High-avidity CD8 Research Articles

CD3 downregulation identifies high-avidity, multipotent SARS-CoV-2 vaccine- and recall antigen-specific Th cells with distinct metabolism.

Functional avidity is supposed to critically shape the quality of immune responses, thereby influencing host protection against infectious agents including SARS-CoV-2. Here we show that after human SARS-CoV-2 vaccination, a large portion of high-avidity spike-specific CD4+ T cells lost CD3 expression after in vitro activation. The CD3- subset was enriched for cytokine-positive cells, including elevated per-cell expression levels, and showed increased polyfunctionality. Assessment of key metabolic pathways by flow cytometry revealed that superior functionality was accompanied by a shift toward fatty acid synthesis at the expense of their oxidation, whereas glucose transport and glycolysis were similarly regulated in SARS-CoV-2-specific CD3- and CD3+ subsets. As opposed to their CD3+ counterparts, frequencies of vaccine-specific CD3- T cells positively correlated with both the size of the naive CD4+ T cell pool and vaccine-specific IgG levels. Moreover, their frequencies negatively correlated with advancing age and were impaired in patients under immunosuppressive therapy. Typical recall antigen-reactive T cells showed a comparable segregation into functionally and metabolically distinct CD3+ and CD3- subsets but were quantitatively maintained upon aging, likely due to earlier recruitment in life. In summary, our data identify CD3- T helper cells as correlates of high-quality immune responses that are impaired in at-risk populations.

Overall avidity declines in TCR repertoires during latent CMV but not EBV infection.

The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown. To address these issues, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCRαβ clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. The parameters involved during the long-term persistence of virus-specific T cell clonotypes were further evaluated by gene expression profiling, phenotype and functional analyses. Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression. These findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data further suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.

CD3 downregulation identifies high-avidity human CD8 T cells.

CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.

Low avidity CD8 +T cells are less exhausted than their high avidity counterparts and provide superior anti-tumor immunity

Abstract The interaction of T cell receptors (TCRs) on T cells with MHC I-peptide complexes on cancer cells elicits the cytotoxic activity of CD8 +T cells. Here we analyze how the strength of this interaction, or T cell avidity, shapes the exhaustion of CD8 +TILs and dictates anti-tumor immunity. We developed a novel tetramer decay assay to isolate T cells based on their TCR avidities. We used this method to study low and high avidity CD8 +TILs responding to the tumor neoantigen PDPR MUTof the murine sarcoma Meth A. Single cell sequencing reveals that high avidity CD8 +TILs are more terminally exhausted (51.41% vs 27.21%) and less effector-like (28.39% vs. 60.0%) than low avidity CD8 +TILs. We used flow cytometry to analyze PDPR MUT-specific CD8 +TILs with low or high avidity from 28 day-old Meth A tumors. There were significantly more TIM3 +PD-1 +cells (Paired t-test; P < 0.0001) and TOX +PD-1 +cells (Paired t-test; P < 0.05) in the high avidity CD8 +TILs than the low avidity CD8 +TILs. We adoptively transferred 1,000 PDPR MUT-specific low or high avidity CD8 +T cells, or control CD8 +T cells into tumor-bearing mice 10 days after tumor challenge. Low avidity T cells significantly improved the survival of mice (Mantel-Cox; P = 0.0044), while high avidity t cells did not do so (Mantel-Cox; P = 0.7081). These results establish a novel correlation between avidity, exhaustion and T cell-mediated tumor control in vivo.

Abstract 608: Low avidity neoepitope-specific CD8+ tumor infiltrating lymphocytes are less exhausted and more effective than their high avidity counterparts in a BALB/c murine fibrosarcoma

Abstract The frequency of CD8+ tumor infiltrating lymphocytes (TILs) has been positively associated with overall survival in several cancers. The interaction of T cell receptors (TCRs) on T cells with Major Histocompatibility Complex (MHC)-peptide complexes on cancer cells elicits the cytotoxic activity of CD8+ T cells. Here we set out to understand how the strength of this interaction, or T cell avidity, shapes the phenotype of CD8+ TILs and how that dictates anti-tumor immunity. We developed a novel tetramer decay assay to isolate T cells based on their TCR avidities. We then used this method to isolate from tumors of Meth A tumor-bearing mice (28 days after tumor challenge), Low (K off < 15 min), Medium (75 min > K off > 45 min), and High (K off > 105 min) avidity CD8+ TILs responding to the tumor rejection neoepitope, PDPRMUT of this fibrosarcoma (Ebrahimi-Nik et al. JCI Insight 2019). Unbiased clustering of single cell RNA sequencing, TCR sequencing, and CITE-seq of these PDPRMUT -specific CD8+ TILs revealed five clusters: Stem-Like (LY108+Tcf7+), Effector-Exhausted (Eff-Exh PD-1Low,CX3CR1+) Terminally-Exhausted-1 (Term-Exh1 PD-1+TIM3High), Terminally-Exhausted-2 (Term-Exh2 PD-1HighToxHigh), and NK-Like (PD-1Low, KlrHigh). The low avidity CD8+ TILs have more than twice the proportion of Eff-Exh (60.0%) when compared to high avidity CD8+ TILs (28.39%). The high avidity CD8+ TILs have more Stem-Like (12.37% vs 6.31%), Term-Exh1 (27.38% vs 21.88%), and Term-Exh2 (24.03% vs 5.33%) cells than low avidity CD8+ TILs. Furthermore, trajectory inference of these clusters, by scVelo, predicts that Stem-Like cells differentiate into Eff-Exh cells that can differentiate into either Term-Exh1 or Term-Exh2. Our data demonstrates that low avidity Eff-Exh cells preferentially differentiate into Term-Exh1, while high avidity Eff-Exh cells differentiate into both Term-Exh1 and Term-Exh2. To validate these results, we isolated CD8+ TILs responding to PDPRMUT with low or high avidity from tumors of 21 Meth A bearing mice 28 days after tumor challenge and analyzed their exhaustion phenotype by flow cytometry. There were significantly more TIM3+PD-1+ cells (Paired t-test; P < 0.0001) and TOX+PD-1+ cells (Paired t-test; P < 0.05) in the high avidity CD8+ TILs than the low avidity CD8+ TILs. We conclude that CD8+ TILs with high avidity are more exhausted and less effector like than low avidity CD8+ TILs. This finding has major implications for T cell-based cancer immunotherapy. Citation Format: Summit Singhaviranon, Joseph P. Dempsey, Adam T. Hagymasi, Pramod K. Srivastava. Low avidity neoepitope-specific CD8+ tumor infiltrating lymphocytes are less exhausted and more effective than their high avidity counterparts in a BALB/c murine fibrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 608.

EBAG9 controls CD8+ T cell memory formation responding to tumor challenge in mice.

Insight into processes that determine CD8+ T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often use high-avidity CD8+ T cells in adoptive-transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon noninflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8+ T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen–mismatched leukocytes. CD8+ memory commitment was analyzed in Ebag9-deficient mice that exhibited enhanced tumor cell lysis. This property endowed Ebag9–/– mice with extended control of Tcl-1 oncogene–induced chronic lymphocytic leukemia progression. In Ebag9–/– mice, an expanded memory population was obtained for anti-HY and anti–SV-40 T antigen–specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8+ T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9+/+ and Ebag9–/– T cells. In Ebag9–/– cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation and anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8+ memory lineage.

TCR-engineered neoantigen-specific CD4+ T cells mediate immunotherapy of a class II-negative murine squamous cell carcinoma

Abstract The expansion of neoantigen (NeoAg)-specific T cells often accompanies clinical responses to immunotherapies such as immune checkpoint blockade (ICB), adoptive cellular therapy (ACT), and personalized cancer vaccines (PCV), highlighting their importance for antitumor immunity. While the functions of NeoAg-specific CD8+ T cells have been characterized, the role of NeoAg-specific CD4+ T cells is less well understood. To study the antitumor mechanisms of NeoAg-specific CD4+ T cells, we sorted single CD4+ T cells specific for a mutated clathrin heavy chain epitope (Cltc H129Q) expressed by the murine squamous cell carcinoma VII (SCC VII) tumor. T cell receptor (TCR) sequencing analysis revealed the presence of four distinct TCR clonotypes and expression of these TCRs in primary cells was sufficient to confer preferential recognition of Cltc H129Q as compared to the corresponding wildtype Cltc epitope. Despite differences in TCR avidity, both low and high avidity CD4+ T cells were capable of proliferating to similar degrees in response to tumor antigen in vivo and enhancing primary tumor immunity in a CD8+ T cell- and CD40L-dependent manner. ACT with Cltc H129Q specific CD4+ T cells also synergized with cyclophosphamide treatment to reduce disease burden in mice with established tumors. Overall, these findings illuminate the mechanistic role of NeoAg-specific CD4+ T cells in tumor immunity, provide insights into the impact of TCR avidity on the helper functions of CD4+ T cells, and highlight the therapeutic potential of ACT with TCR-engineered CD4+ T cells.

Identification of shared tumor epitopes from endogenous retroviruses inducing high-avidity cytotoxic T cells for cancer immunotherapy.

Human endogenous retroviruses (HERVs) represent 8% of the human genome. HERV products may represent tumor antigens relevant for cancer immunotherapy. We developed a bioinformatic approach to identify shared CD8+ T cell epitopes derived from cancer-associated HERVs in solid tumors. Six candidates among the most commonly shared HLA-A2 epitopes with evidence of translation were selected for immunological evaluation. In vitro priming assays confirmed the immunogenicity of these epitopes, which induced high-avidity CD8+ T cell clones. These T cells specifically recognize and kill HLA-A2+ tumor cells presenting HERV epitopes on HLA molecules, as demonstrated by mass spectrometry. Furthermore, epitope-specific CD8+ T cells were identified by dextramer staining among tumor-infiltrating lymphocytes from HLA-A2+ patients with breast cancer. Last, we showed that HERV-specific T cells lyse patient-derived organoids. These shared virus-like epitopes are of major interest for the development of cancer vaccines or T cell–based immunotherapies, especially in tumors with low/intermediate mutational burden.

Immune Response in Moderate to Critical Breakthrough COVID-19 Infection After mRNA Vaccination.

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines.

Consequence of Histoincompatibility beyond GvH-Reaction in Cytomegalovirus Disease Associated with Allogeneic Hematopoietic Cell Transplantation: Change of Paradigm.

Hematopoietic cell (HC) transplantation (HCT) is the last resort to cure hematopoietic malignancies that are refractory to standard therapies. Hematoablative treatment aims at wiping out tumor cells as completely as possible to avoid leukemia/lymphoma relapse. This treatment inevitably co-depletes cells of hematopoietic cell lineages, including differentiated cells that constitute the immune system. HCT reconstitutes hematopoiesis and thus, eventually, also antiviral effector cells. In cases of an unrelated donor, that is, in allogeneic HCT, HLA-matching is performed to minimize the risk of graft-versus-host reaction and disease (GvHR/D), but a mismatch in minor histocompatibility antigens (minor HAg) is unavoidable. The transient immunodeficiency in the period between hematoablative treatment and reconstitution by HCT gives latent cytomegalovirus (CMV) the chance to reactivate from latently infected donor HC or from latently infected organs of the recipient, or from both. Clinical experience shows that HLA and/or minor-HAg mismatches increase the risk of complications from CMV. Recent results challenge the widespread, though never proven, view of a mechanistic link between GvHR/D and CMV. Instead, new evidence suggests that histoincompatibility promotes CMV disease by inducing non-cognate transplantation tolerance that inhibits an efficient reconstitution of high-avidity CD8+ T cells capable of recognizing and resolving cytopathogenic tissue infection.

Immunodominant Cytomegalovirus Epitopes Suppress Subdominant Epitopes in the Generation of High-Avidity CD8 T Cells

CD8+ T-cell responses to pathogens are directed against infected cells that present pathogen-encoded peptides on MHC class-I molecules. Although natural responses are polyclonal, the spectrum of peptides that qualify for epitopes is remarkably small even for pathogens with high coding capacity. Among those few that are successful at all, a hierarchy exists in the magnitude of the response that they elicit in terms of numbers of CD8+ T cells generated. This led to a classification into immunodominant and non-immunodominant or subordinate epitopes, IDEs and non-IDEs, respectively. IDEs are favored in the design of vaccines and are chosen for CD8+ T-cell immunotherapy. Using murine cytomegalovirus as a model, we provide evidence to conclude that epitope hierarchy reflects competition on the level of antigen recognition. Notably, high-avidity cells specific for non-IDEs were found to expand only when IDEs were deleted. This may be a host’s back-up strategy to avoid viral immune escape through antigenic drift caused by IDE mutations. Importantly, our results are relevant for the design of vaccines based on cytomegaloviruses as vectors to generate high-avidity CD8+ T-cell memory specific for unrelated pathogens or tumors. We propose the deletion of vector-encoded IDEs to avoid the suppression of epitopes of the vaccine target.

CBFB-MYH11 fusion neoantigen enables T cell recognition and killing of acute myeloid leukemia.

Proteins created from recurrent fusion genes like CBFB-MYH11 are prevalent in acute myeloid leukemia (AML), often necessary for leukemogenesis, persistent throughout the disease course, and highly leukemia specific, making them attractive neoantigen targets for immunotherapy. A nonameric peptide derived from a prevalent CBFB-MYH11 fusion protein was found to be immunogenic in HLA-B*40:01+ donors. High-avidity CD8+ T cell clones isolated from healthy donors killed CBFB-MYH11+ HLA-B*40:01+ AML cell lines and primary human AML samples in vitro. CBFB-MYH11-specific T cells also controlled CBFB-MYH11+ HLA-B*40:01+ AML in vivo in a patient-derived murine xenograft model. High-avidity CBFB-MYH11 epitope-specific T cell receptors (TCRs) transduced into CD8+ T cells conferred antileukemic activity in vitro. Our data indicate that the CBFB-MYH11 fusion neoantigen is naturally presented on AML blasts and enables T cell recognition and killing of AML. We provide proof of principle for immunologically targeting AML-initiating fusions and demonstrate that targeting neoantigens has clinical relevance even in low-mutational frequency cancers like fusion-driven AML. This work also represents a first critical step toward the development of TCR T cell immunotherapy targeting fusion gene-driven AML.

Revisiting CD8 T-cell 'Memory Inflation': New Insights with Implications for Cytomegaloviruses as Vaccine Vectors.

Murine models of cytomegalovirus (CMV) infection have revealed an exceptional kinetics of the immune response. After resolution of productive infection, transient contraction of the viral epitope-specific CD8 T-cell pool was found to be followed by a pool expansion specific for certain viral epitopes during non-productive ‘latent’ infection. This phenomenon, known as ‘memory inflation’ (MI), was found to be based on inflationary KLRG1+CD62L− effector-memory T cells (iTEM) that depend on repetitive restimulation. MI gained substantial interest for employing CMV as vaccine vector by replacing MI-driving CMV epitopes with foreign epitopes for generating high numbers of protective memory cells specific for unrelated pathogens. The concept of an MI-driving CMV vector is questioned by human studies disputing MI in humans. A bias towards MI in experimental models may have resulted from systemic infection. We have here studied local murine CMV infection as a route that is more closely matching routine human vaccine application. Notably, KLRG1−CD62L+ central memory T cells (TCM) and conventional KLRG1−CD62L− effector memory T cells (cTEM) were found to expand, associated with ‘avidity maturation’, whereas the pool size of iTEM steadily declined over time. The establishment of high avidity CD8 T-cell central memory encourages one to pursue the concept of CMV vector-based vaccines.

Fine tuning CD8+ T cell functional avidity by the PD-1/SHP2 axis

Abstract T cells of high functional avidity recognize low antigen levels and have the most effective cytotoxic responses against viruses and tumor cells. Understanding the molecular regulations of T cell functional avidity is essential to aid novel vaccine design as well as adoptive immunotherapy by improving the T cell quality. Programmed cell death protein 1 (PD-1) is known to negatively regulate the signal strength of T cell receptor (TCR). Activated PD-1 recruits Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (Shp2), which dephosphorylates kinases involved in TCR signaling. However, it is unclear whether PD-1/SHP2 can modulate T cell functional avidity. To address this question, we treated splenocytes from OT-1 transgenic mice with anti-PD-1 or SHP099 (SHP2 inhibitor), or a combination of both for 3 days in a selective culture condition of APC-pulsed stimulation with 10−6 M or 10−9 M SINNFEKL, respectively. Our preliminary data showed that blocking SHP2/PD-1 enhanced functional avidity and antigen sensitivity of CD8+ T cell in vitro when stimulated with limited SINNFEKL antigen. Blocking of PD1/SHP2 also enhanced CD8 beta expression, which has been shown correlated with high functional avidity CD8+ T cells. These data suggest that PD1/SHP2 is involved in regulating T cell functional avidity.

CD3 and CD8 coreceptor down-modulation are inversely associated with CD8 T cell functional avidity in humans

Abstract This study investigated the function of memory CD8 T cells in HIV-infected people durably suppressed on antiretroviral therapy (HIV+ cART). We assessed bulk and virus-specific memory CD8 T cells in HIV+ cART and HIV-seronegative individuals (HIV−) by flow cytometry. We observed a population of CD3+ CD8dim CD14− CD16− (CD8dim) T cells that was expanded as a percentage of total CD8 T cells in both HIV− and CMV-seropositive individuals. Bulk memory CD8dim T cells expressed significantly higher CD69 and less MHC Class I and CD127 ex vivo than CD8bright T cells, suggesting recent activation. CD8dim T cells expressed less GLUT1 and PGC-1α and took up less glucose (2-NBDG) and lipid (Bodipy) than CD8bright T cells, indicating relatively lower metabolic activity. Multimer reactivity was used to examine CMV-, EBV- and HIV-specific CD8 T cells ex vivo. Virus-specific populations were consistently CD8high. However, after peptide stimulation, antigen-specific CD8 T cells down-regulated CD3 and CD8. CMV-specific CD8 T cells down-regulated CD3 and CD8 more than HIV-specific cells. CD3 and CD8 downregulation were strongly correlated with the functional avidity of the response. A strong correlation between GLUT1 down-regulation and CD8 down-regulation was also observed, suggesting an association between CD8 expression and metabolic activity. These results suggest that the expanded CD8dim population in HIV+ cART individuals, who are >90% CMV-seropositive, may be driven by ongoing activation of high-avidity CMV-specific CD8 T cells. They also suggest that different virus-specific CD8 T cell populations differentially downregulate components of the TCR complex and metabolism after antigen stimulation, possibly to avoid excessive activation.

Insufficient Antigen Presentation Due to Viral Immune Evasion Explains Lethal Cytomegalovirus Organ Disease After Allogeneic Hematopoietic Cell Transplantation.

Reactivation of latent cytomegalovirus (CMV) poses a clinical problem in transiently immunocompromised recipients of hematopoietic cell (HC) transplantation (HCT) by viral histopathology that results in multiple organ manifestations. Compared to autologous HCT and to syngeneic HCT performed with identical twins as HC donor and recipient, lethal outcome of CMV infection is more frequent in allogeneic HCT with MHC/HLA or minor histocompatibility loci mismatch between donor and recipient. It is an open question if a graft-vs.-host (GvH) reaction exacerbates CMV disease, or if CMV exacerbates GvH disease (GvHD), or if interference is mutual. Here we have used a mouse model of experimental HCT and murine CMV (mCMV) infection with an MHC class-I mismatch by gene deletion, so that either HCT donor or recipient lack a single MHC class-I molecule, specifically H-2 Ld. This particular immunogenetic disparity has the additional advantage that it allows to experimentally separate GvH reaction of donor-derived T cells against recipient's tissues from host-vs.-graft (HvG) reaction of residual recipient-derived T cells against the transplanted HC and their progeny. While in HvG-HCT with Ld-plus donors and Ld-minus recipients almost all infected recipients were found to control the infection and survived, almost all infected recipients died of uncontrolled virus replication and consequent multiple-organ viral histopathology in case of GvH-HCT with Ld-minus donors and Ld-plus recipients. Unexpectedly, although anti-Ld-reactive CD8+ T cells were detected, mortality was not found to be associated with GvHD histopathology. By comparing HvG-HCT and GvH-HCT, investigation into the mechanism revealed an inefficient reconstitution of antiviral high-avidity CD8+ T cells, associated with lack of formation of protective nodular inflammatory foci (NIF) in host tissue, selectively in GvH-HCT. Most notably, mice infected with an immune evasion gene deletion mutant of mCMV survived under otherwise identical GvH-HCT conditions. Survival was associated with enhanced antigen presentation and formation of protective NIF by antiviral CD8+ T cells that control the infection and prevent viral histopathology. This is an impressive example of lethal viral disease in HCT recipients based on a failure of the immune control of CMV infection due to viral immune evasion in concert with an MHC class-I mismatch.

High Peptide Dose Vaccination Promotes the Early Selection of Tumor Antigen-Specific CD8 T-Cells of Enhanced Functional Competence.

CD8 T-cell response efficiency critically depends on the TCR binding strength to peptide-MHC, i.e., the TCR binding avidity. A current challenge in onco-immunology lies in the evaluation of vaccine protocols selecting for tumor-specific T-cells of highest avidity, offering maximal immune protection against tumor cells and clinical benefit. Here, we investigated the impact of peptide and CpG/adjuvant doses on the quality of vaccine-induced CD8 T-cells in relation to binding avidity and functional responses in treated melanoma patients. Using TCR-pMHC binding avidity measurements combined to phenotype and functional assays, we performed a comprehensive study on representative tumor antigen-specific CD8 T-cell clones (n = 454) from seven patients vaccinated with different doses of Melan-A/ELA peptide (0.1 mg vs. 0.5 mg) and CpG-B adjuvant (1–1.3 mg vs. 2.6 mg). Vaccination with high peptide dose favored the early and strong in vivo expansion and differentiation of Melan-A-specific CD8 T-cells. Consistently, T-cell clones generated from those patients showed increased TCR binding avidity (i.e., slow off-rates and CD8 binding independency) readily after 4 monthly vaccine injections (4v). In contrast, the use of low peptide or high CpG-B doses required 8 monthly vaccine injections (8v) for the enrichment of anti-tumor T-cells with high TCR binding avidity and low CD8 binding dependency. Importantly, the CD8 binding-independent vaccine-induced CD8 T-cells displayed enhanced functional avidity, reaching a plateau of maximal function. Thus, T-cell functional potency following peptide/CpG/IFA vaccination may not be further improved beyond a certain TCR binding avidity limit. Our results also indicate that while high peptide dose vaccination induced the early selection of Melan-A-specific CD8 T-cells of increased functional competence, continued serial vaccinations also promoted such high-avidity T-cells. Overall, the systematic assessment of T-cell binding avidity may contribute to optimize vaccine design for improving clinical efficacy.

Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma

5T4 (trophoblast glycoprotein, TPBG) is a transmembrane tumor antigen expressed on more than 90% of primary renal cell carcinomas (RCC) and a wide range of human carcinomas but not on most somatic adult tissues. The favorable expression pattern has encouraged the development and clinical testing of 5T4-targeted antibody and vaccine therapies. 5T4 also represents a compelling and unexplored target for T-cell receptor (TCR)-engineered T-cell therapy. Our group has previously isolated high-avidity CD8+ T-cell clones specific for an HLA-A2-restricted 5T4 epitope (residues 17–25; 5T4p17). In this report, targeted single-cell RNA sequencing was performed on 5T4p17-specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of T-cell receptor α chain (TRA) and β chain (TRB) genes. Full-length TRA and TRB sequences were cloned into lentiviral vectors and transduced into CD8+ T-cells from healthy donors. Redirected effector T-cell function against 5T4p17 was measured by cytotoxicity and cytokine release assays. Seven unique TRA-TRB pairs were identified. All seven TCRs exhibited high expression on CD8+ T-cells with transduction efficiencies from 59 to 89%. TCR-transduced CD8+ T-cells demonstrated redirected cytotoxicity and cytokine release in response to 5T4p17 on target-cells and killed 5T4+/HLA-A2+ kidney-, breast-, and colorectal-tumor cell lines as well as primary RCC tumor cells in vitro. TCR-transduced CD8+ T-cells also detected presentation of 5T4p17 in TAP1/2-deficient T2 target-cells. TCR-transduced T-cells redirected to recognize the 5T4p17 epitope from a broadly shared tumor antigen are of interest for future testing as a cellular immunotherapy strategy for HLA-A2+ subjects with 5T4+ tumors.

Early primed KLRG1- CMV-specific T cells determine the size of the inflationary T cell pool.

Memory T cell inflation is a process in which a subset of cytomegalovirus (CMV) specific CD8 T cells continuously expands mainly during latent infection and establishes a large and stable population of effector memory cells in peripheral tissues. Here we set out to identify in vivo parameters that promote and limit CD8 T cell inflation in the context of MCMV infection. We found that the inflationary T cell pool comprised mainly high avidity CD8 T cells, outcompeting lower avidity CD8 T cells. Furthermore, the size of the inflationary T cell pool was not restricted by the availability of specific tissue niches, but it was directly related to the number of virus-specific CD8 T cells that were activated during priming. In particular, the amount of early-primed KLRG1- cells and the number of inflationary cells with a central memory phenotype were a critical determinant for the overall magnitude of the inflationary T cell pool. Inflationary memory CD8 T cells provided protection from a Vaccinia virus challenge and this protection directly correlated with the size of the inflationary memory T cell pool in peripheral tissues. These results highlight the remarkable protective potential of inflationary CD8 T cells that can be harnessed for CMV-based T cell vaccine approaches.

Counter-regulation of regulatory T cells by autoreactive CD8+ T cells in rheumatoid arthritis

The mechanisms whereby autoreactive T cells escape peripheral tolerance establishing thus autoimmune diseases in humans remain an unresolved question. Here, we demonstrate that autoreactive polyfunctional CD8+ T cells recognizing self-antigens (i.e., vimentin, actin cytoplasmic 1, or non-muscle myosin heavy chain 9 epitopes) with high avidity, counter-regulate Tregs by killing them, in a consistent percentage of rheumatoid arthritis (RA) patients. Indeed, these CD8+ T cells express a phenotype and gene profile of effector (eff) cells and, upon antigen-specific activation, kill Tregs indirectly in an NKG2D-dependent bystander fashion in vitro. This data provides a mechanistic basis for the finding showing that AE-specific (CD107a+) CD8+ T killer cells correlate, directly with the disease activity score, and inversely with the percentage of activated Tregs, in both steady state and follow-up studies in vivo. In addition, multiplex immunofluorescence imaging analyses of inflamed synovial tissues in vivo show that a remarkable number of CD8+ T cells express granzyme-B and selectively contact FOXP3+ Tregs, some of which are in an apoptotic state, validating hence the possibility that CD8+ Teff cells can counteract neighboring Tregs within inflamed tissues, by killing them. Alternatively, the disease activity score of a different subset of patients is correlated with the expansion of a peculiar subpopulation of autoreactive low avidity, partially-activated (pa)CD8+ T cells that, despite they conserve the conventional naïve (N) phenotype, produce high levels of tumor necrosis factor (TNF)-α and exhibit a gene expression signature of a progressive activation state. Tregs directly correlate with the expansion of this autoreactive (low avidity) paCD8+ TN cell subset in vivo, and efficiently control their differentiation rather their proliferation in vitro. Interestingly, autoreactive high avidity CD8+ Teff cells or low avidity paCD8+ TN cells are significantly expanded in RA patients who would become non-responders or patients who would become responders to TNF-α inhibitor therapy, respectively. These data provide evidence of a previously undescribed role of such mechanisms in the progression and therapy of RA.