K^dM2₈₂ T cells dominate bulk cytotoxicity but D^bM₁₈₇ T cells have superior individual cytotoxicity.

<p>(<b>a)</b> Bulk cytotoxicity of D^bM₁₈₇ and K^dM2₈₂ T cells <i>in vivo</i>. The epitope peptide-loaded and fluorochrome-labeled targets, as well as OVA₂₅₇ peptide-loaded and fluorochrome-labeled controls, were co-transferred into RSV-infected mice at 7dpi, and recovered 3 hours later. The recovery ratio were assessed with flow cytometry and compared with recovery ratio from naïve recipients to calculate epitope-specific lysis. Data represent 5 independent experiments (n = 5/group/experiment). <b>(b)</b> Cytotoxicity of individual D^bM₁₈₇ and K^dM2₈₂ T cells. Ratio of specific lysis of donor targets in (a) was divided by frequency of endogenous D^bM₁₈₇ and K^dM2₈₂ T cells respectively to quantitatively express arbitrary “Killing Unit” of individual cells. Data represent 5 independent experiments (n = 5/group/experiment). All data are shown as mean with independent data point and compared by Student t-test. Each symbol represents one mouse.</p

The D^bM₁₈₇ T cells efficiently control viral replication.

<p><b>(a)</b> Adoptive transfer of pMHC-specific donor cells increases precursor of CD8 T effector cells during early infection. Live D^bM₁₈₇, K^dM2₈₂ and bulk (with neither specificity) CD8 T cells from spleen lymphocytes of RSV-infected mice at 7dpi were sorted with FACS, and transferred into naive recipients respectively. The recipients were then challenged with RSV next day, and were evaluated for the donor D^bM₁₈₇ and K^dM2₈₂ T cell frequencies in the right lung at 4 dpi by flow cytometry. <b>(b)</b> Viral activity in RSV challenged recipients. Left lungs of the RSV-challenged recipients were assessed for virus replication. The virus titers are expressed as log₁₀ PFU/gram of lung tissue. Data are shown as mean with independent data point and compared by Student’s <i>t</i>-test. Data represent 3 independent experiments (n = 4 or 5/group/experiment). Each symbol represents one mouse.</p

The D^bM₁₈₇ T cells express high avidity TCR and signaling pathways promoting cytotoxic function.

<p><b>(a)</b> The TCR avidity was assessed by dissociation of D^bM₁₈₇ and K^dM2₈₂ from CD8 T cells. CD8 T cells were labeled with pMHCs and assessed cell-bound median fluorescence intensity (MFI) at indicated time point by flow cytometry. The MFI at 0 min was defined as the maximum measurement (100%). Data were analyzed with one-phase exponential decay using nonlinear regression, and shown at mean ± SEM of three independent experiments (n = 5/group/experiment). <b>(b)</b> Transcriptional expression of genes that are up-regulated after RSV infection and associated with conventional signaling pathways. The D^bM₁₈₇, K^dM2_82, and bulk CD8 T cells were sorted from spleen lymphocytes by FACS at 7 dpi. The mRNAs were isolated, amplified and labeled, then hybridized onto Illumina Mouse Chips. The quantitative gene expression were analyzed and normalized. Genes with Log₂ Fold Change (FC) > 1.3, p < 0.05 and FDR < 0.25 (listed on left side of the chat) and associated signaling pathways were shown (Pathways 1: Altered T Cell and B Cell Signaling in Rheumatoid Arthritis; 2: T Helper Cell Differentiation; 3: Dendritic Cell Maturation; 4: Type I Diabetes Mellitus Signaling; 5: Roe of NFAT in Regulation of the Immune Response; 6: Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses; 7: IL-10 Signaling; 8: Role of CHK Proteins in Cell Cycle Checkpoint Control; 9: TREM1 Signaling; 10: Communication between Innate and Adaptive Immune Cells; 11: CD40 Signaling; 12: Production of Nitric Oxide and Reactive Oxygen Species in Macrophages; 13: Cell Cycle Control of Chromosomal Replication; 14: Acute Phase Response Signaling; 15: CD28 Signaling in T Helper Cells; 16: PKC Signaling in T Lymphocytes; 17: IL-12 Signaling and Production in Macrophages.). Data were pooled from 10 or 11 individual mice in each group.</p

Activated K^dM2₈₂ T cells up-regulate expression of inhibitory receptors.

<p><b>(a)</b> Transcriptional expression of genes encoding inhibitory receptors with FC > ±1.3, p < 0.05 and FDR < 0.25 were listed. Data were pooled from 10 or 11 individual mice in each group. (<b>b</b>) Post-transcriptional expression of inhibitory receptors at 7 dpi was assessed by flow cytometry. The frequencies are shown as mean with independent data point and compared by Student’s <i>t</i>-test. Data represent 5 independent experiments (n = 5/group/experiment). Each symbol represents one mouse.</p

Activated K^dM2₈₂ T cells are apoptotic.

<p><b>(a)</b> Transcriptional expression of genes encoding pro- and anti-apoptosis molecules with FC > ±1.3, p < 0.05 and FDR < 0.25 were listed. Data were pooled from 10 or 11 individual mice in each group. (<b>b</b>) Apoptotic cells were identified by Annexin V staining, and post-transcriptional expression of Bcl-2 was identified by monoclonal antibody with flow cytometry at 7 dpi. The frequencies are shown as mean with independent data point and compared by Student’s <i>t</i>-test. Data represent 3 or 4 independent experiments (n = 5/group/experiment). Each symbol represents one mouse.</p

Additional file 3: Table S2. of Human newborn bacille Calmette–Guérin vaccination and risk of tuberculosis disease: a case-control study

Genes differentially expressed between cluster 1 and cluster 2 infants from the primary cohort. Gene expression was calculated by subtracting expression in PBMC incubated with medium alone from expression in PBMC incubated with BCG. (XLSX 62 kb

CD160 and PD-1 Co-Expression on HIV-Specific CD8 T Cells Defines a Subset with Advanced Dysfunction

<div><p>Chronic viral infections lead to persistent CD8 T cell activation and functional exhaustion. Expression of programmed cell death-1 (PD-1) has been associated to CD8 T cell dysfunction in HIV infection. Herein we report that another negative regulator of T cell activation, CD160, was also upregulated on HIV-specific CD8 T lymphocytes mostly during the chronic phase of infection. CD8 T cells that expressed CD160 or PD-1 were still functional whereas co-expression of CD160 and PD-1 on CD8 T cells defined a novel subset with all the characteristics of functionally exhausted T cells. Blocking the interaction of CD160 with HVEM, its natural ligand, increased HIV-specific CD8 T cell proliferation and cytokine production. Transcriptional profiling showed that CD160⁻PD-1⁺CD8 T cells encompassed a subset of CD8⁺ T cells with activated transcriptional programs, while CD160⁺PD-1⁺ T cells encompassed primarily CD8⁺ T cells with an exhausted phenotype. The transcriptional profile of CD160⁺PD-1⁺ T cells showed the downregulation of the NFκB transcriptional node and the upregulation of several inhibitors of T cell survival and function. Overall, we show that CD160 and PD-1 expressing subsets allow differentiating between activated and exhausted CD8 T cells further reinforcing the notion that restoration of function will require multipronged approaches that target several negative regulators.</p> </div

Summary of clinical characteristics.

<p>Summary of clinical characteristics.</p

Blocking the interaction between CD160 and HVEM enhances CMV and HIV-specific CD8 T cell proliferation and cytokine production.

<p>HIV-infected individuals(n = 11) were stimulated with HLA-restricted CMV and HIV peptides in the presence of blocking antibodies to HVEM and/or PD-L1. Dying cells were eliminated with an amine-reactive viability dye and PBMCs were stained at day 6 with HLA class I matched-tetramers and mAbs to CD3 and CD8. (A) Representative flow cytometry plots of an HIV-infected patient stimulated for 6 days with a CMV and HIV peptide in the presence of isotype, αPD-L1 and/or αHVEM blocking antibodies. Scatter plots represent the median fold increase in (B) CMV and (C) HIV-specific proliferation (Tetramer⁺/CFSE_low) compared to the isotype control. Each dot represents a CMV or HIV tetramer-specific response. <i>P</i>-values were determined by the Wilcoxon matched pairs test.</p

The frequency of HIV-specific CD8 T cells with a CD160⁺PD-1⁺ phenotype increases with disease progression.

<p>(A) Representative flow cytometry plots of CMV and HIV-specific CD8 T cells (A*02 CMV and A*03 Gag) detected during AHI (<3 months) and CHI (>6 months) within the same individual. Frequencies of 4 distinct CD8 T cell subsets expressing CD160 and/or PD-1 were measured at both time points. Figures represent the frequency of CD160 and/or PD-1 expressing subsets over time for (B) HIV and (C) CMV/EBV-specific responses in 4 HIV-infected subjects. <i>P</i>-values were determined by the Wilcoxon matched pairs test.</p