Quantitative Analysis of T Cell Receptor Complex Interaction Sites Using Genetically Encoded Photo-Cross-Linkers

The T cell receptor (TCR)-cluster of differentiation 3 (CD3) signaling complex plays an important role in initiation of adaptive immune responses, but weak interactions have obstructed delineation of the individual TCR-CD3 subunit interactions during T cell signaling. Here, we demonstrate that unnatural amino acids (UAA) can be used to photo-cross-link subunits of TCR-CD3 on the cell surface. Incorporating UAA in mammalian cells is usually a low efficiency process. In addition, TCR-CD3 is composed of eight subunits and both TCR and CD3 chains are required for expression on the cell surface. Photo-cross-linking of UAAs for studying protein complexes such as TCR-CD3 is challenging due to the difficulty of transfecting and expressing multisubunit protein complexes in cells combined with the low efficiency of UAA incorporation. Here, we demonstrate that by systematic optimization, we can incorporate UAA in TCR-CD3 with high efficiency. Accordingly, the incorporated UAA can be used for site-specific photo-cross-linking experiments to pinpoint protein interaction sites, as well as to confirm interaction sites identified by X-ray crystallography. We systemically compared two different photo-cross-linkersp-azido-phenylalanine (pAzpa) and H-p-Bz-Phe-OH (pBpa)for their ability to map protein subunit interactions in the 2B4 TCR. pAzpa was found to have higher cross-linking efficiency, indicating that optimization of the selection of the most optimal cross-linker is important for correct identification of protein–protein interactions. This method is therefore suitable for studying interaction sites of large, dynamic heteromeric protein complexes associated with various cellular membrane systems

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

<div><p>Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8⁺ and CD4⁺ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression.</p> </div

Peptide-dependent activation of human T cell subsets engineered with specific TCRs.

<p>(<b>A</b>) Upregulation of CD25 expression from T cells expressing gp100-TCR or SL9-TCR in a dose-dependent manner. CD8⁺ or CD4⁺ T cells were activated and transduced with distinct TCRs. Sorted pure T_TCR-gp100 or T_TCR-SL9 cells were cultured with either gp100 or SL9 presented through T2 cells. The frequency of CD25 expressing cells was determined by FACS analysis. The data are representative from three different experiments from multiple donors. (<b>B</b>) Concentration-dependent induction of IL-2 and IFN-γ secretion from gp100-TCR or SL9-TCR-transduced T cells. Cytokines were measured in the supernatant taken from (A) using CBA assay. The data represent the mean±SD from three different donors. (C) Antigen-specific proliferation of gp100- or SL9- TCR engineered T cells as in (A) was monitored. The data are representative from three different experiments from multiple donors.</p

Antigen specific activation and proliferation of naïve human T cells transduced with engineered-TCRs.

<p>(<b>A</b>) Resting CD4⁺ and CD8⁺ CD45RO⁻CD25⁻ T (T_N) cells were cultured in IL-7-containing medium for 7 days followed by ectopic expression of SL9- or gp100-TCR. 7 days later, cells were then activated with either SL9 or gp100 presented by T2 cells. (<b>B</b>) Upregulation of CD25 from T_N cells expressing SL9-TCR upon activation. Cells collected as indicated in (A) were subject to CD25 staining followed by FACS analysis. The data are representative from three different experiments from multiple donors. (<b>C, D</b>) CD8⁺ and CD4⁺ T_N cells expressing SL9-TCR proliferate and expand via activation through SL9 peptide presented by T2 cells. SL9-TCR-transduced cells as indicated in (A) were labeled with CellTrace Violet and the proliferation was monitored at day 4 post activation and the expansion of T cells was determined at day 4 and day 8 post activation. (<b>E</b>) Induction of CD25 and cytotoxicity from gp100-TCR-transduced CD8⁺ T_N cells at 1st TCR-stimulation and during reactivation. gp100-TCR-overexpressing CD8⁺ T_N cells were co-cultured with T2 cells in the presence of gp100. The frequency of CD25⁺ cells and the cytotoxicity were determined 1 day after 1st activation. Activated cells were kept in culture for an extra 2 weeks and were then restimulated again with gp100 presenting T2 cells (2^nd activation). CD25 levels and cytotoxicity were determined 1 day after reactivation. The data are representative from three different experiments from multiple donors.</p

Antigen-specific cytotoxicity of T cells expressing engineered-TCRs.

<p>CD8⁺ and CD4⁺ T cells expressing gp100-TCR or SL9-TCR are cytotoxic in a peptide concentration-dependent fashion. Activated CD8⁺ (<b>A</b>) or CD4⁺ (<b>B</b>) T cells ectopically expressing gp100-TCR or SL9-TCR were sorted based on GFP or RFP expression. Sorted cells were cultured with T2 cells plus different peptides at the indicated concentrations. The portion of viable T2 cells was analyzed by FACS after activation, as shown on the Y-axis, whereas the percentage of T cells is shown on the X-axis (<b>A, B</b>). MART-1 (1000 nM) was used as a control. The data are representative from three different experiments from multiple donors. (<b>C</b>) The percent killing of T2 cells (% Cytotoxicity) by CD8⁺ T cells. We calculated % Cytotoxicity by comparing percentage of T2 cell death from peptide-cultured group to those from no peptide control group. The data represent the mean±SD from three different donors. (<b>D</b>) CD8_TCR-SL9 effector cells kill HIV-infected CD4⁺ T cells. HLA-A*0201⁺ CD4⁺ T cells were infected with VSVG.HIV, and were co-cultured with CD8_TCR-SL9 at different CD8⁺: CD4⁺ cell ratios as indicated. The upregulation of CD25 on CD8⁺ T cells (Effector) as well as the % cell death of CD4⁺ T cells (Target), normalized to a CD4-HIV T cell-only group, was determined by FACS analysis. The data are representative from three different experiments from multiple donors.</p

MOESM1 of Impact of aging on host immune response and survival in melanoma: an analysis of 3 patient cohorts

Additional file 1: Table S1. A Association of SEER clinicopathologic features with age. B Association of IMCG clinicopathologic features with age and tumor infiltrating lymphocytes. C Association of TCGA clinicopathologic features with age and lymphocyte score

MOESM2 of Impact of aging on host immune response and survival in melanoma: an analysis of 3 patient cohorts

Additional file 2: Table S2. Clinicopathologic features of 84 IMCG and 43 TCGA primary melanomas in gene expression analysis

MOESM3 of Impact of aging on host immune response and survival in melanoma: an analysis of 3 patient cohorts

Additional file 3: Table S3. A Significant gene expression differences with aging in the IMCG primary melanoma cohort (n = 84). B Significant gene expression differences with aging in the TCGA primary melanoma cohort (n = 43)

MOESM4 of Impact of aging on host immune response and survival in melanoma: an analysis of 3 patient cohorts

Additional file 4: Table S4. Immunoregulatory gene expression of 84 IMCG primary melanomas

MOESM8 of Baseline antibody profiles predict toxicity in melanoma patients treated with immune checkpoint inhibitors

Additional file 8: Table S7. Functions of protein targets of treatment termination-associated antibodies. Functional analysis of protein targets for top 15 DE toxicity-associated antibodies for each of the anti-CTLA-4, anti-PD-1, and combination treatment groups. Associations of each antibody target with immune toxicity are given, based on literature findings