Mycobacterium tuberculosis-Specific CD4 T Cells Expressing Transcription Factors T-Bet or RORγT Associate with Bacterial Control in Granulomas.

Despite the extensive research on CD4 T cells within the context of Mycobacterium tuberculosis (Mtb) infections, few studies have focused on identifying and investigating the profile of Mtb-specific T cells within lung granulomas. To facilitate the identification of Mtb-specific CD4 T cells, we identified immunodominant epitopes for two Mtb proteins, namely, Rv1196 and Rv0125, using a Mauritian cynomolgus macaque model of Mtb infection, thereby providing data for the synthesis of MHC class II tetramers. Using tetramers, we identified Mtb-specific cells within different immune compartments, postinfection. We found that granulomas were enriched sites for Mtb-specific cells and that tetramer+ cells had increased frequencies of the activation marker CD69 as well as the transcription factors T-bet and RORγT, compared to tetramer negative cells within the same sample. Our data revealed that while the frequency of Rv1196 tetramer+ cells was positively correlated with the granuloma bacterial burden, the frequency of RORγT or T-bet within tetramer+ cells was inversely correlated with the granuloma bacterial burden, thereby highlighting the importance of having activated, polarized, Mtb-specific cells for the control of Mtb in lung granulomas. IMPORTANCE Tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, kills 1.5 million people each year, despite the existence of effective drugs and a vaccine that is given to infants in most countries. Clearly, we need better vaccines against this disease. However, our understanding of the immune responses that are necessary to prevent tuberculosis is incomplete. This study seeks to understand the functions of T cells that are specific for M. tuberculosis at the site of the disease in the lungs. For this, we developed specialized tools called MHC class II tetramers to identify those T cells that can recognize M. tuberculosis and applied the tools to the study of this infection in nonhuman primate models that mimic human tuberculosis. We demonstrate that M. tuberculosis-specific T cells in lung lesions are associated with control of the bacteria only when those T cells are expressing certain functions, thereby highlighting the importance of combining the identification of specific T cells with functional analyses. Thus, we surmise that these functions of specific T cells are critical to the control of infection and should be considered as a part of the development of vaccines against tuberculosis.

High and Sustained Ex Vivo Frequency but Altered Phenotype of SARS-CoV-2-Specific CD4+ T-Cells in an Anti-CD20-Treated Patient with Prolonged COVID-19.

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145-164) epitope-specific CD4+ T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4+ T-cell response in both patients, with higher frequencies of virus-specific CD4+ T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4+ T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4+ T-cells with CD45RA- CXCR5+ PD-1+ circulating T follicular helper cell (cTFH) phenotype. Furthermore, we observed a delayed contraction of CD127- virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4+ T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell-T-cell interaction, a robust virus-specific CD4+ T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection.

Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis.

BACKGROUND: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. RESULTS: Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-ß, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. CONCLUSIONS: Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA 'at risk' subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.

Inventories of naive and tolerant mouse CD4 T cell repertoires reveal a hierarchy of deleted and diverted T cell receptors.

Deletion or Treg cell differentiation are alternative fates of autoreactive MHCII-restricted thymocytes. How these different modes of tolerance determine the size and composition of polyclonal cohorts of autoreactive T cells with shared specificity is poorly understood. We addressed how tolerance to a naturally expressed autoantigen of the central nervous system shapes the CD4 T cell repertoire. Specific cells in the tolerant peripheral repertoire either were Foxp3+ or displayed anergy hallmarks and, surprisingly, were at least as frequent as in the nontolerant repertoire. Despite this apparent lack of deletional tolerance, repertoire inventories uncovered that some T cell receptors (TCRs) were lost from the CD4 T cell pool, whereas others mediated Treg cell differentiation. The antigen responsiveness of these TCRs supported an affinity model of central tolerance. Importantly, the contribution of different diverter TCRs to the nascent thymic Treg cell population reflected their antigen reactivity rather than their frequency among precursors. This reveals a multilayered TCR hierarchy in CD4 T cell tolerance that separates deleted and diverted TCRs and assures that the Treg cell compartment is filled with cells of maximal permissive antigen reactivity.

MHC class II tetramer analyses in AE37-vaccinated prostate cancer patients reveal vaccine-specific polyfunctional and long-lasting CD4(+) T-cells.

Realizing the basis for generating long-lasting clinical responses in cancer patients after therapeutic vaccinations provides the means to further ameliorate clinical efficacy. Peptide cancer vaccines stimulating CD4(+) T helper cells are often promising for inducing immunological memory and persistent CD8(+) cytotoxic T cell responses. Recent reports from our clinical trial with the AE37 vaccine, which is a HER2 hybrid polypeptide, documented its efficacy to induce CD4(+) T cell immunity, which was associated with clinical improvements preferentially among HLA-DRB1*11(+) prostate cancer patients. Here, we performed in-depth investigation of the CD4(+) T cell response against the AE37 vaccine. We used the DR11/AE37 tetramer in combination with multicolor flow cytometry to identify and characterize AE37-specific CD4(+) T cells regarding memory and Tregs phenotype in HLA-DRB1*11(+) vaccinated patients. To verify vaccine-specific immunological memory in vivo, we also assessed AE37-specific CD4(+) T cells in defined CD4(+) memory subsets by cell sorting. Finally, vaccine-induced AE37-specific CD4(+) T cells were assessed regarding their functional profile. AE37-specific memory CD4(+) T cells could be detected in peptide-stimulated cultures from prostate cancer patients following vaccination even 4 y post-vaccination. The vast majority of vaccine-induced AE37-specific CD4(+) T cells exhibited a multifunctional, mostly Th1 cytokine signature, with the potential of granzyme B production. In contrast, we found relatively low frequencies of Tregs among AE37-specific CD4(+) T cells. This is the first report on the identification of vaccine-induced HER2-specific multifunctional long-lasting CD4(+) T cells in vaccinated prostate cancer patients.

Production of NY-ESO-1 peptide/DRB1*08:03 tetramers and ex vivo detection of CD4 T-cell responses in vaccinated cancer patients.

We established CD4 T-cell clones, Mz-1B7, and Ue-21, which recognized the NY-ESO-1 121-138 peptide from peripheral blood mononuclear cells (PBMCs) of an esophageal cancer patient, E-2, immunized with an NY-ESO-1 protein and determined the NY-ESO-1 minimal epitopes. Minimal peptides recognized by Mz-1B7 and Ue-21 were NY-ESO-1 125-134 and 124-134, respectively, both in restriction to DRB1*08:03. Using a longer peptide, 122-135, and five other related peptides, including either of the minimal epitopes recognized by the CD4 T-cell clones, we investigated the free peptide/DR recognition on autologous EBV-B cells as APC and peptide/DR tetramer binding. The results showed a discrepancy between them. The tetramers with several peptides recognized by either Mz-1B7 or the Ue-21 CD4 T-cell clone did not bind to the respective clone. On the other hand, unexpected binding of the tetramer with the peptide not recognized by CD4 T-cells was observed. The clone Mz-1B7 did not recognize the free peptide 122-135 on APC, but the peptide 122-135/DRB1*08:03 tetramer bound to the TCR on those cells. The failure of tetramer production and the unexpected tetramer binding could be due to a subtly modified structure of the peptide/DR tetramer from the structure of the free peptide/DR molecule. We also demonstrated that the NY-ESO-1 123-135/DRB1*08:03 tetramer detected ex vivo CD4 T-cell responses in PBMCs from patients after NY-ESO-1 vaccination in immunomonitoring.

Specific immunotherapy modifies allergen-specific CD4(+) T-cell responses in an epitope-dependent manner.

BACKGROUND: Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T-cell epitope level is critical for the design of new allergy vaccine strategies. OBJECTIVE: We sought to characterize allergen-specific T-cell responses linked with allergy or peripheral tolerance and to determine how CD4(+) T-cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy. METHODS: Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy, and phenotype of the DR04:01-restricted TGP-specific T-cell responses in 10 subjects with grass pollen allergy, 5 nonatopic subjects, and 6 allergy vaccine-treated subjects was determined by using an ex vivo peptide-MHC class II tetramer approach. RESULTS: CD4(+) T cells in allergic subjects are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. Allergen-specific immunotherapy was associated with preferential deletion of allergen-specific TH2 cells and without a significant change in the frequency of TH1/TR1 cells. CONCLUSIONS: Preferential allergen-specific TH2 cell deletion after repeated high-dose antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy.

Detection of Foreign Antigen-specific CD4(+)Foxp3(+) Regulatory T Cells by MHC Class II Tetramer and Intracellular CD154 Staining.

The unrestricted population of CD4(+)Foxp3(+) regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific CD4(+)Foxp3(+) Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect CD4(+)Foxp3(+) Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV GP66-77-specific CD4(+) T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV GP66-77-specific CD4(+) T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP66-77-specific CD4(+)Foxp3(+) Tregs using Foxp3(GFP) knock-in mouse, and found that LCMV GP66-77-specific CD4(+)Foxp3(+) Tregs and polyclonal CD4(+)Foxp3(+) Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4(+)Foxp3(+) Treg in various models.

Detection of Foreign Antigen-specific CD4+Foxp3+ Regulatory T Cells by MHC Class II Tetramer and Intracellular CD154 Staining

The unrestricted population of CD4+Foxp3+ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific CD4+Foxp3+ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect CD4+Foxp3+ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV GP66-77-specific CD4+ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV GP66-77-specific CD4+ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP66-77-specific CD4+Foxp3+ Tregs using Foxp3GFP knock-in mouse, and found that LCMV GP66-77-specific CD4+Foxp3+ Tregs and polyclonal CD4+Foxp3+ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4+Foxp3+ Treg in various models.