Memory CD8⁺ T cell heterogeneity is primarily driven by pathogen-specific cues and additionally shaped by the tissue environment

SummaryFactors that govern the complex formation of memory T cells are not completelyunderstood. A better understanding of thedevelopment of memory Tcell hetero-geneity is however required to enhance vaccination and immunotherapy ap-proaches. Here we examined the impact of pathogen- and tissue-specific cueson memory CD8+T cell heterogeneity using high-dimensional single-cell mass cy-tometry and a tailored bioinformatics pipeline. We identified distinct populationsof pathogen-specific CD8+T cells that uniquely connected to a specific pathogenor associated to multiple types of acute and persistent infections. In addition, thetissue environment shaped the memory CD8+T cell heterogeneity, albeit to alesser extent than infection. The programming of memory CD8+T cell differenti-ation during acute infection is eventually superseded by persistent infection.Thus, the plethora of distinct memory CD8+T cell subsets that arise upon infec-tion is dominantly sculpted by the pathogen-specific cues and further shaped by the tissue environment.</p

OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy.</p

The Breadth of Synthetic Long Peptide Vaccine-Induced CD8⁺ T Cell Responses Determines the Efficacy against Mouse Cytomegalovirus Infection

<div><p>There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ⁺, TNF⁺, IL-2⁺) CD8⁺ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8⁺ T cells (KLRG1^hi, CD44^hi, CD127^lo, CD62L^lo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8⁺ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8⁺ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8⁺ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease.</p></div

Phenotypic heterogeneity of SLP vaccine-induced CD8⁺ T cells.

<p><b>(A)</b> Peripheral blood from either SLP immunized (day 7 after booster peptide vaccination) or MCMV infected C57BL/6 and BALB/c mice (day 7 post infection) were stained with MHC class I tetramers and for cell surface molecules. Representative plots show CD127 versus KLRG1 expression on tetramer-positive CD8⁺ T cells. <b>(B)</b> Cell-surface characteristics of tetramer-positive CD8⁺ T cells in blood and spleen at day 8 post booster SLP vaccination and day 8 after MCMV infection. <b>(C)</b> Peripheral blood from either SLP immunized (day 60 after booster peptide vaccination) or MCMV infected C57BL/6 and BALB/c mice (day 60 post infection) were stained with MHC class I tetramers and for cell surface molecules. Representative plots show CD127 versus KLRG1 expression on tetramer-positive CD8⁺ T cells. <b>(D)</b> Cell-surface characteristics of antigen-specific CD8⁺ T cells in blood and spleen at day 60 post booster SLP vaccination and day 60 after MCMV infection. Data represents mean values, and are representative of three independent experiments (n = 6 per group).</p

PD-L1 blockade engages tumor-infiltrating lymphocytes to co-express targetable activating and inhibitory receptors

Background: The clinical benefit of immunotherapeutic approaches against cancer has been well established although complete responses are only observed in a minority of patients. Combination immunotherapy offers an attractive avenue to develop more effective cancer therapies by improving the efficacy and duration of the tumor-specific T-cell response. Here, we aimed at deciphering the mechanisms governing the response to PD-1/PD-L1 checkpoint blockade to support the rational design of combination immunotherapy. Methods: Mice bearing subcutaneous MC-38 tumors were treated with blocking PD-L1 antibodies. To establish high-dimensional immune signatures of immunotherapy-specific responses, the tumor microenvironment was analyzed by CyTOF mass cytometry using 38 cellular markers. Findings were further examined and validated by flow cytometry and by functional in vivo experiments. Immune profiling was extended to the tumor microenvironment of colorectal cancer patients. Results: PD-L1 blockade induced selectively the expansion of tumor-infiltrating CD4+ and CD8+ T-cell subsets, co-expressing both activating (ICOS) and inhibitory (LAG-3, PD-1) molecules. By therapeutically co-targeting these molecules on the TAI cell subsets in vivo by agonistic and antagonist antibodies, we were able to enhance PD-L1 blockade therapy as evidenced by an increased number of TAI cells within the tumor micro-environment and improved tumor protection. Moreover, TAI cells were also found in the tumor-microenvironment of colorectal cancer patients. Conclusions: This study shows the presence of T cell subsets in the tumor micro-environment expressing both activating and inhibitory receptors. These TAI cells can be targeted by combined immunotherapy leading to improved survival.</p

SLP vaccination elicits polyfunctional CD8⁺ T cells.

<p>Following SLP vaccination or MCMV infection the cytokine polyfunctionality of splenic CD8⁺ T cells was determined after peptide restimulation. Representative plots show IFN-γ versus TNF production at <b>(A)</b> day 8 (acute phase) and <b>(B)</b> day 60 (memory phase) post booster vaccination and post MCMV infection. Pie charts depict the percentages of the single (IFN-γ), double (IFN-γ/TNF) and triple (IFN-γ/TNF/IL-2) cytokine producers of each antigen-specific T cell population upon peptide stimulation. Data represents mean values, and are representative of three independent experiments (n = 4–5 per group). Statistics of the results depicted in these pie charts are reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005895#ppat.1005895.s005" target="_blank">S4 Fig</a>.</p

Secondary expansion potential of SLP-induced CD8⁺ T cells upon MCMV challenge.

<p>M45_985-993 and m139_419-426 epitope specific CD8⁺ T cells were isolated from the spleen at day 60 after booster vaccination and infection of SLP vaccinated and MCMV infected CD45.1 mice. 1 × 10⁴ antigen-specific CD8⁺ T cells were adoptively transferred into naive C57BL/6 (CD45.2) recipient mice. Recipient mice were subsequently infected i.p. with 5 × 10⁴ PFU MCMV-Smith. The total numbers of the donor derived M45_985-993 and m139_419-426 CD8⁺ T cells were determined in the spleen at day 5 post challenge. Data represent mean values + SEM (n = 5). Experiments were performed twice with similar outcome. *, P<0.05; **, P<0.01.</p

The T cell precursor frequency but not the functional avidity of the SLP vaccine-induced CD8⁺ T cells predicts the magnitude of the response.

<p><b>(A)</b> Splenocytes from SLP vaccinated (n = 4–5 mice per epitope) and MCMV infected C57BL/6 mice (n = 4–5 mice) were isolated during the acute (day 8 post booster SLP vaccination and MCMV infection) and memory (day 60 post booster SLP vaccination and MCMV infection) phase and were restimulated with various peptide concentrations in the presence of brefeldin A. The percentage of IFN-γ producing CD8⁺ T cells was measured and normalized to the response at the highest peptide concentration. <b>(B)</b> Similar description as in (A) for experiments performed with BALB/c mice. Note that the M45_507-515 CD8⁺ T cell response during MCMV infection was too low to obtain accurate results. Shown are the functional avidity curves and are representative of at least 2 independent experiments. Data represents mean values. <b>(C)</b> Absolute numbers of epitope specific T cell precursors present in spleen and lymph nodes of naive C57BL/6 and BALB/c mice were determined by tetramer staining. Each symbol represents an individual mouse. Data represents mean values ± SEM (n = 4–6 per group). Experiments were performed twice with similar outcome.</p

Prime-boost SLP vaccination provokes the induction of robust CD8⁺ T cell responses analogous to MCMV infection.

<p><b>(A)</b> The magnitude of the CD8⁺ T cell responses specific to the indicated epitopes was determined in blood by MHC class I tetramer staining at day 7 post booster vaccination with SLPs and at day 7 post MCMV infection in C57BL/6 mice and in BALB/c mice. Representative flow cytometry plots show MHC class I tetramer (Tm) staining within the CD8⁺ T cell population. Numbers represent the percentage of Tm+ cells within the total CD8⁺ T cell population. <b>(B)</b> Longitudinal analysis of the epitope-specific CD8⁺ T cell responses induced by either SLP vaccination or MCMV infection in blood. Data represents mean values ± SEM (n = 6 per group). <b>(C)</b> Percentages and total numbers of splenic SLP and MCMV-specific CD8⁺ T cells during the acute phase (at day 7 post booster vaccination and day 8 and after MCMV infection) and memory phase (at day 60 post booster vaccination and day 60 post MCMV infection) are shown. Data represents mean values + SEM (n = 6 per group), and are representative of three independent experiments. *, P< 0.05; **, P<0.01; ***, P<0.001.</p

Efficacy of single SLP vaccines against acute MCMV infection.

<p>Unvaccinated (naive), SLP vaccinated and MCMV infected C57BL/6 and BALB/c mice were challenged at day 60 post vaccination/infection with 5 × 10⁴ PFU and 5 × 10³ PFU salivary gland-derived MCMV Smith, respectively. At day 5 post challenge, spleen, liver, and lungs were isolated and the viral genome copies were determined by qPCR. The viral titres of individual <b>(A)</b> C57BL/6 and <b>(B)</b> BALB/c mice are depicted (n = 5–8 per group). Mean ± SEM is indicated. Dashed line represents the detection limit as measured in naive mice. Experiments were performed twice with similar outcome. *, P< 0.05; **, P<0.01; ***, P<0.001; ns, not significant.</p