Targeting the MR1-MAIT cell axis improves vaccine efficacy and affords protection against viral pathogens.

Mucosa-associated invariant T (MAIT) cells are MR1-restricted, innate-like T lymphocytes with tremendous antibacterial and immunomodulatory functions. Additionally, MAIT cells sense and respond to viral infections in an MR1-independent fashion. However, whether they can be directly targeted in immunization strategies against viral pathogens is unclear. We addressed this question in multiple wild-type and genetically altered but clinically relevant mouse strains using several vaccine platforms against influenza viruses, poxviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), a riboflavin-based MR1 ligand of bacterial origin, can synergize with viral vaccines to expand MAIT cells in multiple tissues, reprogram them towards a pro-inflammatory MAIT1 phenotype, license them to bolster virus-specific CD8+ T cell responses, and potentiate heterosubtypic anti-influenza protection. Repeated 5-OP-RU administration did not render MAIT cells anergic, thus allowing for its inclusion in prime-boost immunization protocols. Mechanistically, tissue MAIT cell accumulation was due to their robust proliferation, as opposed to altered migratory behavior, and required viral vaccine replication competency and Toll-like receptor 3 and type I interferon receptor signaling. The observed phenomenon was reproducible in female and male mice, and in both young and old animals. It could also be recapitulated in a human cell culture system in which peripheral blood mononuclear cells were exposed to replicating virions and 5-OP-RU. In conclusion, although viruses and virus-based vaccines are devoid of the riboflavin biosynthesis machinery that supplies MR1 ligands, targeting MR1 enhances the efficacy of vaccine-elicited antiviral immunity. We propose 5-OP-RU as a non-classic but potent and versatile vaccine adjuvant against respiratory viruses.

Discordant rearrangement of primary and anamnestic CD8+ T cell responses to influenza A viral epitopes upon exposure to bacterial superantigens: Implications for prophylactic vaccination, heterosubtypic immunity and superinfections.

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vß makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.

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

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 "immunodominance" in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Bacterial Superantigens Expand and Activate, Rather than Delete or Incapacitate, Preexisting Antigen-Specific Memory CD8+ T Cells.

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 ß-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.

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

The interactions between programmed death-1 (PD-1) and its ligands hamper tumor-specific CD8+ T cell (TCD8) responses, and PD-1-based "checkpoint inhibitors" have shown promise in certain cancers, thus revitalizing interest in immunotherapy. PD-1-targeted therapies reverse TCD8 exhaustion/anergy. However, whether they alter the epitope breadth of TCD8 responses remains unclear. This is an important question because subdominant TCD8 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 TCD8 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 TCD8, 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 TCD8 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 TCD8 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 TCD8 responses by relieving their lysis-dependent suppression by immunodominant TCD8 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.

Quantification of Alloantibody-Mediated Cytotoxicity In Vivo.

BACKGROUND: 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. METHODS: C57BL/6 (H-2b) mice were sensitized with H-2 kidney epithelial cells, and a cytofluorimetric killing assay was tailored to the measurement of allocytotoxicity. We took cell/complement depletion, costimulation blockade, and serum transfer approaches to reveal the mediators of cytotoxicity. "Third-party" controls and a skin allotransplantation model were used to confirm DSAs' specificity for allo-major histocompatibility complex. We validated our experimental approach in other mouse strains primed with different allogeneic cell types, including endothelial cells. To demonstrate the usefulness of our model/method for drug efficacy testing, we examined the effect of CTLA4-Ig and rapamycin on DSA-mediated cytolysis. RESULTS: Allosensitization of MHC-disparate mouse strains with costimulation-deficient cells led to robust cytotoxicity mediated by complement-fixing DSAs and phagocytic cells. This response was independent of CTLs, natural killer or natural killer T cells. It required CD4 T cell help, CD40 signaling and CD28-based costimulation during allosensitization and could be reversed by sustained rapamycin treatment. CONCLUSIONS: The unique model described herein should enable mechanistic studies on sensitization and effector phases of humoral alloreactivity as well as efficacy testing of future immunotherapies to prevent DSA-induced pathology.

Suppression of immunodominant antitumor and antiviral CD8+ T cell responses by indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme known to suppress antitumor CD8(+) T cells (TCD8). The role of IDO in regulation of antiviral TCD8 responses is far less clear. In addition, whether IDO controls both immunodominant and subdominant TCD8 is not fully understood. This is an important question because the dominance status of tumor- and virus-specific TCD8 may determine their significance in protective immunity and in vaccine design. We evaluated the magnitude and breadth of cross-primed TCD8 responses to simian virus 40 (SV40) large T antigen as well as primary and recall TCD8 responses to influenza A virus (IAV) in the absence or presence of IDO. IDO(-/-) mice and wild-type mice treated with 1-methyl-D-tryptophan, a pharmacological inhibitor of IDO, exhibited augmented responses to immunodominant epitopes encoded by T antigen and IAV. IDO-mediated suppression of these responses was independent of CD4(+)CD25(+)FoxP3(+) regulatory T cells, which remained numerically and functionally intact in IDO(-/-) mice. Treatment with L-kynurenine failed to inhibit TCD8 responses, indicating that tryptophan metabolites are not responsible for the suppressive effect of IDO in our models. Immunodominant T antigen-specific TCD8 from IDO(-/-) mice showed increased Ki-67 expression, suggesting that they may have acquired a more vigorous proliferative capacity in vivo. In conclusion, IDO suppresses immunodominant TCD8 responses to tumor and viral antigens. Our work also demonstrates that systemic primary and recall TCD8 responses to IAV are controlled by IDO. Inhibition of IDO thus represents an attractive adjuvant strategy in boosting anticancer and antiviral TCD8 targeting highly immunogenic antigens.