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

Image based machine learning for identification of macrophage subsets

Macrophages play a crucial rule in orchestrating immune responses against pathogens and foreign materials. Macrophages have remarkable plasticity in response to environmental cues and are able to acquire a spectrum of activation status, best exemplified by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at the two ends of the spectrum. Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time consuming, require large numbers of cells and are resource intensive. In this study, we used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Fluorescent microscopy was used to assess cell morphology of different cell types which were stained for nucleus and actin distribution using DAPI and phalloidin respectively. By only analysing their morphology we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes with an average accuracy of 90%. Thus we suggest high-content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers

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

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

Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization.

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair