Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells

SHP-1 phosphatase activity counteracts increased T cell receptor affinity.

Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K(d) < 1 μM) lead to drastic functional declines. Using human CD8(+) T cells engineered with TCRs of incremental affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity-associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K(d) = 5 to 1 μM), those with supraphysiological affinity (K(d) = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8(+) T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity-dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8(+) T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell-mediated immunity

Single cell analysis reveals similar functional competence of dominant and nondominant CD8 T-cell clonotypes.

Immune protection from infectious diseases and cancer is mediated by individual T cells of different clonal origin. Their functions are tightly regulated but not yet fully characterized. Understanding the contribution of each T cell will improve the prediction of immune protection based on laboratory assessment of T-cell responses. Here we developed techniques for simultaneous molecular and functional assessment of single CD8 T cells directly ex vivo. We studied two groups of patients with melanoma after vaccination with two closely related tumor antigenic peptides. Vaccination induced T cells with strong memory and effector functions, as found in virtually all T cells of the first patient group, and fractions of T cells in the second group. Interestingly, high functionality was not restricted to dominant clonotypes. Rather, dominant and nondominant clonotypes acquired equal functional competence. In parallel, this was also found for EBV- and CMV-specific T cells. Thus, the nondominant clonotypes may contribute similarly to immunity as their dominant counterparts

Molecular Targets for 17α-Ethynyl-5-Androstene-3β,7β,17β-Triol, an Anti-Inflammatory Agent Derived from the Human Metabolome

HE3286, 17α-ethynyl-5-androstene-3β, 7β, 17β-triol, is a novel synthetic compound related to the endogenous sterol 5-androstene-3β, 7β, 17β-triol (β-AET), a metabolite of the abundant adrenal steroid dehydroepiandrosterone (DHEA). HE3286 has shown efficacy in clinical studies in impaired glucose tolerance and type 2 diabetes, and in vivo models of types 1 and 2 diabetes, autoimmunity, and inflammation. Proteomic analysis of solid-phase HE3286-bound bead affinity experiments, using extracts from RAW 264.7 mouse macrophage cells, identified 26 binding partners. Network analysis revealed associations of these HE3286 target proteins with nodes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for type 2 diabetes, insulin, adipokine, and adipocyte signaling. Binding partners included low density lipoprotein receptor-related protein (Lrp1), an endocytic receptor; mitogen activated protein kinases 1 and 3 (Mapk1, Mapk3), protein kinases involved in inflammation signaling pathways; ribosomal protein S6 kinase alpha-3 (Rsp6ka3), an intracellular regulatory protein; sirtuin-2 (Sirt2); and 17β-hydroxysteroid dehydrogenase 1 (Hsd17β4), a sterol metabolizing enzyme

Purification, characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity

A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773. The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation. The purified enzyme showed three subunits in SDS gel, and the gene sequence revealed that this enzyme belongs to the molybdopterin binding oxidoreductase family containing molybdopterins, FAD, and iron-sulfur clusters

T-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection.

The clinical course of autoimmune and infectious disease varies greatly, even between individuals with the same condition. An understanding of the molecular basis for this heterogeneity could lead to significant improvements in both monitoring and treatment. During chronic infection the process of T-cell exhaustion inhibits the immune response, facilitating viral persistence. Here we show that a transcriptional signature reflecting CD8 T-cell exhaustion is associated with poor clearance of chronic viral infection, but conversely predicts better prognosis in multiple autoimmune diseases. The development of CD8 T-cell exhaustion during chronic infection is driven both by persistence of antigen and by a lack of accessory 'help' signals. In autoimmunity, we find that where evidence of CD4 T-cell co-stimulation is pronounced, that of CD8 T-cell exhaustion is reduced. We can reproduce the exhaustion signature by modifying the balance of persistent stimulation of T-cell antigen receptors and specific CD2-induced co-stimulation provided to human CD8 T cells in vitro, suggesting that each process plays a role in dictating outcome in autoimmune disease. The 'non-exhausted' T-cell state driven by CD2-induced co-stimulation is reduced by signals through the exhaustion-associated inhibitory receptor PD-1, suggesting that induction of exhaustion may be a therapeutic strategy in autoimmune and inflammatory disease. Using expression of optimal surrogate markers of co-stimulation/exhaustion signatures in independent data sets, we confirm an association with good clinical outcome or response to therapy in infection (hepatitis C virus) and vaccination (yellow fever, malaria, influenza), but poor outcome in autoimmune and inflammatory disease (type 1 diabetes, anti-neutrophil cytoplasmic antibody-associated vasculitis, systemic lupus erythematosus, idiopathic pulmonary fibrosis and dengue haemorrhagic fever). Thus, T-cell exhaustion plays a central role in determining outcome in autoimmune disease and targeted manipulation of this process could lead to new therapeutic opportunities

Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma

BACKGROUND: Multiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity. METHODS: To test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4. RESULTS: Temporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies. CONCLUSIONS: These data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40425-014-0043-z) contains supplementary material, which is available to authorized users

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells