress HIV replication in vitro unlike antigen-specific CTL from chronic progressors. Viral suppression correlates with target cell elimination suggesting a critical role for contact-dependent killing.
Results: To identify factors that define superior CTL efficacy downstream of the TCR, we generated TCR-identical CD8+ CTL clones specific for a single epitope, HIV-1 FLKEKGGL (FL8) Nef (90-97) restricted by HLA B*0801, from individuals with HLA B*08-FL8 tetramer specific responses dominated by the use of a single TCR. Remarkably, clones that used the same TCR and bound tetramer with identical avidity, indicating the same surface expression of TCR, had distinct abilities to suppress HIV-1 in vitro. The mechanism of in vitro suppression correlated with target cell elimination demonstrating TCR-identical CTL clones can have distinct contact-dependent killing efficiency. Effective clones were highly responsive at low antigen densities, as might be encountered during physiological viral infection, with more rapid onset of signalling demonstrated by higher phospho-ERK MFI after 10 minutes of stimulation, faster onset of degranulation, and a higher final proportion of cells responding with CD107a expression, a marker of degranulation, at one hour. These novel distinctions in a reductionist in vitro model enabled us to dissect virus suppressive activity beyond initial TCR-antigen contact. On transcriptome examination we identified novel putative coreceptors containing ITAM (XX receptor to be revealedXX) or ITIM (XX two receptors to be revealed XX) motifs that were consistently over or under expressed on CTL clones that demonstrated superior contact-dependent killing.
Furthermore, ex vivo RT-PCR analysis of TCR-identical single cells sorted from the HLA B*08-FL8 tetramer positive population from which the CTL clones were generated, identified two populations of CTL, defined by expression of these novel ITAM and ITIM receptors. Lentiviral transduction of Jurkat cells with the novel ITAM receptor enhanced MFI phospho-ERK expression at 10 minutes when activated using plate bound OKT3, to generate a TCR signal, suggesting that this ITAM receptor may act as a TCR co-stimulatory molecule. T-cell poise may be critical to efficient contact-dependent killing, particularly if antigen or CTL cell numbers are limiting, by modulation of the probability of target cell death during each CTL-TCR engagement with its cognate peptide- MHC complex. We also demonstrated that TCR-identical CTL clones from one individual had distinct epigenetic regulation of TNF secretion.
Conclusion: Our results suggest that poise of the signalling cascade and intrinsic ontogenetic regulation of functions are critical to antiviral activity and identify putative novel check-point targets for therapeutic development.

Disclosures
Mead: BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.