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There is growing interest in HLA‐E‐restricted T‐cell responses as a possible novel, highly conserved, vaccination targets in the context of infectious and malignant diseases. The developing field of HLA multimers for the detection and study of peptide‐specific T cells has allowed the in‐depth study of TCR repertoires and molecular requirements for efficient antigen presentation and T‐cell activation. In this study, we developed a method for efficient peptide thermal exchange on HLA‐E monomers and multimers allowing the high‐throughput production of HLA‐E multimers. We optimized the thermal‐mediated peptide exchange, and flow cytometry staining conditions for the detection of TCR and NKG2A/CD94 receptors, showing that this novel approach can be used for high‐throughput identification and analysis of HLA‐E‐binding peptides which could be involved in T‐cell and NK cell‐mediated immune responses. Importantly, our analysis of NKG2A/CD94 interaction in the presence of modified peptides led to new molecular insights governing the interaction of HLA‐E with this receptor. In particular, our results reveal that interactions of HLA‐E with NKG2A/CD94 and the TCR involve different residues. Altogether, we present a novel HLA‐E multimer technology based on thermal‐mediated peptide exchange allowing us to investigate the molecular requirements for HLA‐E/peptide interaction with its receptors. [ABSTRACT FROM AUTHOR]

High serum levels of interleukin‐6 (IL‐6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. To address this, we developed a human papillomavirus type 16 (HPV16)‐associated tumor model expressing IL‐6 to investigate the impact of tumor‐expressed IL‐6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor‐produced IL‐6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor‐produced IL‐6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T‐cell‐mediated killing. Furthermore, no overt differences in the frequencies of tumor‐infiltrating subsets of T cells or CD11b+ myeloid cells were observed. IL‐6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II+ myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL‐6‐mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL‐6‐mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer. What's new? Interleukin‐6 (IL‐6) cytokine has multiple effects on hematopoiesis and immune function and typically circulates at low levels. In cancer, however, IL‐6 serum levels are significantly elevated, with suspected impacts on tumor behavior. In this study, using a mouse model of human papillomavirus‐induced cancer with IL‐6 expression, the authors show that tumor‐produced IL‐6 confers resistance to both chemotherapy and immunotherapy. Resistance was associated with impaired myeloid cell maturation, with no evidence of involvement of mechanisms intrinsic to tumor cells. Resistance was overcome by combining chemotherapy and immunotherapy, providing insight into a potentially effective therapeutic approach for cancers with IL‐6‐mediated resistance. [ABSTRACT FROM AUTHOR]

Abstract: HLA‐E presented antigens are interesting targets for vaccination given HLA‐Es’ essentially monomorphic nature. We have shown previously that Mycobacterium tuberculosis (Mtb) peptides are presented by HLA‐E to CD8+ effector T cells, but the precise phenotype and functional capacity of these cells remains poorly characterized. We have developed and utilized in this study a new protocol combining HLA‐E tetramer with intracellular staining for cytokines, transcription factors and cytotoxic molecules to characterize these cells in depth. We confirm in this study the significantly increased ex vivo frequency of Mtb‐peptide/HLA‐E‐TM+ CD8+ T cells in the circulation of patients with active tuberculosis (TB). HLA‐E restricted CD8+ T cells from TB patients produced more IL‐13 than cells from controls or subjects with latent tuberculosis infection (LTBI). Compared to total CD8+ T cells, HLA‐E restricted cells produced more IFNγ, IL‐4, IL‐10, and granulysin but less granzyme‐A. Moreover, compared to “classical” Mtb specific HLA‐A2 restricted CD8+ T cells, HLA‐E restricted CD8+ T cells produced less TNFα and perforin, but more IL‐4. In conclusion, HLA‐E restricted‐ Mtb specific cells can produce Th2 cytokines directly. [ABSTRACT FROM AUTHOR]

Latent tuberculosis infection (LTBI) is evidence of immunological control of tuberculosis. Dormancy survival regulator (DosR) regulon-encoded proteins may have a role in the maintenance of LTBI. T cell responses to Rv1733c, Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins were found to be most frequent among household contacts of TB cases from Uganda compared to other DosR proteins, but antibody responses were not described. We characterized antibody responses to these proteins in individuals from Uganda. Antibodies to Rv1733c, Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins were measured in 68 uninfected individuals, 62 with LTBI, and 107 with active pulmonary tuberculosis (APTB) cases. There were no differences in the concentrations of antibodies to Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins between individuals with LTBI and APTB and those who were uninfected. LTBI was associated with higher concentrations of antibodies to Rv1733c in female participants [adjusted geometric mean ratio: 1.812, 95% confidence interval (CI): 1.105 2.973, and p = 0.019] but not in males (p value for interaction = 0.060). Antibodies to the four DosR regulon-encoded proteins investigated may not serve as good biomarkers of LTBI in the general population. More of the M.tb proteome needs to be screened to identify proteins that induce strong antibody responses in LTBI. [ABSTRACT FROM AUTHOR]

Latent tuberculosis infection (LTBI) is evidence of immunological control of tuberculosis. Dormancy survival regulator (DosR) regulon-encoded proteins may have a role in the maintenance of LTBI. T cell responses to Rv1733c, Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins were found to be most frequent among household contacts of TB cases from Uganda compared to other DosR proteins, but antibody responses were not described. We characterized antibody responses to these proteins in individuals from Uganda. Antibodies to Rv1733c, Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins were measured in 68 uninfected individuals, 62 with LTBI, and 107 with active pulmonary tuberculosis (APTB) cases. There were no differences in the concentrations of antibodies to Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins between individuals with LTBI and APTB and those who were uninfected. LTBI was associated with higher concentrations of antibodies to Rv1733c in female participants [adjusted geometric mean ratio: 1.812, 95% confidence interval (CI): 1.105 2.973, and p = 0.019] but not in males (p value for interaction = 0.060). Antibodies to the fourDosR regulon-encoded proteins investigatedmay not serve as good biomarkers of LTBI in the general population.More of the M.tb proteome needs to be screened to identify proteins that induce strong antibody responses in LTBI. [ABSTRACT FROM AUTHOR]

The stable incidence of new leprosy cases suggests that transmission of infection continues despite worldwide implementation of MDT. Thus, specific tools are needed to diagnose early stage Mycobacterium leprae infection, the likely sources of transmission. M. leprae antigens that induce T-cell responses in M. leprae exposed and/or infected individuals thus are major targets for new diagnostic tools. Previously, we showed that ML1601c was immunogenic in patients and healthy household contacts (HHC). However, some endemic controls (EC) also recognized this protein. To improve the diagnostic potential, IFN-γ responses to ML1601c peptides were assessed using PBMC from Brazilian leprosy patients and EC. Five ML1601c peptides only induced IFN-γ in patients and HHC. Moreover, 24-hour whole-blood assay (WBA), two ML1601c peptides could assess the level of M. leprae exposure in Ethiopian EC. Beside IFN-γ, also IP-10, IL-6, IL-1β, TNF-α, and MCP-1 were increased in EC from areas with high leprosy prevalence in response to these ML1601c peptides. Thus, ML1601c peptides may be useful for differentiating M. leprae exposed or infected individuals and can also be used to indicate the magnitude of M. leprae transmission even in the context of various HLA alleles as present in these different genetic backgrounds. [ABSTRACT FROM AUTHOR]

More than 2 billion individuals are latently infected with Mycobacterium tuberculosis ( Mtb). Knowledge of the key Mtb antigens and responding T-cell subsets mediating protection against Mtb is critical for developing improved tuberculosis (TB) vaccines. We previously reported that Mtb DosR-regulon-encoded antigens are recognized well by human T cells in association with control of Mtb infection. The characteristics of the responding T-cell subsets, however, remained unidentified. We have therefore studied the cytokine production and memory phenotypes of Mtb DosR-regulon-encoded antigen-specific T cells from individuals who had been infected with Mtb decades ago, yet never developed TB (long-term latent Mtb-infected individuals). Using multi-parameter flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α and IL-2, we found double and single cytokine-producing CD4+ as well as CD8+ T cells to be the most prominent subsets, particularly IFN-γ+ TNF-α+ CD8+ T cells. The majority of these T cells comprised effector memory and effector T cells. Furthermore, CFSE labeling revealed strong CD4+ and CD8+ T-cell proliferative responses induced by several 'immunodominant' Mtb DosR antigens and their specific peptide epitopes. These findings demonstrate the prominent presence of double- and monofunctional CD4+ and CD8+ T-cell responses in naturally protected individuals and support the possibility of designing Mtb DosR antigen-based TB vaccines. [ABSTRACT FROM AUTHOR]

The article discusses a study which identified citrullinated vimentin peptides present in HLA-DRB1*0401-restricted T cells. The study included HLA-DR4-transgenic mice that were immunized with all citrulline-containing peptide from vimentin. It discovered a citrulline-speeific response in two peptides analyzed in the transgenic mice. It also observed T cell reactivity against the peptides.

CD4 T cells are important for CD8 T-cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4 T cells to influence CD8 T-cell responses induced by activated DC. Surprisingly, mice depleted for CD4 cells were able to generate stronger antigen-specific CD8 T-cell responses after DC vaccination than non-depleted mice. The same observation was made when mice were vaccinated with MHC class II DC, indicating the presence of a MHC class II-dependent CD4 T-cell population inhibiting CD8 T-cell responses. Recently we described the expansion of DX5CD4 T cells, a T-cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8 T-cell induction and expansion of DX5CD4 T cells as the latter cells did not expand after vaccination with MHC class II DC. In vitro, DX5CD4 T cells were able to limit proliferation, modulate cytokine production and induce Foxp3 expression in OVA-specific CD8 T cells. Together, our data show an inhibitory role of CD4 T cells on the induction of CD8 T-cell responses by activated DC and indicate the involvement of DX5CD4, but not CD4CD25, T cells in this process. [ABSTRACT FROM AUTHOR]

The immune response against human adenovirus (HAdV) has gained interest because of the application of HAdV-based vectors in gene therapy and the high incidence of infections in pediatric recipients of allogeneic stem cell grafts. Because antiviral medication is frequently ineffective, the option of adoptive transfer of HAdV-specific donor-derived T cells in these immunocompromised patients is investigated. To generate good manufacturing practice-compatible reagents, a panel of 63 long, overlapping, peptides of the hexon protein was screened for recognition by T cells. Five conserved peptides of 30 amino acids were identified that were recognized by the majority of adult donors. CD4 T cells from long-term cultures of PBMC, stimulated with this set of five peptides, recognized cells infected with HAdV serotypes belonging to different species. These data demonstrate that adult human T cells preferentially recognize conserved sequences of amino acid residues from a structural protein of HAdV. In the context of gene therapy, this observation may limit the beneficial effect of switching to HAdV-based vectors derived from less common serotypes of HAdV in an attempt to circumvent pre-existing immunity. However, this cross-reactivity benefits the application of HAdV-specific T cells for adoptive immunotherapy in immunocompromised transplant recipients. [ABSTRACT FROM AUTHOR]