Ex vivo monitoring of antigen-specific CD4+ T cells after recall immunization with tetanus toxoid

Abstract

To monitor antigen-specific CD4+ T cells during a recall immune response to tetanus toxoid (TT), a sequential analysis including ex vivo phenotyping and cytokine flow cytometry, followed by cloning and T-cell-receptor (TCR) spectratyping of cytokine-positive CD4+ T cells, was performed. Grossly, twice as many TT-specific CD4+ T-cell clones, ex vivo derived from the CCR7+/- CD69+ interleukin-2-positive (IL-2+) CD4+ subsets, belonged to the central memory (T(CM); CD62L+ CD27+ CCR7+) compared to the effector memory population (T(EM); CD62L- CD27- CCR7-). After the boost, a predominant expansion of the T(CM) population was observed with more limited variations of the T(EM) population. TCR beta-chain-variable region (BV) spectratyping and sequencing confirmed a large concordance between most frequently expressed BV TCR-CDR3 from ex vivo-sorted CCR7+/- CD69+ IL-2+ CD4+ subsets and BV usage of in vitro-derived TT-specific CD4+ T-cell clones, further demonstrating the highly polyclonal but stable character of the specific recall response to TT. Taken together, ex vivo flow cytometry analysis focused on the CCR7+/- CD69+ IL-2+ CD4+ subsets appears to target the bulk of antigen-specific T cells and to reach an analytical power sufficient to adequately delineate in field trials the profile of the antigen-specific response to vaccine.

FIG. 1.

Ex vivo phenotypic characterization of total CD4⁺ and TT-stimulated IL-2⁺ CD4⁺ T-cell population. (A) Steady-state, unstimulated total PBMC (from donor 4) were stained ex vivo for the expression of CCR7, CD45RO, CD27, CD62L, CD69, and CD4. Four-color flow cytometry was used to delineate naive (CCR7⁺ CD45RO⁻ CD62L⁺ CD4⁺; CD27⁺ CD45RO⁻ CD62L⁺ CD4⁺), T_CM (CCR7⁺ CD45RO⁺ CD62L^int CD4⁺; CD27⁺ CD45RO⁺ CD62L^int CD4⁺), and T_EM (CCR7⁻ CD45RO⁺ CD62L⁻ CD4⁺; CD27⁻ CD45RO⁺ CD62L⁻ CD4⁺) T-cell populations. Values in quadrants represent the percentage of each subpopulation of gated CD4⁺ T cells. There were a total of 10⁵ analyzed events. (B) PBMC (from donor 4) were stimulated for 16 h with TT at 100 μg/ml, stained for IL-2 and cell surface markers, and analyzed as in panel A. Values in the quadrants represent the percentage of each subpopulation of gated IL-2⁺ CD4⁺ T cells. There were a total of 10⁶ analyzed events. (C) PBMC cultured in the absence of antigen (from donor 4) were analyzed as in panel B. Similar experiments were performed in parallel with the three other donors at four different time points with similar results.

FIG. 2.

Sorting and cloning strategy for activated TT-specific CD4⁺ T cells. Freshly prepared PBMC were first stimulated for 16 h with TT at 100 μg/ml; stained for IFN-γ, IL-2, and cell surface markers CD4, CD69, and CCR7; and then sorted by cytofluorimetry and cloned. (A) Stimulated cells were first gated on the IFN-γ⁺ CD4⁺ or IL-2⁺ CD4⁺ subpopulations and then sorted according to CD69 expression to isolate CD69^+/− IFN-γ⁺ CD4⁺ T cells or CD69^+/− IL-2⁺ CD4⁺ T cells for cloning. (B) Stimulated cells were first gated on the IL-2⁺ CD4⁺ subpopulation and then sorted according to CCR7 and CD69 expression to identify the CCR7^+/− CD69^+/− IL-2⁺ CD4⁺ T-cell subpopulations for cloning. A similar approach was performed for the IFN-γ⁺ CD4⁺ subpopulation. For abundant cell populations, an average number of 400 cells were cloned, and at least 100 cells were evaluated for smaller populations.

FIG. 3.

Kinetics of CD4⁺ T-cell proliferation and IL-2 secretion. (A) Proliferation of TT-specific T cells. Pre- and post-TT boost PBMC were stimulated for 4 days with TT at 50 μg/ml. The data (means of triplicates) are expressed as stimulation indexes. (B) IL-2 secretion by TT-specific T cells. PBMC were stimulated for 16 h with TT at 100 μg/ml. Supernatants were collected, and IL-2 was detected by a functional IL-2-dependent CTL-L cell line assay. The data represent means of triplicates. (C) T_CM versus T_EM IL-2⁺ CD4⁺ T cells before and after TT immunization. PBMC were stimulated as in panel B; stained for IL-2, CCR7, CD4, CD45RO, and CD69; and analyzed as in Fig. 1. The data represent the percentage of CCR7^+/− CD69⁺ IL-2⁺ CD4⁺ T cells from gated IL-2⁺ CD4⁺ T cells. D0, day 0; D7, day 7, etc.

FIG. 4.

TCR BV usage of a TT-specific repertoire is broad and stable over time. (A) Spectratype analysis of ex vivo-sorted CD4⁺ T cells. PBMC sampled before (day 0 [D0]) or after (days 14 and 35 [D14 and D35]) a TT boost were stimulated for 16 h with TT at 100 μg/ml and stained for IL-2, CCR7, CD69, and CD4. CCR7^+/− CD69⁺ IL-2⁺ CD4⁺ T cells were sorted, and TCR BV spectratypes were performed by RT-PCR (21 BV families [top grids]; detected BVs are indicated in boldface, and nondetected BVs are in italics). In the gel, two BV PCR products were loaded per slot. The virtual horizontal bar delineates short and long sizes PCR products in the upper part (U) and lower part (L), respectively. This is a representative experiment with a CCR7⁺ CD69⁺ IL-2⁺ CD4⁺ T-cell population from donor 4; similar results were obtained with the three other donors. Filled dots designate TCRs present at all time points, open squares indicate TCRs detected after TT boost only. (B) For comparison, TCR BV spectratype analysis of a polyclonal, non-antigen-specific CD4⁺ T-cell population with the expected Gaussian distribution for BV3 and BV13. (C) In vitro-generated TT-specific T-cell clones from ex vivo-sorted CCR7⁺ CD69⁺ IL-2⁺ CD4⁺ T cells are found within the original population (from donor 2). Specific T-cell clone TCR BV usage was determined by RT-PCR and compared to the original sorted CD4⁺ T-cell population for the corresponding TCR. Analysis is limited to a representative example with TT-specific T-cell clones restricted to TCR BV9. “Total sorted cells,” spectratype of the whole population of originally sorted T cells at days 0, 7, 14, and 280. (D) Similar analysis as in panel C for donor 1, with TT-specific T-cell clones restricted to TCR BV8, BV5.1, and BV22 and corresponding total sorted cells (ex vivo, left lanes).

FIG. 5.

Longitudinal analysis of TCR BV spectratype usage of CCR7^+/− CD69⁺ IL-2⁺ CD4⁺ sorted T cells and of TT-specific derived T-cell clones. (A) Pre- and post-TT boost PMBC were treated as described in Fig. 4. TCR BV spectratypes were performed on sorted CCR7^+/− CD69⁺ IL-2⁺ CD4⁺ T cells. The diagram indicates the presence (filled rectangles) of TCR-CDR3 at different time points before and after the TT boost for the 21BV families. (B) Number of TT-specific T-cell clones and TCR BV expression analysis. TT-specific T-cell clones were generated in vitro from sorted CCR7^+/− CD69⁺ IL-2⁺ CD4⁺ T cells, and their BV expression was determined by RT-PCR. Panels A and B are examples from donor 3; similar data were obtained with the other donors. (C) TCR diversity in T_CM CCR7⁺ and T_EM CCR7⁻ populations. CD69⁺ IL-2⁺ CD4⁺ T_CM and T_EM populations were ex vivo sorted and analyzed as described in Fig. 4. Diversity is expressed as the total number of TCR-CDR3 obtained in each population. *, sample not available and not tested at this time point. D0, day 0; D7, day 7, etc.