Profile of a Serial Killer: Cellular and Molecular Approaches to Study Individual Cytotoxic T-Cells following Therapeutic Vaccination

T-cell vaccination may prevent or treat cancer and infectious diseases, but further progress is required to increase clinical efficacy. Step-by-step improvements of T-cell vaccination in phase I/II clinical studies combined with very detailed analysis of T-cell responses at the single cell level are the strategy of choice for the identification of the most promising vaccine candidates for testing in subsequent large-scale phase III clinical trials. Major aims are to fully identify the most efficient T-cells in anticancer therapy, to characterize their TCRs, and to pinpoint the mechanisms of T-cell recruitment and function in well-defined clinical situations. Here we discuss novel strategies for the assessment of human T-cell responses, revealing in part unprecedented insight into T-cell biology and novel structural principles that govern TCR-pMHC recognition. Together, the described approaches advance our knowledge of T-cell mediated-protection from human diseases

Assessing ageing of individual T lymphocytes: Mission impossible?

Effector T lymphocytes are the progeny of a limited number of antigen- specific precursor cells and it has been estimated that clonotypic human T cells may expand million fold on their way reaching high cell numbers that are sufficient for immune protection. Moreover, memory T cell responses are characterized by repetitive expansion of antigen-specific T cell clonotypes, and limitations in the proliferative capacity could lead to immune senescence. Because telomeres progressively shorten as a function of cell division, telomere length is a powerful indicator of the replicative in vivo history of human T lymphocytes. In this review, we summarize observations made over the last decade on telomere length dynamics of well-defined T cell populations derived from healthy donors and patients with infectious disease or cancer. We focus on T cell differentiation, T cell ageing, and natural and vaccine induced immune responses. We also discuss the scientific evidence for in vivo replicative senescence of antigen-specific T cells, and evaluate the available methods for measuring telomere lengths and telomerase activity, and their potential and limitations to increase our understanding of T cell physiology. (C) 2007 Elsevier Ireland Ltd. All rights reserved

<i>Ex</i><i>vivo</i> gene-expression profiling of single EBV antigen-specific CD8 T cells following cell differentiation from patient LAU 1013.

<p><b>A</b>. Schedule of the treatment regimen for patient LAU 1013, who received two rounds of TLD (depicted as grey boxes). The grey arrows indicate leukapheresis (Leuka I and Leuka II) and reinfusion of PBMCs, respectively. Blood samples were taken at day 45 (post-Leuka I) and at day 15/22 (post-Leuka II) after reinfusion. <b>B</b>. Phenotype of EBV BMFL1-specific CD8 T cells (left panel) and total CD8 T cells (right panel) showing the proportions of early-differentiated (EM28^pos; CCR7^negCD45RA^negCD28^pos) and late-differentiated (comprising of EM28^neg; CCR7^negCD45RA^negCD28^neg and EMRA; CCR7^negCD45RA^posCD28^neg) subsets in PBMCs taken from patient LAU 1013 at Leuka I (n = 2) and Leuka II (n = 3) time points. Error bars (mean +/- SD) represent independent experimental replicates. <b>C</b>. Direct <i>ex </i><i>vivo</i> cumulative expression of memory/homing-associated genes (<i>CD62L/SELL</i>, <i>CCR5</i>, <i>IL7R</i>, <i>CD27</i> and <i>EOMES</i>) and effector-related genes (<i>PRF1</i>, <i>KLRD1/CD94</i>, <i>IFNG</i> and <i>GZMB</i>). Single EBV-specific CD8 T cells were sorted from early-differentiated EM28^pos and late-differentiated EMRA at Leuka I (n = 266) and Leuka II (n = 162) time-points and processed for global cDNA amplification as described in Materials and Methods. <i>P</i>-values were performed by one-way ANOVA test; ns, not significant. </p

Direct <i>ex</i><i>vivo</i> analysis of HLA-A*0201/BMFL1 specific CD8 T cells from healthy donors and melanoma patients.

<p><b>A</b>. Schematic representation of non-myeloablating lympho-depleting treatment followed by ACT of PBMCs for melanoma patients. Blood samples were analyzed for all patients at the leukapheresis time-point (Leuka) before TLD chemotherapy and post-ACT (time-point T1), which corresponded to day 32 (patient LAU 1144), day 45 (LAU 1013), or day 60 (LAU 618, LAU 936, and LAU 672). <b>B</b>. Left panel; total counts of CD8 T cells from five melanoma patients at Leuka and post-ACT. Right panel; phenotype of total CD8 T cells showing proportions of early-differentiated (EM28^pos; CCR7^negCD45RA^negCD28^pos) and late-differentiated (comprising of EM28^neg; CCR7^negCD45RA^negCD28^neg and EMRA; CCR7^negCD45RA^posCD28^neg) subsets from four healthy donors (HDs) and from five melanoma patients at Leuka and post-ACT. <b>C</b>. Left panel; total counts of EBV-specific CD8 T cells in peripheral blood from five melanoma patients at Leuka and post-ACT time points. Right panel; phenotype of EBV-specific CD8 T cells showing proportions of early-differentiated (EM28^pos; CCR7^negCD45RA^negCD28^pos) and late-differentiated (comprising of EM28^neg; CCR7^negCD45RA^negCD28^neg and EMRA; CCR7^negCD45RA^posCD28^neg) subsets from four healthy donors and from five melanoma patients at Leuka and post-ACT. <b>D</b>. EBV-specific antibody levels measured in plasma samples from five melanoma patients at various time-points before and after lympho-depleting treatment. Day 0 on the x-axis represents the day of ACT. Results are represented as arbitrary units on a relative index scale. Plasma level evolution is shown separately for each anti-EBV antibody (anti-VCA IgM and IgG, anti-EBNA IgG and anti-EA IgG), and the grey bar represents the cut-off between negative and positive status for each marker.</p

Co-expression of memory- and effector-related genes by individual EBV antigen-specific CD8 T cells following two rounds of TLD and immune reconstitution.

<p><i>Ex </i><i>vivo</i> gene expression polyfunctionality was determined as a measure of co-expression of the five memory/homing-associated and the four effector-associated gene transcripts in single cell samples from the EM28^pos subset at the indicated time-points (n = 332). Colors of the pie arcs depict the co-expression of individual memory or effector genes, whereas the color in the pie depicts the number of co-expressed memory- or effector-associated genes, as determined by SPICE 5.2. Increased polyfunctional gene co-expression (from 0 to 4 or 5) is shown as progressive grey gradients (from white to black). <i>P</i>-values of the permutation test are shown. Of note, at D45 (post-Leuka I) and D15/22 (post-Leuka II), EBV antigen-specific T cells were not sufficiently frequent to allow separation into defined subsets, thus data represent individual virus-specific T cells sorted from the total BMFL1/tetramer-specific population. </p

Co-expression of memory-related genes by <i>ex</i><i>vivo</i> sorted dominant EBV antigen-specific TCR clonotypes before and following transient lympho-depletion.

<p><b>A</b>. Cumulative gene expression analysis of the dominant BV14c1 clonotype from patient LAU 618 at Leuka and post-Leuka (D60) time-points. Expression of memory-associated genes was determined on 5-cell cDNA samples sorted from early-differentiated EM28^pos EBV-specific CD8 T cells (n = 28). <b>B</b>. Quantification of co-dominant TCR clonotypes from patient LAU 1013 at Leuka I (n = 130) and Leuka II (n = 107) time-points, within the early-differentiated EM28^pos subset, shown in frequencies (in %). <b>C</b>. Cumulative memory/homing gene expression profile of single cell samples for each of the four dominant clonotypes at Leuka I time-point. Individual EBV-specific CD8 T cell clonotypes from patient LAU 1013 were sorted from the early-differentiated EM28^pos subset (n = 94). <i>P</i>-values were performed by one-way ANOVA test. <b>D</b>. Gene co-expression polyfunctionality was determined on single cell samples representing individual TCR clonotypes from EM28^pos EBV-specific CD8 T cell subset at Leuka I (n = 94) and Leuka II (n = 83) from patient LAU 1013. Colors of the pie arcs depict the co-expression of individual memory genes, whereas the color in the pie depicts the number of co-expressed memory-associated genes, as determined by SPICE 5.2. Increased polyfunctional gene co-expression (from 0 to 5) is shown as progressive grey gradients (from white to black). <i>P</i>-values of the permutation test are shown.</p

<i>In</i><i>vivo</i> selection of public TCR clonotypes in healthy donors and melanoma patients.

<p><b>A</b>. Compilation of the 14 public amino acid TCR beta-domain sequences detected among three healthy individuals and five melanoma patients. Each TCR beta-chain clonotype is described by the TRBV segment, CDR3-beta sequence, TRBJ segment, number of nucleotide sequence variants identified for each amino acid sequence, as well as the proportion of healthy donors and patients from which each sequence was identified. <b>B</b>. Distribution of public sequences within the overall EBV-specific CD8 T cells in healthy donors and patients according to their relative frequency; dominant clonotypes (with frequency >5%) are represented in black, low dominant (1-5%) in grey and unique sequences in white. </p