Costimulation of naive human CD4 T cells through intercellular adhesion molecule-1 promotes differentiation to a memory phenotype that is not strictly the result of multiple rounds of cell division

Abstract

The process by which naive T cells become activated, differentiate into effector cells and ultimately generate long-lived memory cells is dependent upon a number of factors, including the costimulatory signals received by the T cell. To best understand the multiple events involved, it is important to understand the potential contributions by individual signalling proteins using both in vitro and in vivo studies. Here, the potential for costimulation through intercellular adhesion molecule-1 (ICAM-1; CD54), resident on the surface of naive human T cells, to influence differentiation was investigated. Costimulation of naive T cells through ICAM-1 resulted in expansive cell division, high interleukin-2 production, and protection from apoptosis. Prolonged culture led to outgrowth of a subpopulation of cells with a highly differentiated CD45RA- CD11a(hi) CD27- phenotype. In this respect, costimulation through ICAM-1 was similar to costimulation through CD28 and different from costimulation through leucocyte function-associated antigen-1. The CD45RA- CD11a(hi) CD27- cells responded to suboptimal stimulation through the T-cell receptor alone with a more robust proliferative response compared with naive cells from the same subject. These cells also secreted higher levels of T helper type 1 cytokines in response to lower levels of stimulation than their naive counterparts. The surface phenotype and more sensitive response characteristics suggest the creation of a memory T-cell subpopulation as a result of costimulation through ICAM-1. Finally, generation of this memory population was the result of specific costimulatory signals, and not merely because of a high number of cell divisions. These data reveal a new role for resident ICAM-1 to influence the differentiation of naive T cells.

Figure 1

Sustained and efficient activation of naive CD4⁺ T cells following costimulation through ICAM-1. (a) Purified human naïve CD4⁺ T cells (2 × 10⁵/well) were loaded with CFSE and stimulated with anti-CD3 in combination with anti-ICAM-1, anti-LFA-1, or anti-CD28. Cell division was assessed at day 7 and the data (mean ± SEM) are presented as the percentage of cells within each generation relative to the entire population. Representative of >10 experiments. (b) Naive CD4⁺ T cells (2 × 10⁵/well) were stimulated with anti-CD3 in combination with anti-ICAM-1, anti-LFA-1, or anti-CD28. Cells were harvested on the days indicated and the total number of viable cells (mean ± SEM) was measured by flow cytometry. Representative of four experiments. (c) To directly measure apoptosis, naive CD4⁺ T cells were labelled with CFSE and stimulated for 7 days with anti-CD3 in combination with anti-ICAM-1, anti-LFA-1, or anti-CD28. The percentage of apoptotic cells (AnnexinV⁺ and PI^–) within each generation is displayed. Representative of eight experiments.

Figure 2

Costimulation of naive CD4⁺ T cells through ICAM-1 induced high IL-2 production. Triplicate cultures of naive CD4⁺ T cells were left not stimulated or stimulated with anti-CD3 alone or in combination with anti-ICAM-1, anti-LFA-1, or anti-CD28. Cell culture supernatants were harvested on days 1, 2, 3, 5 and 7, and IL-2 secretion was measured by ELISA. Values for all stimuli were plotted but some were too low to be visible. Data are presented as the mean of triplicate samples. Representative of four experiments.

Figure 3

T cells with a differentiated phenotype were generated from naive CD4⁺ cells following costimulation through ICAM-1. Naive CD4⁺ T cells were left not stimulated or stimulated for 14 days with anti-CD3 in combination with anti-ICAM-1, anti-LFA-1, or anti-CD28. Purity of the starting naive population (CD45RA⁺ CD11a^dim CD27⁺) was confirmed by staining non-stimulated cells on day 1 (upper left panel, Ø). Results shown for stimulated cells were gated on CD45RA^– cells before analysis (remaining three panels). The percentage of cells matching a stringent memory phenotype (CD4⁺ CD45RA^– CD11a^hi CD27^–) in response to costimulation is depicted in the lower right quadrant of each appropriate panel. Representative of eight experiments.

Figure 4

Memory-like cells generated following costimulation through ICAM-1 were pure and capable of responding to stimulation. (a) Cells costimulated through ICAM-1 were isolated by negative magnetic sorting using antibodies against CD45RA and CD27, and this routinely yielded a highly purified population (98%) of cells with the CD45RA^– CD11a^hi CD27^– phenotype. (b) Purified memory cells (CD45RA^– CD11a^hi CD27^–) and freshly isolated naive cells (CD45RA⁺ CD11a^lo CD27⁺) from the same subject were stimulated for 8 hr with anti-CD3 alone (1 μg/ml) and assessed for expression of the activation marker CD69 by flow cytometry. Representative of four experiments.

Figure 5

Memory-like cells generated following costimulation through ICAM-1 were more responsive than corresponding naive cells. (a) As used in Fig. 4, purified, ICAM-1-induced memory-like cells and freshly isolated naive cells from the same subject were stimulated with increasing concentrations (0·005–5·0 μg/ml) of anti-CD3 alone for 72 hr. Proliferation was measured by [³H]thymidine incorporation and data are presented as the mean c.p.m. ± SEM. Representative of four experiments. Purified memory cells and freshly isolated naive cells were tested for their capacity to produce (b) IL-2 following stimulation with PHA for 6 and 24 hr or (c) IFN-γ in response to stimulation through the TCR (antibody at 1 μg/ml) or PHA at 24 hr. Cytokine concentrations are listed as the mean ± SEM. Representative of three experiments.

Figure 6

Generation of CD45RA^– CD11a^hi CD27^– cells was dependent on costimulatory signalling rather than number of cell divisions. Naive CD4⁺ T cells were costimulated for 10 days with anti-CD3 plus either (a) anti-ICAM-1 or (b) anti-LFA-1 as a costimulatory signal, washed and rested for 2 days, purified over Ficoll to remove dead cells and costimulated a second time with the same costimulatory regimen. The generation of CD45RA^– CD11a^hi CD27^– effector cells was assessed by flow cytometry 10 days following the second stimulation and the percentages are shown in the lower right quadrant. (c) Parallel cultures were labelled with CFSE before the first stimulation and cell division was assessed at the conclusion of both the first costimulation (ICAM-1, open bars and LFA-1 closed bars) and the second costimulation (ICAM-1, shaded bars, and LFA-1, hatched bars). (d) Naive CD4⁺ T cells were costimulated first for 10 days with LFA-1 as the costimulatory signal, rested and re-purified as described above, and costimulated a second time with ICAM-1 as the costimulatory signal. The generation of CD45RA^– CD11a^hi CD27^– effector cells was assessed by flow cytometry 10 days following the second stimulation and the percentage is noted. Representative of three experiments.