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Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8<sup>+</sup> T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Western University

Preexisting, donor-specific antibodies (DSAs) are culprits of hyperacute rejection. Donor-specific antibodies are also formed de novo, and their role in acute and chronic rejection is increasingly appreciated. However, it is difficult to assess damage inflicted exclusively by DSAs when alloreactive T cell and B cell responses coincide. We reasoned that allosensitization with "costimulation-deficient" cells should induce DSA synthesis but not naive cytotoxic T lymphocyte (CTL) precursors' priming via direct allorecognition. Accordingly, we have developed a novel model to quantify DSA-mediated cytotoxicity in vivo.

Quantification of Alloantibody-Mediated Cytotoxicity In Vivo.

The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8 T cell (T) responses, and PD-1-based "checkpoint inhibitors" have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1-targeted therapies reverse T exhaustion/anergy. However, whether they alter the epitope breadth of T responses remains unclear. This is an important question because subdominant T are more likely than immunodominant clones to escape tolerance mechanisms and may contribute to protective anticancer immunity. We have addressed this question in an in vivo model of T responses to well-defined epitopes of a clinically relevant oncoprotein, large T Ag. We found that unlike other coinhibitory molecules (CTLA-4, LAG-3, TIM-3), PD-1 was highly expressed by subdominant T, which correlated with their propensity to favorably respond to PD-1/PD-1 ligand-1 (PD-L1)-blocking Abs. PD-1 blockade increased the size of subdominant T clones at the peak of their primary response, and it also sustained their presence, thus giving rise to an enlarged memory pool. The expanded population was fully functional as judged by IFN-γ production and MHC class I-restricted cytotoxicity. The selective increase in subdominant T clonal size was due to their enhanced survival, not proliferation. Further mechanistic studies utilizing peptide-pulsed dendritic cells, recombinant vaccinia viruses encoding full-length T Ag or epitope mingenes, and tumor cells expressing T Ag variants revealed that anti-PD-1 invigorates subdominant T responses by relieving their lysis-dependent suppression by immunodominant T To our knowledge, our work constitutes the first report that interfering with PD-1 signaling potentiates epitope spreading in tumor-specific responses, a finding with clear implications for cancer immunotherapy and vaccination.

PD-1 Blockade Promotes Epitope Spreading in Anticancer CD8 T Cell Responses by Preventing Fratricidal Death of Subdominant Clones To Relieve Immunodomination.

Superantigens (SAgs) released by common Gram-positive bacterial pathogens have been reported to delete, anergize, or activate mouse T cells. However, little is known about their effects on preexisting memory CD8+ T cell (TCD8) pools. Furthermore, whether SAgs manipulate human memory TCD8 responses to cognate antigens is unknown. We used a human peripheral blood mononuclear cell culture system and a nontransgenic mouse model in which the impact of stimulation by two fundamentally distinct SAgs, staphylococcal enterotoxin B and Mycoplasma arthritidis mitogen, on influenza virus- and/or cytomegalovirus-specific memory TCD8 could be monitored. Bacterial SAgs surprisingly expanded antiviral memory TCD8 generated naturally through infection or artificially through vaccination. Mechanistically, this was a T cell-intrinsic and T cell receptor β-chain variable-dependent phenomenon. Importantly, SAg-expanded TCD8 displayed an effector memory phenotype and were capable of producing interferon-γ and destroying target cells ex vivo or in vivo. These findings have clear implications for antimicrobial defense and rational vaccine design.

Courtney E. Meilleur

Department of Microbiology and Immunology

Western University

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8⁺ T Cell Responses

Courtney E. Meilleur

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology and Immunology

Western University

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Department of Microbiology and Immunology

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8⁺ T Cell Responses