Signal transduction by different forms of the γδ T cell-specific pattern recognition receptor WC1

Abstract

WC1 coreceptors are scavenger receptor cysteine-rich (SRCR) family members, related to T19 in sheep, SCART in mice, and CD163c-α in humans, and form a 13-member subfamily in cattle exclusively expressed on γδ T cells. Subpopulations of γδ T cells are defined by anti-WC1 mAbs and respond to different pathogen species accordingly. In this study, variegated WC1 gene expression within subpopulations and differences in signaling and cell activation due to endodomain sequences are described. The endodomains designated types I to III differ by a 15- or 18-aa insert in type II and an additional 80 aa containing an additional eight tyrosines for type III. Anti-WC1 mAbs enhanced cell proliferation of γδ T cells when cross-linked with the TCR regardless of the endodomain sequences. Chimeric molecules of human CD4 ectodomain with WC1 endodomains transfected into Jurkat cells showed that the tyrosine phosphorylation of the type II was the same as that of the previously reported archetypal sequence (type I) with only Y24EEL phosphorylated, whereas for type III only Y199DDV and Y56TGD were phosphorylated despite conservation of the Y24EEL/Y24QEI and Y199DDV/I tyrosine motifs among the three types. Time to maximal phosphorylation was more rapid with type III endodomains and sustained longer. Differences in tyrosine phosphorylation were associated with differences in function in that cross-linking of type III chimeras with TCR resulted in significantly greater IL-2 production. Identification of differences in the signal transduction through the endodomains of WC1 contributes to understanding the functional role of the WC1 coreceptors in the γδ T cell responses.

FIGURE 1.

Alignments of 13 WC1 endodomain-deduced amino acid sequences. WC1 endodomain-deduced amino acid sequences were aligned with ClustalW2 using the default parameters and visualized with JalView. Analysis includes all nonredundant genomic sequences as described previously (16, 19). Identity is indicated by periods (.) and gaps by hyphens (-). The small vertical lines above the sequence indicate approximate exon boundaries that may occur within the codon for some amino acids.

FIGURE 2.

Subpopulations of WC1⁺ γδ T cells defined by expression of WC1 molecules with different types of WC1 endodomains. (A) Ab CACT21A was evaluated for reactivity against the myc-tagged domain 1 proteins of 13 WC1 molecules in ELISA and immunoblotting. Negative controls included no Ag (no Ag). The OD values represent the mean ± SEM for three independent experiments. (B) PBMC stained with anti-WC1.1 mAb BAG25A, anti-WC1.2 mAb CACTB32A, and anti-WC1.3 mAb CACT21A were analyzed via flow cytometry (100,000 events). The left panel shows the PBMC staining with mAb BAG25A and mAb CACTB32A. The right panel shows the PBMC staining with mAb BAG25A and mAb CACT21A. The gated populations are indicated in the figure. (C) Zmol of WC1 gene transcripts present in the WC1.1⁺/WC1.2⁻ subpopulation (BAG25A⁺/CACTB32A⁻, empty bars), the WC1.1⁻/WC1.2⁺ subpopulation (BAG25A⁻/CACTB32A⁺, gray bars), the WC1.3 subpopulation (mAb CACT21A⁺, black bars), and the WC1.1⁺/WC1.2⁺ subpopulation (BAG25A⁺/CACTB32A⁺ striped bars). Mean values ± SE for n = 5 replicates from two independent experiments are represented, and significant differences (p < 0.05, one-way ANOVA with Bonferroni posttest, Prism 5, GraphPad) between any 2 of 13 WC1 transcripts within a subpopulation are indicated by asterisks.

FIGURE 3.

Proliferation of ex vivo primary bovine γδ T cell subpopulations induced by Abs that react with WC1 with various endodomains. (A) A representation of the gating strategy to determine dividing cells in the various subpopulations is shown using Dye670-loaded PBMC and culturing with Con A. (B) Dye670-PBMC were cultured for 3 d with medium only, plate-bound anti-TCR mAb (1 μg/ml) only, or plate bound anti-TCR (1 μg/ml) plus anti-WC1 mAb (anti-WC1.1 mAb BAG25A, 10 μg/ml; anti-WC1.2 mAb CACTB32A, 10 μg/ml; anti-WC1.3 mAb CACT21A, 10 μg/ml; or anti-pan WC1⁺ mAb ILA29, 10 μg/ml). Cells were gated for viability and WC1.1⁺ (BAG25A⁺), WC1.2⁺ (CACTB32A⁺), and WC1.3⁺ (CACT21A⁺) subpopulations before plotting for Dye670 intensity. These results are representative of two experiments. (C) Summary of the proliferation of WC1.1⁺ (BAG25A⁺), WC1.2⁺ (CACTB32A⁺), and WC1.3⁺ (CACT21A⁺) subpopulations stimulated by Abs that react with WC1 gene products, as detailed in Table I.

FIGURE 4.

Tyrosine motifs of WC1 type I, II, and III endodomains. (A) Deduced amino acid sequence from transmembrane regions and intracytoplasmic tails of WC1-9, WC1-11, and the archetypal WC1 sequence (WC1-3) were aligned using ClustalW2 using the default parameters and refined manually. GenBank accession numbers (https://www.ncbi.nlm.nih.gov/genbank/) for amino acid sequences used for comparison are as follows: WC1-3 type I endodomain (archetypal WC1.1; X63723; WC1-3), WC1-9 type II endodomain (FJ031208; WC1-9), and WC1-11 type III endodomain (FJ031209; WC1-11). Identities are indicated by periods (.) and gaps by hyphens (-). The five tyrosines shared by WC1-3 and WC1-9 (rectangles) and the eight tyrosine residues in WC1-11 (arrowheads) are indicated. Amino acids are numbered from the beginning of the cytoplasmic tail. The tyrosine residues that were found to be the main targets of tyrosine kinases in this study were indicated by asterisks. (B) Jurkat cells were infected with lentivirus encoding CD4/WC1-3 WT, CD4/WC1-9 WT, CD4/WC1-9 Y24F (second tyrosine mutated to phenylalanine), CD4/WC1-11 WT, or CD4/WC1-11 Y199F (eighth tyrosine mutated to phenylalanine) individually. Empty lentiviral vector was used as a control. Transduced cells were enriched by flow cytometry based on their GFP expression and stained with PE-conjugated mouse anti-human CD4. (C) Cell membrane extracts of 293T cells transfected with 2 μg each of pBK-CMV CD4/WC1-9 and CD4/WC1-11 constructs were immunoblotted with anti-phosphotyrosine mAb 4G10. Membranes were stripped and reprobed with rabbit anti-human CD4 Ab to determine the amount of CD4/WC1 proteins expressed. The amount of phosphotyrosine signal was normalized to the amount of CD4/WC1 proteins detected by anti-CD4 Ab. The phosphorylation intensity of each chimeric protein was measured, and the ratio of anti-phosphotyrosine signal to anti-CD4 signal (area density ratio) was determined and represented as mean ± SD for three experiments. Significant differences between WT and mutant CD4/WC1, as determined by t test (one tailed), are marked by asterisks, and each corresponding p value is shown above the bar.

FIGURE 5.

Kinetics of tyrosine phosphorylation varies depending upon the type of WC1 endodomain expressed. Jurkat T cells expressing CD4/WC1-3 (type I), CD4/WC1-9 (type II), or CD4/WC1-11 (type III) were cross-linked by anti-CD3 plus anti-CD4 or cross-linked with anti-CD4 alone. The intensity of phosphorylation at the indicated time points was normalized to the amount of CD4/WC1 chimeric protein and annotated as fold increase over the normalized phosphorylation intensity at the 0-min time point. The images shown are representative of at least two experiments. Bar graphs show the relative phosphorylation intensity of three different chimeric proteins (CD4/WC1-3, CD4/WC1-9, and CD4/WC1-11) at the indicated time points after cross-linking by anti-CD3 plus anti-CD4 mAb or cross-linking by anti-CD4 mAb alone. Values are means ± SD for three experiments. Significant differences (p < 0.05, one-way ANOVA with Bonferroni posttest) among the three different chimeric proteins are indicated by asterisks.

FIGURE 6.

The enhancement of IL-2 production varies among Jurkat T cells expressing three different types of WC1 endodomains. Jurkat T cells (2 × 10⁵) transduced with empty vector (control) or CD4/WC1 chimeric proteins were stimulated with PMA (50 ng/ul) and ionomycin (200 ng/ml) (A), or plate-bound anti-CD3 mAb (1 μg/ml) only, or plate-bound anti-CD3 (1 μg/ml) plus anti-CD4 mAb (5 ug/ml) for 48 h (B and C). IL-2 levels in supernatants were measured from triplicate cultures. Means ± SD for three independent experiments using populations of infected cells are plotted. Significant differences between WT CD4/WC1-transduced cells stimulated with anti-CD3 alone or by anti-CD3 and anti-CD4 or between WT CD4/WC1-transduced cells and mutant CD4/WC1 transduced stimulated by anti-CD3 and anti-CD4, as determined by a t test (two-tailed), are marked by asterisks (p < 0.05). (C) Enhanced IL-2 production is calculated by subtracting IL-2 production by control cells cross-linked with anti-CD3 plus anti-CD4 from IL-2 production by cells expressing CD4/WC1 proteins cross-linked with anti-CD3 plus anti-CD4. Significant differences between CD4/WC1-transduced cells stimulated with anti-CD3 and anti-CD4, as determined by a t test (two tailed), are marked by asterisks (p < 0.05).