T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice - PubMed (original) (raw)

T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice

Sarah J Jarmin et al. J Clin Invest. 2008 Mar.

Abstract

The establishment of T cell-mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110delta contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110delta displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110delta activity. These observations suggest that pharmacological targeting of p110delta activity is a viable strategy for the therapy of T cell-mediated pathology.

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Figures

Figure 1

Figure 1. PI3K P110δ does not contribute to constitutive T cell migration.

(A) HY-specific H2-Ab–restricted WT and P110δD910A T cells (5 × 105/well) were seeded onto untreated syngeneic female-derived EC monolayers grown on 3-μm-pore Transwells, and T cell migration was monitored as described in Methods. Results are expressed as a percentage of migrated T cells at the given time points and reported as the average of 3 experiments of identical design. Error bars are shown. (B) HY-specific H2-Ab–restricted WT and P110δD910A T cells (5 × 105/well) were seeded onto ICAM-1–coated (2 μg/ml) 35-mm dishes, and their migration was observed for 25–30 minutes by time-lapse microscopy. A representative example of a series of 3 independent experiments with similar design is shown. Mean migration speed ± SEM is shown. (C and D) PKH26-labeled HY-specific H2-Ab–restricted CD4+ WT and P110δD910A T cells were injected i.v. into syngeneic female mice. T cell localization in the indicated tissues was assessed 24 hours later by wide-field fluorescence microscopy. To minimize the effect of arbitrary choice of field, tissue infiltration was quantified by randomly selecting ten ×10-magnified fields from tissue samples from at least 3 animals and assessing the number of fluorescent cells in each field. Each panel shows a representative tissue image. The mean T cell infiltration ± SD observed in samples from at least 3 animals is shown in D.

Figure 2

Figure 2. PI3K p110δ is not required for chemokine-induced T cell migration.

(A and B) HY-specific H2-Ab–restricted WT and P110δD910A CD4+ T cells (5 × 105/well) were exposed to CXCL10 (300 ng/ml; filled symbols, A and D), CXCL12 (50 ng/ml; filled symbols, B and E), or CCL5 (100 ng/ml; filled symbols, C and F) through 5-μm-pore Transwells. In the experiments analyzing migratory responses to CXCL12, T cells were stimulated with plastic-bound anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml) for 72 hours to induce CXCR4 expression. Spontaneous migration in chemotaxis medium (RPMI 0.5% FCS) alone was also measured (open symbols). The number of migrated T cells was monitored at the indicated time points. Results are expressed as the percentage of input T lymphocytes that had migrated through the filters at any given time point and represent the mean of at least 3 independent experiments ± SEM. *P < 0.05 at all time points except 2 hours (B, C, and E) and 24 hours. (G–J) C57BL/6 female mice received an i.p. injection of CXCL10 (1,200 ng). One hour later, PKH26-labeled HY-specific H2-Ab–restricted WT and P110δD910A T cells (5 × 105/well) T cells were injected i.v. After 6 hours, localization of PKH26-labeled T cells in the peritoneal cavity was assessed by flow cytometry. The panels (representative of 1 experiment) show the number of PKH26-labeled T cells in the CD4-gated T cell population. The mean T cell numbers ± SEM observed in samples from at least 3 animals are summarized in K. *P < 0.02.

Figure 3

Figure 3. PI3K P110δ is instrumental to antigen-induced T cell recruitment.

(A–C) HY-specific H2-Ab–restricted WT and p110δD910A CD4+ T cells or HY-specific H2-Kk–restricted CD8+ C6 T cells were seeded onto IFN-γ–treated female- and male-derived syngeneic EC monolayers grown on 3-μm-pore Transwells. T cell migration was assessed as described in Figure 1. Error bars for 3 averaged experiments are shown. *P < 0.05 versus migration through female ECs at all time points except 2 hours. (D and E) Male and female C57BL/6 mice were injected i.p. with 600 U IFN-γ or PBS. Two days later, mice received an i.v. injection of PKH26-labeled HY-specific CD4+ T cells (107/mouse). The presence of labeled cells in the peritoneal membrane (D) and cavity (E) was assessed 24 hours later by wide-field fluorescence microscopy and flow cytometry, respectively. A cluster of 3 PKH26-labeled cells is visible in the membrane of male recipient of WT HY-specific T cells. Due to the presence of an autofluorescent population of non-T cells often detected in FL-2 (also in control mice that received IFN-γ but no T cells; far-left dot plot, “saline”), cells were double stained with an APC-conjugated anti-CD4 antibody following harvesting, and the percentage of PKH26 (FL-2)–labeled T cells gated in the CD4+ T cell population is shown in the dot plots and the graph representing cumulative data from at least 3 animals. The mean ± SEM observed in samples from at least 3 animals is shown on the right. *P < 0.04 versus female mice.

Figure 4

Figure 4. PI3K p110δ activation by TCR but not CD28 is required for antigen-dependent T cell recruitment.

(A and B) H2-Ab–restricted HY-specific WT (black bars), CD28Y170F (white bars), and p110δD910A (light gray bars) CD4+ T cells were injected i.v. into male mice (107/mouse) that had received an i.p. injection of IFN-γ (600 U) 48 hours earlier. The following day, mice were sacrificed, and the presence of fluorescently labeled cells in the peritoneal cavity (A) and membrane (B) was assessed by flow cytometry and wide-field fluorescence microscopy, respectively. To facilitate visualization by flow cytometry, cells were double stained with an APC-conjugated anti-CD4 antibody following harvesting. The mean T cell numbers ± SEM observed in samples from at least 3 animals are shown. A: *P < 0.04, CD28Y170F versus WT T cells; **P < 0.001, p110δD910A versus WT T cells; P < 0.005, p110δD910A versus CD28Y170F T cells. B: *P < 0.03, CD28Y170F versus WT T cells; **P < 0.002, p110δD910A versus WT T cells; P < 0.007, p110δD910A versus CD28Y170F T cells. (C and D) HY-specific WT or p110δD910A CD4+ T cells that had either undergone antibody-mediated CD28 ligation (30 minutes at 37°C, PKH26-labeled) or had been pretreated with an antibody isotype control (IsC) (CFSE-labeled) were injected i.v. (107/mouse) into male mice that had received an i.p. injection of IFN-γ 48 hours earlier. The presence of fluorescently labeled cells in the peritoneal cavity (C) and membrane (D) was assessed 24 hours later as described for A and B. The mean T cell number ± SEM observed in samples from at least 3 animals is shown. C: *P < 0.02 versus WT + IsC; D: *P < 0.02 versus WT + IsC.

Figure 5

Figure 5. PI3K p110δ is required for tissue infiltration by antigen-specific T cells.

Female and male C57BL/6 mice were treated i.p. with 600 U IFN-γ. After 48 hours, 3 × 106 PKH26-labeled WT or p110δD910A HY-specific H2-Ab–restricted CD4+ T cells (107/mouse) were injected i.p. The presence of labeled T cells in the peritoneal membrane and lavage was analyzed after 24 hours by wide-field fluorescence microscopy (A and B) and flow cytometry (C and D), respectively. In addition, the presence of labeled cells in the spleen was quantified by wide-field fluorescence microscopy (E). Original magnification, ×10. Tissue infiltration was quantified by randomly selecting ten ×10-magnified fields and assessing the number of fluorescent cells in each field. The mean values ± SD observed in samples from at least 3 animals are summarized in B and E (infiltration of the peritoneal membrane and spleen, respectively) and D (cells retrieved in the peritoneal lavage). B and D: *P < 0.004 versus p110δD910A; E: *P < 0.02 versus p110δD910A.

Figure 6

Figure 6. Pharmacologic inhibition of PI3K p110δ prevents antigen-dependent T cell localization to antigenic sites.

HY-specific H2-Ab–restricted CD4+ T cells were treated with 5 μM IC87114 or with medium containing the vehicle (1% DMSO) for 30 minutes at room temperature and subsequently washed before use. (A and B) Untreated (filled symbols) and treated (open symbols) T cells (5 × 105/well) were seeded onto syngeneic female- or male-derived IFN-γ–treated EC monolayers. T cell migration was monitored as described in Figure 1. The percentage of migrated T cells at the given time points is reported as the average of 3 experiments of identical design. *P < 0.05, except at 2 and 4 hours. (C) PKH26-labeled HY-specific untreated (black bars) or IC87114-treated (white bars) T cells were injected i.v. into syngeneic female mice. Infiltration of the indicated tissues was assessed 24 hours later as described in Figure 1, B and C. The mean T cell infiltration ± SD observed in samples from at least 3 animals is shown. (D–G) Female and male C57BL/6 mice were treated i.p. with 600 U IFN-γ. After 48 hours, 107 labeled untreated (black bars) or IC87114-treated (white bars) HY-specific T cells were injected i.v. (107 T cells/mouse, D and E) or i.p. (3 × 106 T cells/mouse, F and G). The presence of labeled T cells in the peritoneal membrane and cavity was analyzed after 24 hours as described in the legends to Figures 3 and 5, respectively. The mean values ± SD observed in samples from at least 3 animals are shown. *P < 0.02 (D), *P < 0.05 (E), *P < 0.02 (F), *P < 0.03 (G) versus untreated T cells.

Figure 7

Figure 7. Lack of PI3K p110δ activity prevents T cell localization into HY-mismatched skin grafts.

C57BL/6 female mice received 3 skin grafts: one from a syngeneic male mouse (A), one from a syngeneic female mouse (B), and the third from a CBA/Ca male mouse (C). Four days following grafting, HY-specific Ab-restricted WT (PKH26-labeled) CD4+ T cells (107/mouse), were injected i.v. Graft infiltration by labeled T cells was assessed 24 hours later by wide-field fluorescence microscopy, as described in the legend to Figure 1, C and D. Original magnification, ×10. The mean values ± SD observed in samples from at least 3 animals are summarized in D. *P < 0.03 versus female and CBA/Ca graft infiltration. Parallel experiments were run in which C57BL/6 female mice engrafted with C57BL/6 male-derived skin 4 days earlier were coinjected with either WT (PKH26-labeled, red) or p110δD910A (CFSE-labeled, green) HY-specific CD4+ T cells (E and F) or with untreated (PKH26-labeled, red) or IC87114-treated (CFSE-labeled, green) HY-specific CD4+ T cells (G and H). Grafts were removed 24 hours later, and tissue infiltration was quantified by randomly selecting ten ×10-magnified fields from tissue samples from at least 3 animals and assessing the number of fluorescent cells in each field. The few CFSE-labeled cells are indicated by an arrowhead. Nuclei are stained by DAPI (blue). A–C show representative ×10-magnified tissue images, while the images shown in E and G were taken at a ×20 magnification. The mean T cell infiltration ± SD observed in samples from at least 3 animals is shown; *P < 0.03 (F); *P < 0.01 (H).

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