T cell receptor–induced phosphoinositide-3-kinase p110δ activity is required for T cell localization to antigenic tissue in mice (original) (raw)
PI3K p110δ activity is not required for antigen-independent memory T cell transendothelial migration, chemotaxis, and constitutive trafficking. Before investigating the effect of PI3K-mediated signals on TCR-driven T cell trafficking, the ability p110δD910A T cells to undergo antigen-independent migration in steady state and in response to inflammatory stimuli was analyzed. Memory HY-specific H2-Ab–restricted CD4+ T cells were generated by immunization of female C57BL/6 WT and p110δD910A mice with syngeneic WT male splenocytes followed by in vitro expansion. The 2 T cell populations displayed similar phenotypes, as assessed 7 days after restimulation with male splenocytes and recombinant IL-2 (rIL-2) (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI33267DS1). Following further resting in medium alone and subsequent antibody stimulation with anti-CD3 and anti-CD28, HY-specific WT and p110δD910A CD4+ T cells equally upregulated surface expression of CD25, CD69, and the chemokine receptor CXCR4 (Supplemental Figure 2, A–D). However, fewer p110δD910A CD4+ T cells were induced to produce IFN-γ (Supplemental Figure 2E), consistent with previous results (21). HY-specific WT and p110δD910A CD4+ T cells migrated equally through resting (Figure 1A) and TNF-α–treated (data not shown) syngeneic, female-derived EC monolayers and displayed similar migration speed over plastic-bound recombinant ICAM-1 (Figure 1B) in vitro. Thus, p110δ does not appear to regulate basal motility in absence of cognate antigen.
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.
To investigate whether constitutive memory T cell trafficking was affected by lack of p110δ activity in physiological settings, WT female mice were injected with either PKH26-labeled WT or p110δD910A T cells (107/mouse), and their localization in several tissues was analyzed 16–24 hours later by wide-field microscopy. Both WT and p110δD910A T cells recirculated normally and could be detected in the spleen, lung, liver, and kidney of recipient mice in similar numbers (Figure 1, C and D). Notably, WT and p110δD910A T cells displayed similar patterns of distribution within the various organs and localized in the liver, kidney, and heart in a scattered pattern, while they clustered in restricted areas in lymph nodes. These observations suggest that p110δ signaling is not involved in constitutive memory T cell trafficking. The inefficient localization of both WT and p110δD910A T cells to the lymph nodes as compared to the spleen was likely due to lack of expression of CD62L and CCR7 (Supplemental Figure 1; ref. 22).
As PI3K activity is associated with chemokine-induced T cell migration (14), we also compared the recruitment of WT and p110δD910A T cells in response to chemokines. WT and p110δD910A T cell migration was equally enhanced by the chemokines CXCL10, CXCL12, and CCL5 in vitro (Figure 2, A–F). As both WT and p110δD910A T cells were found to express CXCR4 only following antibody-mediated stimulation with plastic-bound anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml) (Supplemental Figure 2), in the experiments assessing migration to CXCL12, T cells were used following exposure to these antibodies for 72 hours in vitro. In these experiments, the increase in migration in response to CXCL12 was quite low, possibly due to the low expression of CXCR4, but p110δ and WT T cells responded similarly.
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.
As chemokine-induced T cell adhesion and subsequent transendothelial migration is regulated by shear flow (23), the potential contribution of PI3K p110δ activity to T cell migratory response to chemokines in vivo was investigated. HY-specific PKH26-labeled CD4+ WT or p110δD910A T cells (107/mouse) were injected i.v. into syngeneic female mice that had received an i.p. injection of CXCL10 (1,200 ng/mouse) 1 hour earlier. CXCL10 was used in these experiments because of the high level of expression of its counterreceptor CXCR3 by both WT and p110δD910A T cells (Supplemental Figure 1) and because CXCL10 elicited strong chemotactic responses in vitro. Control mice received an equal volume of saline solution i.p. T cell migration to the peritoneal cavity was assessed 6 hours later by flow cytometric analysis of the peritoneal lavage. As shown in Figure 2, G–K, WT and p110δD910A CD4+ T cells localized to the peritoneal cavity in response to chemokine challenge with similar efficiency. In line with previous observations (15, 16), these data suggest that PI3K p110δ activity is not required for constitutive or chemokine-induced T cell migration and that the machinery required to leave the circulation and cross the endothelial barrier in response to a chemokine gradient is intact in p110δD910A T cells.
PI3K p110δ activity is essential for antigen-dependent T cell recruitment. We and others have previously reported that antigen displayed by the endothelium contributes to the recruitment of specific T cells into the antigenic tissue. As PI3K activity is regulated by TCR signaling (9) and has the potential to regulate cytoskeletal rearrangements (24), we sought to investigate whether its activity contributes to antigen-dependent T cell recruitment. To explore this, the effect on migration after cognate recognition of syngeneic WT male- or female-derived endothelial cells by HY-specific WT and p110δD910A T cells on their migration was first compared using an in vitro transendothelial migration assay that we have previously described (25). T cells were purified on a Ficoll gradient and seeded onto IFN-γ–treated EC monolayers (derived from female or male WT mice) grown on a Transwell insert, and the number of transmigrated T cells recovered in the bottom well was monitored hourly. As shown in Figure 3, A–C, WT HY-specific T cells displayed a much greater rate of migration through MHC class II H2-Ab–expressing male-derived ECs (Figure 3A). By contrast, migration of p110δD910A T cells through male-derived EC monolayers was not enhanced when compared with the migration through female-derived ECs (Figure 3B). The male and female EC monolayers mediated comparable levels of migration of third-party control MHC class I–restricted CD8+ T cells (HY-specific Kk-restricted C6 T cells; Figure 3C).
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.
To assess the physiological relevance of the dependence of p110δ on antigen-mediated transendothelial migration, we compared the recruitment of HY-specific CD4+ WT and p110δD910A T cells from the circulation into the peritoneum of male and female WT mice. In this model, HY-specific T cells localize in the peritoneal membrane and cavity of male but not female mice following induction of local MHC molecule upregulation (and subsequently antigen presentation) by i.p. injection of IFN-γ (4). PKH26-labeled WT or p110δD910A H2-Ab–restricted CD4+ T cells (107/mouse) were injected i.v. into male or female WT C57BL/6 mice that had received an i.p. injection of IFN-γ (600 U) 48 hours earlier. Significant amounts of WT T cells were detected in the peritoneal membrane (Figure 3D) and cavity (Figure 3E) of male mice. By contrast, HY-specific p110δD910A T cells failed to localize to the peritoneal membrane and were not found in the peritoneal lavage of IFN-γ–treated male mice. As expected, neither WT nor p110δD910A T cells migrated the peritoneal membrane or cavity of IFN-γ–treated female mice. Similar observations were made when TCR-dependent recruitment of HY-specific CD8+ T cells was analyzed, suggesting that PI3K p110δ activity is required for antigen-driven localization of both CD4+ and CD8+ T cells (Supplemental Figure 3).
TCR-induced PI3K recruitment selectively mediates antigen-dependent T cell localization. We have previously shown that the PI3K binding motif in CD28 is necessary for the efficient localization of CD8+ memory T cells to nonlymphoid tissue (22). However, this is also required for Grb2 and Gads binding, and hence a requirement of PI3K had been implied but not proven. Constitutive low expression of CD80 molecules by murine endothelium provided an opportunity to discriminate the relative contribution of TCR- and CD28-induced PI3K activity in antigen-dependent T cell localization. To test the role of PI3K specifically in the context of CD28 signaling, we used HY-specific H2-Ab–restricted CD4+ CD28Y170F T cells (which carry a mutation in the cytoplasmic tail of CD28 that abrogates PI3K recruitment without leading to defects in clonal expansion; ref. 26) and compared their responses to those by p110δD910A T cells, where PI3K signaling is uncoupled from both TCR and CD28 signaling.
First, localization of HY-specific WT, p110δD910A, and CD28Y170F CD4+ T cells to the peritoneal membrane and cavity of male mice was compared by the antigen-dependent peritoneal recruitment model described above. PKH26-labeled HY-specific CD4+ T cells derived from WT, CD28Y170F, or p110δD910A T cells (107/mouse) were injected i.v. into male C57BL/6 mice that had received an i.p. injection of IFN-γ (600 U) 72 hours earlier. Infiltration of the peritoneal membrane by labeled T cells and enrichment of HY-specific T cells in the peritoneal lavage were assessed 24 hours later as described for the experiments in Figure 3, D and E. WT T cells were readily identified from the peritoneal membrane and in the peritoneal lavage, demonstrating efficient recruitment from the circulation. The recruitment of HY-specific CD28Y170F CD4+ T cells to the peritoneal cavity of male mice was moderately impaired, whereas the recruitment of p110δD910A T cells was virtually undetectable (Figure 4, A and B). In line with our previous findings, these observations suggest that CD28- and TCR-mediated PI3K activation can independently regulate T cell migration.
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.
We have previously shown that anti-CD28 can stimulate trafficking of T cells into nonlymphoid tissues and that this was dependent on the tyrosine within a YMNM motif in the cytoplasmic domain of CD28 (13). As this tyrosine can bind the SH2 domains of Grb2 and Gads in addition to the SH2 domains of p85, we sought to independently determine whether PI3K p110δ is required for CD28-dependent trafficking. To this end, HY-specific CD4+ WT or p110δD910A T cells (107/mouse) that had either undergone CD28 ligation (PKH26-labeled) or had been pretreated with an isotype control antibody (CFSE-labeled) were coinjected i.v. into male C57BL/6 mice that had received an i.p. injection of IFN-γ (600 U) 72 hours earlier, as previously described (13). Tissue infiltration by labeled T cells and enrichment of HY-specific T cells in the peritoneal lavage were assessed 24 hours later as described above and in Methods. CD28 ligation significantly enhanced the recruitment of WT T cells, which were detected in the peritoneal membrane and lavage (Figure 4, C and D). By contrast, peritoneal recruitment of HY-specific p110δD910A T cells was not reconstituted by CD28 triggering. Isotype control antibody–treated and untreated T cells displayed similar localization (data not shown). These results show that CD28 through its YMNM motif signals via p110δ to promote tissue infiltration.
PI3K p110δ activity is instrumental in retention of antigen-specific T cells into antigenic tissue. Once T cells have reached the antigenic sites, they are retained in the tissue by mechanisms involving antigen presentation by resident antigen-presenting cells (27). To investigate the contribution of TCR-induced PI3K p110δ activity on this effect, we used an established model of antigen-dependent tissue infiltration (22, 25). In this model, tissue infiltration is strictly dependent on recognition of HY antigen within the peritoneal tissue. As in the model described above, male or female C57BL/6 mice were injected with 600 U IFN-γ. Two days later, PKH26-labeled, HY-specific WT or p110δD910A T cells (3 × 106 cells/mouse) were injected directly into the peritoneum. The following day mice were sacrificed, and T cells that had infiltrated the peritoneal membrane or remained in the peritoneal lavage were detected by wide-field fluorescence microscopy and flow cytometry, respectively. HY-specific WT T cells efficiently localized into the peritoneal membrane of male recipients (Figure 5, A and B) and were depleted from the peritoneal cavity (Figure 5, C and D), whereas only a few p110δD910A T cells could be detected in the peritoneal membrane (Figure 5, A and B) and were instead readily recovered from the peritoneal lavage of male recipients (Figure 5, C and D).
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.
As expected, HY-specific WT and p110δD910A T cells were not detected in the peritoneal membrane but were readily recovered from the peritoneal cavity of female recipients. In female recipient mice, the few T cells that had entered the peritoneum also entered the general circulation as evidenced by their recovery from the spleens (Figure 5E). By contrast, in male recipients, few WT T cells could be recovered from the spleens, reflecting their retention in the peritoneal wall. In contrast, p110δD910A T cells were readily detected in the spleens of male recipient mice as well. We conclude that p110δ controls both the recruitment and retention of T cells in nonlymphoid tissues without affecting passive entry into the general circulation. These findings raise the possibility that p110δ-mediated signals may be required for the establishment of antigen-dependent stable interactions with conventional antigen-presenting cells.
Pharmacological inhibition of PI3K p110δ abrogates antigen-dependent T cell recruitment and retention into antigenic tissue. Having established its role in mediating TCR-dependent T cell recruitment, we sought to investigate the effectiveness of pharmacological inhibition of p110δ activity on this effect. The IC87114 compound has been reported to selectively inhibit p110δ at doses up to 5–10 μM both in vitro and in vivo (28). In all the experiments described below, control T cells were treated with PBS containing vehicle (1% DMSO).
First we carried out studies to establish whether IC87114 might affect the viability of ECs or their ability to mediate migration of T cells. IC87114-treated ECs did not display increased cell death or permeability following exposure to up to 5 μM of the inhibitor for 30 minutes to 24 hours (data not shown). In addition, compared with untreated endothelium, female-derived, IFN-γ–treated ECs were equally capable of mediating migration of HY-specific T cells following exposure of ECs to 5 μM IC87114, suggesting that EC function was not adversely affected by inhibition of PI3K p110δ activity (data not shown). Nonetheless, in order to exclude effects that IC87114 might have on other cell types, T cells were treated with the inhibitor and subsequently washed prior to use. In addition, exposure of T cells to the inhibitor (5 μM) for up to 48 hours did not affect their viability (data not shown).
Migration of untreated and IC87114-treated HY-specific T cells through IFN-γ–treated EC monolayers derived from C57BL/6 male or female mice and grown on a Transwell insert in vitro was compared. As shown in Figure 6, A and B, inhibition of PI3K p110δ activity prevented antigen-induced enhancement of HY-specific T cell migration, while it did not affect T cell migration through female-derived ECs.
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.
To assess the effect of pharmacological inhibition of PI3K p110δD910A on constitutive T cell trafficking in vivo, we compared the recruitment of HY-specific untreated and IC87114-treated T cells from the circulation into various tissues as described in Figure 1. PKH26-labeled untreated or IC87114-treated (5 μM, 1 hour at 37°C) H2-Ab–restricted CD4+ T cells (107/mouse) were injected i.v. into C57BL/6 WT female mice. Similar numbers of T cells localized in lymphoid or different nonlymphoid tissues, suggesting that pharmacological inhibition of PI3K p110δ does not affect constitutive memory T cell trafficking (Figure 6C).
Next, to assess the effect of pharmacological inhibition of PI3K p110δD910A on antigen-dependent T cell localization to antigenic sites in vivo, we compared the recruitment of HY-specific untreated and IC87114-treated T cells from the circulation into the peritoneum of male and female mice as described above. PKH26-labeled untreated or IC87114-treated (5 μM, 1 hour at 37°C, CFSE-labeled) H2-Ab–restricted CD4+ T cells (107/mouse), were injected i.v. into male or female C57BL/6 mice that had received an i.p. injection of IFN-γ (600 U) 48 hours earlier to induce local HY antigen presentation in male recipients. As shown in Figure 6, D and E, significant numbers of untreated T cells were detected in the peritoneal membrane and cavity of male mice. By contrast, HY-specific IC87114-inhibited T cells failed to localize to the peritoneal membrane and lavage of male mice. As expected, neither untreated nor IC87114-treated T cells localized in the peritoneal membrane and lavage of IFN-γ–treated female mice.
Finally, the effect of pharmacological inhibition of p110δ activity on tissue infiltration by HY-specific CD4+ T cells was investigated by experiments similar to those described in Figure 5. T cells were treated with IC87114 prior to i.p. injection into male and female C57BL/6 mice that had received an i.p. injection of IFN-γ (600 U) 48 hours earlier. As shown in Figure 6, F and G, pharmacologic inactivation of PI3K p110δ dramatically inhibited tissue infiltration by HY-specific T cells in male mice, which remained localized in the peritoneal cavity.
Taken together, these observations suggest that selective pharmacological inhibition of PI3K p110δ activity is effective in preventing the recruitment of antigen-specific T cells and their localization into target tissue.
Genetic and pharmacological inactivation of PI3K p110δ activity prevents HY-specific T cell infiltration of HY-mismatched skin grafts. Having established that p110δ-mediated signals are required for antigen-dependent T cell recruitment into — and infiltration of — antigenic nonlymphoid tissue, we investigated the effect of PI3K p110δ inactivation on the development of HY-specific T cell–mediated inflammation in HY-mismatched skin grafts.
First, we assessed the effect that antigen display exerts on T cell localization in this model. C57BL/6 female mice received 3 skin grafts: one from a syngeneic female mouse, one from a syngeneic male mouse (usually rejected between days 19 and 31 in WT female mice), and the third from a CBA/Ca male mouse (usually rejected by day 12). Four to 9 days following grafting, mice were injected i.v. with PKH26-labeled HY-specific CD4+ T cells (107/mouse), and T cell infiltration in the grafts was assessed the following day (after sacrifice). It has to be noted that CBA/Ca-derived grafts bear MHC molecules of the H2-K haplotype, while the HY-specific T cells used here are H2-Ab restricted. As shown in Figure 7, A–D, HY-specific T cells selectively infiltrated antigenic (male) grafts, suggesting that antigen location plays a major role in determining primed T cell localization. Graft infiltration was assessed 24 and 48 hours after T cell transfer with similar results (data not shown).
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).
Next, we investigated the effect of p110δ inactivation on the infiltration of HY-mismatched skin grafts by HY-specific H2-Ab–restricted CD4+ T cells. C57BL/6 female mice that had received syngeneic male mouse skin graft were coinjected i.v. with PKH26-labeled, HY-specific WT CD4+ T cells (107/mouse) together with either CFSE-labeled HY-specific p110δD910A T cells (Figure 7, E and F) or CFSE-labeled IC87114-pretreated (5 μM, 1 hour at 37°C; Figure 7, G and H) WT T cells. Genetic (Figure 7, E and F) or pharmacological (Figure 7, G and H) inhibition of PI3K p110δ strongly inhibited male skin graft infiltration by HY-specific CD4+ T cells (the very few green-labeled cells are indicated by arrows in the figure). No differences were observed in T cell infiltration of female-derived skin grafts, which was virtually absent (data not shown).