The T cell CD6 receptor operates a multitask signalosome with opposite functions in T cell activation

Abstract

To determine the respective contribution of the LAT transmembrane adaptor and CD5 and CD6 transmembrane receptors to early TCR signal propagation, diversification, and termination, we describe a CRISPR/Cas9-based platform that uses primary mouse T cells and permits establishment of the composition of their LAT, CD5, and CD6 signalosomes in only 4 mo using quantitative mass spectrometry. We confirmed that positive and negative functions can be solely assigned to the LAT and CD5 signalosomes, respectively. In contrast, the TCR-inducible CD6 signalosome comprised both positive (SLP-76, ZAP70, VAV1) and negative (UBASH3A/STS-2) regulators of T cell activation. Moreover, CD6 associated independently of TCR engagement to proteins that support its implication in inflammatory pathologies necessitating T cell transendothelial migration. The multifaceted role of CD6 unveiled here accounts for past difficulties in classifying it as a coinhibitor or costimulator. Congruent with our identification of UBASH3A within the CD6 signalosome and the view that CD6 constitutes a promising target for autoimmune disease treatment, single-nucleotide polymorphisms associated with human autoimmune diseases have been found in the Cd6 and Ubash3a genes.

Figure 1.

Mouse primary CD4⁺ T cells amenable to fast-track AP-MS characterization of the LAT signalosome. (A) An sgRNA was designed to introduce a double-strand break 12 bp 3′ of the stop codon found in the last exon of the Lat gene, and an 843-bp-long dsDNA was used as a template for HDR (see Fig. S1, A and B). Following HDR, CD4⁺ T cells are expected to coexpress LAT^OST and CD90.1 molecules. (B) Workflow used for editing, selecting, and expanding CD4⁺ T cells expressing LAT^OST molecules amenable to AP-MS. (C) T cells were analyzed for expression of CD90.1 3 d after nucleofection with vehicle alone (None), sgRNA, or sgRNA plus the HDR template (sgRNA + template). Also shown is the expression of CD4 and TCRβ on CD5⁺ CD90.1⁺ T cells. (D) Sorted CD90.1⁺ CD4⁺ T cells and WT CD4⁺ T cells were expanded in vitro and analyzed for expression of CD90.1 before AP-MS analysis. Data in C and D are representative of at least three experiments. (E) PCR genotyping schematics of sorted CD90.1⁺ CD4⁺ T cells expressing LAT^OST molecules. The two specified PCR primer pairs provide diagnostic bands for each junction. Correct targeting was further confirmed by sequencing the 5′ and 3′ junction fragments (Fig. S1, C and D). Also shown are the expected sizes of the PCR amplicons. (F) PCR genotyping was performed on WT CD4⁺ T cells (Ctrl) and on sorted CD90.1⁺ CD4⁺ T cells (LAT^OST) using the PCR primer pairs specified in E. (G) Immunoblot analysis of equal amounts of proteins from WT and LAT^OST CD4⁺ T cell lysates that were either directly analyzed (Total lysate) or subjected to affinity purification on Strep-Tactin Sepharose beads followed by elution of proteins with D-biotin (Pull down), and both were probed with antibody to anti-LAT or anti-VAV1 (loading control). The long-term expanded LAT^OST CD4⁺ T cells showed an even representation of WT and LAT^OST alleles. (H) Immunoblot analysis of equal amounts of proteins from total lysates of WT and LAT^OST CD4⁺ T cells left unstimulated or stimulated for 30 s or 120 s with anti-CD3 and anti-CD4 and probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-VAV1 (loading control). (I) Immunoblot analysis of equal amounts of lysates of WT and of LAT^OST CD4⁺ T cells stimulated as in H and subjected to affinity purification on Strep-Tactin Sepharose beads, followed by elution of proteins with D-biotin, and probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-LAT. Left margin in G–I, molecular size in kilodaltons. Data in H and I are representative of at least two independent experiments. PAM, protospacer-adjacent motif. GSG, Gly-Ser-Gly spacer.

Figure S1.

Structure of the HDR template used to edit the Lat gene and sequences of the resulting junctions. (A) Structure of the dsDNA HDR template used to edit the Lat gene. The specified sequence elements correspond to a 5′ homology arm, an OST tag flanked on both sides by a Gly-Ser-Gly spacer, a self-cleaving P2A peptide, a sequence coding for CD90.1, and a 100-bp-long 3′ homology arm. The Gly-Ser-Gly spacers are intended to give flexibility to the OST-P2A polypeptide. (B) Sequence of the HDR template used to edit the Lat gene. The encoded elements are color-coded as in A. (C) Sequence of the 5′ junction of the intended insertion confirmed that proper HDR occurred in the CD4⁺ T cells sorted for the expression of CD90.1. The sequencing primers correspond to Lat-1 and Cd.90.1-1 (Table S2). (D) Sequence of the 3′ junction of the intended insertion confirmed that proper HDR occurred in the CD4⁺ T cells sorted for the expression of CD90.1. The sequencing primers correspond to Lat-2 and Cd90.1-2 (Table S2).

Figure 2.

Composition and dynamics of the LAT signalosome of long-term–expanded LAT^OST-expressing CD4⁺ T cells. (A) Volcano plot showing proteins significantly enriched after affinity purification in CD4⁺ T cells expressing LAT^OST molecules compared with affinity purification in control CD4⁺ T cells expressing similar levels of WT (untagged) LAT proteins before (t_0s) and at 30 s (t_30s) and 120 s (t_120s) after TCR plus CD4 stimulation. (B) Volcano plot showing proteins significantly enriched after affinity purification in LAT^OST-expressing CD4⁺ T cells 30 and 120 s after TCR engagement compared with affinity purification in unstimulated LAT^OST-expressing CD4⁺ T cells. In A and B, the SLP-76, GRB2, GRAP, GRAP2, THEMIS, ITK, PLC-γ1, SHIP1, SOS1, and MAP4K1 proteins are shown in red, and the x and y axes show the average fold change (in log₁₀ scale) in protein abundance and the statistical significance, respectively. (C) Dot plot showing the interaction stoichiometry of LAT with its 10 high-confidence preys over the course of TCR stimulation. For each LAT–prey interaction, the interaction stoichiometry has been row-normalized to its maximum value observed over the course of TCR stimulation (see normalized stoichiometry key). The 10 preys showed maximal binding to LAT after 30 s of activation. Also shown is the P value of the specified interactions (see P value key). (D) Stoichiometry plot of the LAT interactome. The LAT bait is specified by a yellow dot, and its 10 preys are shown as red dots. For each of these LAT–prey interactions, the ratio of prey to bait cellular abundance (“abundance stoichiometry” in log₁₀ scale) was plotted as a function of the maximal interaction stoichiometry reached by the considered LAT-prey interaction over the course of TCR stimulation (“interaction stoichiometry” in log₁₀ scale). For instance, LAT (41,443 copies per T cell; column G of the LAT tab in Data S2) is more abundant than SOS1 (2,562 copies per T cell), giving a ratio of prey to bait cellular abundance of −2.2 in log₁₀ scale. Moreover, the maximum stoichiometry of the LAT–SOS1 interaction is reached at t_30s and corresponds to 0.023 (−1.6 in log₁₀ scale; column D of the LAT tab in Data S2). Therefore, 953 (41,443 × 0.023) molecules of LAT are complexed to SOS1 at t_30s. As a result, 37% (953/2,562 × 100) of the available SOS1 proteins are complexed to LAT 30 s after TCR engagement. The area including the LAT–prey interaction involving >10% of the available prey molecules is indicated in light gray and includes SOS1, GRB2, and GRAP. The limit imposed on interaction stoichiometries by the relative LAT–prey cellular abundances is shown by a dashed diagonal line that delimits a “forbidden” area (dark gray). Prey dot size is commensurate to its maximal protein enrichment over the course of stimulation.

Figure S2.

Comparison of the fraction of cell mass occupied by the proteins quantified in both short-term– and long-term–expanded CD4⁺ T cells. Using the “proteomic ruler” method, which relies on the MS signal of histones as an internal standard (Wiśniewski et al., 2014), we were able to quantify protein abundance for 5,773 protein groups in long-term–expanded CD4⁺ T cells (Materials and methods and Data S1), among which 5,045 could be mapped to protein groups previously quantified in short-term–expanded CD4⁺ T cells (Voisinne et al., 2019). Long-term–expanded CD4⁺ T cells had an increased cell mass (238 pg) compared with that (37 pg) of short-term–expanded CD4⁺ T cells, resulting in copy numbers per T cell generally higher in long-term–expanded CD4⁺ T cells. Comparing the fraction of cell mass corresponding to each protein present in both proteomes—a value reflecting their respective cellular concentration—showed that the fraction of cell mass corresponding to 79% of the proteins quantified in both interactomes differed by less than fourfold. However, as previously reported (Howden et al., 2019), long-term CD4⁺ T cell expansion does not scale up evenly all proteins. For instance, among the proteins relevant to the present study, some occupied a greater fraction of cell mass in long-term– than in short-term–expanded CD4⁺ T cells (CBL: 1.7-fold; ZAP70: 2.5-fold, UBASH3A: 4.7-fold), whereas other showed a converse pattern (CD5: 3.3-fold, CD6 9.8-fold, CBLB: 17.7-fold). CBLB loss has been associated with a reduced requirement for CD28 costimulation during TCR-induced cell proliferation (Bachmaier et al., 2000), and its decrease in long-term–expanded CD4⁺ T cells might confer to them a selective advantage during long-term in vitro expansion. Some of the proteins discussed in the Results are highlighted in red.

Figure S3.

Mouse primary CD4⁺ T cells amenable to fast-track AP-MS characterization of the CD6 signalosome. (A) An sgRNA was designed to introduce a double-strand break (DSB) in the last Cd6 exon 4 bp 5′ from the first nucleotide of the STOP codon, and an 843-bp-long dsDNA template was used for HDR. Following proper HDR, CD4⁺ T cells are expected to coexpress CD6-OST and CD90.1 molecules at their surface. (B) Sequence of the dsDNA HDR template used to edit the Cd6 gene. (C) CD4⁺ T cells were analyzed for expression of CD6 and CD90.1 3 d after nucleofection with vehicle alone (None), sgRNA, or sgRNA plus HDR template (sgRNA + template). Note that in the absence of an HDR template, inappropriate sealing of the DSB resulted in part in the loss of the Cd6 open reading frame and in a corresponding decrease in CD6 mean fluorescence intensity. Also shown is the expression of CD4 and TCRβ on CD6⁺CD90.1⁺ CD4⁺ T cells. The percentage of CD4⁺TCRβ⁺cells is indicated. (D) Sorted CD90.1⁺ CD4⁺ T cells expressing CD6^OST molecules (CD6^OST) and WT (Control) CD4⁺ T cells were expanded in vitro and analyzed for expression of CD6 and CD90.1 before AP-MS analysis. In C and D, data are representative of at least three experiments. (E) Sequences of the 5′ junction corresponding to the intended insertion confirmed that proper HDR occurred in the primary CD4⁺ T cells sorted for the expression of CD90.1. The sequencing primers correspond to Cd90.1-1 and Cd6-1 (Table S2). (F) Sequences of the 3′ junction corresponding to the intended insertion confirmed that proper HDR occurred in the primary CD4⁺ T cells sorted for the expression of CD90.1. The sequencing primers correspond to Cd6-2 and Cd90.1-2 (Table S2). (G) PCR genotyping schematics of sorted CD90.1⁺ CD4⁺ T cells expressing CD6^OST molecules. The two specified PCR primer pairs provide diagnostic bands for each junction. Also shown are the expected sizes of the PCR amplicons. (H) PCR genotyping was performed on WT CD4⁺ T cells (Control) and on sorted CD90.1⁺ CD4⁺ T cells (CD6^OST) using the PCR primer pairs specified in G. (I) Immunoblot analysis of equal amounts of proteins from total lysates of WT (Control) and CD6^OST CD4⁺ T cells left unstimulated or stimulated for 30 or 120 s with anti-CD3 and anti-CD4, probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) and anti-VAV1 (loading control). Left margin, molecular size in kilodaltons. (J) Immunoblot analysis of equal amounts of lysates of WT (Control) and of CD6^OST CD4⁺ T cells stimulated as in I, subjected to affinity purification on Strep-Tactin Sepharose beads followed by elution of proteins with D-biotin, and probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-CD6 (affinity purification control). Data in I and J are representative of at least two independent experiments. PAM, protospacer-adjacent motif.

Figure 3.

Composition and dynamics of the CD6 signalosome of long-term expanded CD6^OST CD4⁺ T cells. (A) Volcano plot showing proteins significantly enriched after affinity purification in CD4⁺ T cells expressing CD6^OST molecules compared with affinity purification in control CD4⁺ T cells expressing similar levels of WT (untagged) CD6 proteins before (t_0s) and at 30 s (t_30s) and 120 s (t_120s) after TCR plus CD4 stimulation. (B) Volcano plot showing proteins significantly enriched after affinity purification in CD6^OST-expressing CD4⁺ T cells 30 and 120 s after TCR engagement compared with affinity purification in unstimulated CD6^OST-expressing CD4⁺ T cells. For A and B, see description in Fig. 2 B. (C) Dot plot showing the interaction stoichiometry over the course of TCR stimulation of CD6 with its seven high-confidence preys, the interaction stoichiometry of which changed following TCR engagement. See description in Fig. 2 C. (D) Stoichiometry plot of the CD6 interactome. The CD6 bait is shown as a yellow dot. Red and blue dots correspond to preys that showed increased or decreased binding following TCR stimulation. The purple dot corresponds to a prey whose association was not regulated by TCR stimulation. See description in Fig. 2 D. (E) Biochemical validation of the CD6–UBASH3A and CD6–GRK6 interactions predicted on the basis of AP-MS analysis. Long-term–expanded WT (Control) and CD6^OST-expressing CD4⁺ T cells were left unstimulated (0) or were stimulated for 30 and 120 s with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates (Total lysate) were immunoblotted with antibodies specific for UBASH3A and GRK6. Equal amounts of lysates were subjected to AP on Strep-Tactin Sepharose beads (Pull down), followed by elution with D-biotin. Eluates were immunoblotted and probed with antibodies specific for UBASH3A and GRK6. Molecular masses are shown. Data are representative of at least two experiments.

Figure S4.

Normal development and function of T cells of mice expressing endogenous CD5 molecules tagged with an OST tag (CD5^OST). (A) Flow cytometry analysis of thymus and lymph nodes. WT and CD5^OST thymocytes were analyzed for expression of CD4 and CD8 (left) and TCRαβ and TCRγδ (right). Numbers adjacent to outlined areas indicate percentage of CD4⁺CD8⁺ double-positive cells, CD4⁺ single-positive cells, CD8⁺ single-positive cells, and double-negative CD4⁻CD8⁻ cells (left) and of TCRαβ and TCRγδ T cells (right). CD4⁺ (CD19⁻CD5⁺CD4⁺) and CD8⁺ (CD19⁻CD5⁺CD8⁺) T cells from WT and CD5^OST lymph nodes were analyzed for CD44 and CD62L expression. Numbers in quadrants indicate percent naive T cells (CD44^lowCD62L^high) and effector (CD44^highCD62L^low) and central (CD44^highCD62L^high) memory T cells. Data are representative of at least two experiments with three mice per group. (B) Negatively purified CD4⁺ T cells from WT and CD5^OST mice were activated in vitro with plate-bound anti-CD3 antibody in the absence or presence of soluble anti-CD28 antibody or with PMA and ionomycin (P/I). After 48 h of culture, ATP content was assessed by luminescence as a measure of the extent of cell proliferation. Data are representative of at least three experiments with two mice per group (mean and SEM are shown). (C) Purified CD4⁺ T cells from WT and CD5^OST mice were stained with anti-CD5 and analyzed by flow cytometry. Dashed curves: isotype-matched control antibody (negative control). Data are representative of at least three experiments with two mice per group. (D) Immunoblot analysis of equal amounts of proteins from total lysates of short-term–expanded T cells from WT and CD5^OST mice left unstimulated (0) or stimulated with anti-CD3 plus anti-CD4 for 30, 120, and 300 s and probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-VAV1 (loading control). (E) Immunoblot analysis of equal amounts of proteins from total lysates of cells as in D, subjected to affinity purification on Strep-Tactin Sepharose beads followed by elution of proteins with D-biotin, and probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-CD5 (affinity purification control). Left margins of D and E: molecular size in kilodaltons. In D and E, data are representative of three independent experiments.

Figure 4.

Composition and dynamics of the CD5 interactome of short-term–expanded CD4⁺ T cells from CD5^OST mice. (A) Volcano plot showing proteins significantly enriched after affinity purification in CD4⁺ T cells expressing CD5^OST molecules compared with affinity purification in control CD4⁺ T cells expressing similar levels of WT (untagged) CD5 proteins before (t_0s) and at 30 s (t_30s), 120 s (t_120s), and 300 s (t_300s) after TCR plus CD4 stimulation. (B) Volcano plot showing proteins significantly enriched after affinity purification in CD5^OST-expressing CD4⁺ T cells 30, 120, and 300 s after TCR engagement compared with affinity purification in unstimulated CD5^OST-expressing CD4⁺ T cells. For A and B, see description in Fig. 2 B. (C) The dot plot shows the interaction stoichiometry over the course of TCR stimulation of CD5 with its four high-confidence interactors, the interaction stoichiometry of which changed following TCR engagement. See description in Fig. 2 C. (D) Stoichiometry plot of the CD5 interactome. It shows that hundreds of copies of CD5–CBLB, CD5–UBASH3A, and CD5–ANKRD13A complexes formed per T cell as early as 30 s of TCR stimulation, mobilizing close to 10% of the cellular pool of ANKRD13A, CBLB, and UBASH3A. The CD5 bait is shown as a yellow dot. Red dots correspond to preys that show increased binding following TCR stimulation. See description in Fig. 2 D. Mybbp1a; MYB-binding protein 1A.

Figure 5.

T cell development and function in CD5-CD6 doubly deficient mice compared with mice lacking either CD5 or CD6. (A) Expression of CD5 and CD6 on CD4⁺ T cells from WT mice and from mice lacking either CD5 (Cd5^−/−) or CD6 (Cd6^−/−) and both CD5 and CD6 (Cd5^−/−Cd6^−/−) analyzed by flow cytometry. Note that Cd6^−/− T cells expressed levels of CD5 comparable to that of WT mice, whereas increased levels of CD6 were found on Cd5^−/− T cells as reported (Orta-Mascaró et al., 2016). (B) Numbers of CD4⁺ and CD8⁺ T cells found in the spleen of the specified mice. (C) Numbers of T reg cells, γδ T cells, and effector memory CD4⁺ and CD8⁺ T cells found in the spleen of the specified mice. (D) T cells were purified by immunomagnetic negative selection from lymph nodes of Cd5^−/−, Cd6^−/−, Cd5^−/−Cd6^−/−, and WT mice activated in vitro with the specified concentrations of plate-bound anti-CD3 in the absence (−) or presence (+) of soluble anti-CD28 (1 µg/ml). After 48 h of culture, ATP content was assessed using luminescence as a measure of the extent of cell proliferation. The histogram on the right shows the extent of cell proliferation in response to PMA plus ionomycin (PMA/IM). (E) Profiles of CTV-labeled CD4⁺ (upper panel) and CD8⁺ (lower panel) T cells isolated from the specified mice and stimulated with anti-CD3 plus anti-CD28 antibodies for 72 h. The percentages of proliferating CTV^low T cells are indicated. (F) Numbers of CD4⁺ T cells found in the spleen and lungs of WT, Lat^Y136F, Lat^Y136F Cd5^−/−, Lat^Y136F Cd6^−/−, and Lat^Y136F Cd5^−/−Cd6^−/− mice. Data in A–E are representative of at least three independent experiments, whereas data in F correspond to the pool of three independent experiments. In B and C, each dot corresponds to a mouse, and the mean (horizontal bar) is indicated. n.s., nonsignificant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Error bars correspond to the mean and SD.

Figure S5.

Augmented TCR-mediated activation in primary WT CD4⁺ T cells rendered CD5 deficient. (A) Workflow used for CD5, CD6, or EGFP gene deletion in CD4⁺ T cells from Cas9-expressing mice. T cells from Cas9-expressing mice (Platt et al., 2014) also express EGFP and were used as control after EGFP gene inactivation. (B) Expression of CD5, CD6, and EGFP on CD4⁺ T cells from Cas9-expressing mice nucleofected with sgRNA targeting CD5, CD6, or EGFP (see Table S1). Cells were analyzed by flow cytometry 3 d after nucleofection, and histograms show the levels of CD5, CD6, and EGFP on CD4⁺ T cells nucleofected with the specified sgRNA. Percentages of EGFP⁻, CD5⁻, and CD6⁻ cells are shown. (C) Percentages of CTV^low (left) and CD69⁺ (right) cells in CD4⁺ T cells nucleofected with sgCD5⁻, sgCD6⁻, or sgGFP 7 d before and stimulated for 48 h with the specified concentrations of plate-bound anti-CD3 in the presence of soluble anti-CD28 antibodies (1 µg/ml). Data are representative of at least two independent experiments. Error bars correspond to the mean and SD. Sg, single guide.

Figure 6.

Transcriptional changes occurring in primary CD4⁺ T cells after engaging the TCR–CD28 pathways in the absence of LAT, of CD5, of CD6, or of both CD5 and CD6. (A) PCA of gene expression in WT, Cd5^−/−, Cd6^−/−, and Cd5^−/−Cd6^−/− primary CD4⁺ T cells that were left unstimulated (0 h) or stimulated for 20 h with anti-CD3 plus anti-CD28. (B) PCA of gene expression in WT and Lat^−/− primary CD4⁺ T cells that were left unstimulated (0 h) or stimulated for 20 h with anti-CD3 plus anti-CD28 (20 h). (C) Quantification of the genes significantly induced (UP) or repressed (DOWN) in WT, Cd5^−/−, Cd6^−/−, and Cd5^−/−Cd6^−/− CD4⁺ T cells after stimulation for 20 h with anti-CD3 plus anti-CD28. (D) Quantification of the genes significantly induced (UP) or repressed (DOWN) in WT and Lat^−/− CD4⁺ T cells after stimulation for 20 h with anti-CD3 plus anti-CD28. See Data S3 for details on the induced or repressed genes quantified in C and D. In A–D, three independent samples were prepared for each condition. In A and B, numbers shown in parentheses on the PCA1 and PCA2 axes indicate the percentage of overall variability in the dataset along each PC axis. Lat^−/− CD4⁺ T cells were obtained using Lat^fl-dtr maT-Cre mice (Roncagalli et al., 2014).

Figure 7.

A quantitative model of early TCR signal diversification integrating interactomics and transcriptomics. (A) Tyrosine residues (red dots) present in the intracytoplasmic segments of CD5, CD6, and LAT are phosphorylated by the LCK or ZAP70 PTK that associate with active TCR (dashed yellow arrows). Following 30 s of TCR engagement, distinct signalosomes nucleated around the tyrosine phosphorylated CD5 and CD6 transmembrane receptors and the LAT transmembrane adaptor. The number of copies per T cell of CD5, CD6, and LAT is indicated. For instance, 41,443 copies of LAT are expressed per T cell. The maximum copies per T cell of high-confidence bait–prey complexes reached over the course of TCR stimulation is also shown and specified over the arrows connecting the baits and the preys. For instance, the maximum number of CD5–UBASH3A complexes reached per T cell over the course of TCR stimulation is ∼989 (see Fig. 2 D legend and Data S2). Solid arrows correspond to TCR-induced interactions, whereas dotted arrows correspond to constitutive interactions. Consistent with former studies (Roncagalli et al., 2014; Voisinne et al., 2019), after cutoff values were slightly relaxed, VAV1 was found to interact significantly and in a TCR-inducible manner with both CD6 (5.0-fold enrichment; P value: 3.2 × 10⁻⁵) and LAT (8.5-fold enrichment; P value: 4.8 × 10⁻⁶), reaching in both cases maximum binding 30 s after TCR engagement (Data S2). CD5 showed a transient binding with MYB-binding protein 1A (MYBBP1A), a transcriptional regulator that shuttles between the cytoplasm and the nucleus, and the role within the CD5 interactome remains to be determined. Inset: Lifetime of the TCR-induced interactions involving CD5, CD6, and LAT with most of their interactors. (B) The Venn diagram illustrates the commonalities and differences between the CD5, CD6, and LAT signalosomes as well as the functional outcomes expected to result from their engagement at the IS.