LFA-1-mediated T cell costimulation through increased localization of TCR/class II complexes to the central supramolecular activation cluster and exclusion of CD45 from the immunological synapse

Abstract

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.

FIGURE 1

The cSMAC is formed in the absence of LFA-1/ICAM interactions. Previously activated WT (black bars) and CD18KO (grey bars) T cells were mixed with an equal number of antigen-pulsed ProAd (Ad), ProAd-ICAM (ICAM), ProAd-B7 (B7), and A20 APC, co-pelleted to initiate cell:cell contact, plated on poly(L)lysine coated coverslips, and stained for MHC class II. A, The frequency and standard deviation of T cell:APC adhesion, represented as the percentage of APC that had formed a conjugate with a T cell. The differences in adhesion between WT and CD18KO T cells with ICAM-negative APC (ProAd and ProAd-B7) are not statistically significant (P>0.05), whereas the differences in adhesion between WT and CD18KO T cells with ICAM-positive APC (ProAd-ICAM and A20) are highly significant (P<0.001). B, The frequency and standard deviation of conjugates containing a cSMAC region, identified by the clustering of MHC class II molecules in the T cell:APC contact region. None of the differences in frequency between WT and CD18KO T cells with either ICAM-positive or ICAM-negative APC are statistically significant (P>0.05). The number of conjugates analyzed ranged from 28 to 32, except for CD18KO T cells with ProAd-ICAM (n=22), CD18KO T cells with A20 (n=41), and WT T cells with A20 (n=57).

FIGURE 2

TCR signaling is not sufficient to form extensive T cell:APC interactions. WT and CD18KO (18KO) T cell conjugates with antigen-pulsed ProAd-ICAM APC were stained for talin (green) and class II (red). Differential interference contrast (DIC) and fluorescent images are shown. Both WT and CD18KO T cells have formed a cSMAC, as indicated by the clustering of class II molecules in the APC. In the presence of LFA-1/ICAM-1 interactions (WT T cells with ProAd-ICAM APC) all of the conjugates analyzed formed a large cell:cell contact area as illustrated in the example shown. In contrast, in T cell conjugates where only TCR was engaged (CD18KO T cells with ProAd-ICAM APC and WT or CD18KO T cells with ProAd APC) about half of the conjugates displayed a reduced contact region with a distended membrane tether extending from the T cell to the APC, as illustrated in the example shown.

FIGURE 3

LFA-1 is required to organize talin into a pSMAC structure. A and B, WT (A) and CD18KO (B) T cell conjugates with antigen-pulsed A20 APC were stained for talin (green) and class II (red). The images are all orientated so that the T cell is the top cell in the conjugate. Differential interference contrast images are shown on the left. Note that the conjugates between A20 and either WT or CD18KO T cells exhibit a similar morphology, including a large cell:cell contact area. Mid-section, deconvolved images and 3-dimensional projections of the immunofluorescent staining are shown in the middle and right, respectively. C, The frequency and standard deviation of conjugates displaying an organized pSMAC structure. An organized pSMAC structure is based on the 3-dimensional projections and is defined as a central localization of class II staining surrounded by a region of talin staining, with little or no co-localization of class II and talin. Note that in CD18KO T cells, class II was often not centralized (top conjugate in B) and talin was typically not excluded from the region of class II staining (top and middle conjugates in B). In the few CD18KO T cells that were scored as displaying an organized pSMAC structure in panel C, even though class II was centrally localized and did not co-localize with talin, talin was not efficiently recruited to the synapse and/or not well distributed across the pSMAC region(see bottom conjugate in B). The different frequencies of pSMAC formation between WT and CD18KO T cells was highly significant (p<0.001, n=30 for WT and n=18 for CD18KO T cells).

FIGURE 4

LFA-1 enhances the amount of class II that is recruited to the cSMAC. WT and CD18KO T cell conjugates with antigen-pulsed A20 cells were stained for class II. The relative increase in class II concentration (determined by fluorescence intensity) within the cSMAC, compared to the APC cell surface that was not in contact with the T cell is shown for individual conjugates with WT and CD18KO T cells. The horizontal bar represents the mean. The difference in class II accumulation in WT and CD18KO T cell conjugates was statistically significant (P<0.01). The number of conjugates analyzed for WT T cells was 30 and for CD18KO T cells was 18.

FIGURE 5

LFA-1 is required to exclude CD45 from the pSMAC. WT and CD18KO T cell conjugates with antigen-pulsed A20 cells were stained for CD45 (green) and class II (red). A, Representative interface projections of the immunological synapse from WT (left) and CD18KO (center and right) T cells illustrate the three relative distributions of CD45 that were detected. Major, CD45 was excluded from most of the synapse. Minor, CD45 was localized within the synapse, but largely excluded from the cSMAC. None, CD45 was present across the synapse and significantly colocalized with TCR/class II complexes in the cSMAC. The fluorescence intensity profile of a midplane section through the center of the cSMAC is shown below each micrograph. B, The percentage of WT and CD18KO T cell conjugates displaying these distributions of CD45 are shown. The increased frequency of major CD45 exclusion and the increased frequency of any CD45 exclusion (major + minor) seen in WT compared to CD18KO T cells are highly significant (p=0.001, n=32 for WT and n=34 for CD18KO T cells). C, The relative fluorescence intensity of CD45 within the cSMAC compared to the remainder of the T cell surface is shown for individual conjugates with WT and CD18KO T cells. The horizontal bar represents the mean. The difference in CD45 exclusion from the cSMAC in WT and CD18KO T cell conjugates was statistically significant (P<0.001). The number of conjugates analyzed for WT T cells was 15 and for CD18KO T cells was 13.

FIGURE 6

Measurement of T cell calcium responses in T cell:APC conjugates. In vitro primed, resting DO11.10 T cells were loaded with Indo-1-AM and mixed with antigen-pulsed, Alexa 633-labeled A20 cells. The cells were analyzed by flow cytometry to established a baseline of Indo-1 fluorescence and then briefly pelleted to induce T cell:APC conjugate formation (arrow on left in C). The cells were resuspended, rapidly returned to the flow cytometer, and maintained at 37°C. T cell:APC conjugates were gated based on coincident Indo-1 and Alexa 633 fluorescence (A). The percent of T cell:APC conjugates is shown as an inset. The change in the ratio of violet to blue fluorescence of Indo-1, indicative of an increase in intracellular calcium, was monitored over time (shown in seconds in C). The data are represented as the median of the ratio of calcium-bound (violet) to calcium-free (blue) Indo 1 in the T cell:APC and T cells alone populations. Note the rapid rise in intracellular calcium in T cell:APC conjugates (red line in C), but not in T cells that have not formed conjugates with APC (green line in C). To assure that the calcium response that was detected was mediated only by T cell:APC conjugates that formed at the initiation of the assay, and not by new conjugates formed during the analysis, additional antigen-pulsed A20 cells, labeled with a different dye (Alexa 488), were added after the T cell:APC conjugates were resuspended, just before the sample was returned to the flow cytometer. Note that some T cell:APC conjugates do form with APC while in suspension (B), but these T cells do not flux calcium (blue line in C). After 10 minutes, ionomycin was added (arrow on right in C), to assure that all T cells were effectively loaded with Indo-1.

FIGURE 7

LFA-1/ICAM interactions enhance calcium signaling in T cell:APC conjugates. A and B, WT and CD18KO T cells were analyzed for T cell calcium responses as described in Fig. 6. Calcium responses were initiated by pelleting T cells and APC (arrow on left) and monitored for 5 minutes before the addition of ionomycin (arrow at right). A, WT T cell:A20 conjugates (red line) demonstrated a significantly higher calcium response than CD18KO T cell:A20 conjugates (blue line). B, To control for possible intrinsic defects in CD18KO T cells, calcium responses were also measured in conjugates with ProAd-B7 cells, that do not express ICAM. Both WT (red line) and CD18KO (blue line) T cells gave identical calcium responses. T cells that do not form conjugates (T cells alone) do not flux calcium. The data are represented as the median of the ratio of calcium-bound (violet) to calcium-free (blue) Indo 1. C, The mean and standard deviation of the median calcium response from WT and CD18KO T cells conjugates with A20 cells (p <0.005, n=4).