Computational reconstruction of CD4 evolution

The maximum likelihood phylogenetic tree clusters mammalian CD4 sequences. The tree highlights ancestral reconstructions of CD4 sequences from marsupials (kangaroo silhouette), Atlantogenata (elephant), and Boreoeutheria (wildcat). The logoplot of extant eutherian (Atlantogenata and Boreoeutheria) CD4 sequences show sequence conservation over evolutionary time. In these plots, the height of symbols indicates the relative frequency of each amino acid at that specific position. The mouse CD4 numbering (uniprot) is used as a reference, and residues are color-coded based on sidechain polarity. Evolutionary insertion or deletion events are indicated by dashes (-). Most recent common ancestor (MRCA) sequences are shown at each node in the tree (Node 1-4). As in our previous study (Lee et al., 2022), the ratio of synonymous (dS) and nonsynonymous (dN) substitution rates was calculated. Black dots indicate dN/dS ratios that are significantly below 1 across the entire dataset. Red dots indicate residues under purifying selection in the mammalian only dataset. Previously identified motifs are indicated by boxes, while the intracellular domain helix is shaded gray. The arrow at position 422 indicates where CD4 was truncated (CD4-T1), while TMD and T1-Palm show the mutations studied in this study.

Motifs and mutants analyzed in this study

Truncating CD4 does not reduce CD3ζ phosphorylation

(A) Representative IL-2 production is shown in response to a titration of MCC peptide from one experiment (left). Experiments were performed in triplicate and each symbol equals the mean +/− SEM at that peptide concentration. AUC analysis for the dose response is shown as a measure of the response magnitude for the average of three independent experiments performed with one independently generated set of cell lines (center). The average response to a low dose (41nM) of peptide is shown as a measure of sensitivity for three independent experiments performed with one independently generated set of cell lines (right). The data are representative of those obtained with 4 (WT vs T1) or 2 (WT vs T1Δbind) independently generated sets of cell lines.

(B) Phosphorylation intensity of CD3ζ (pCD3ζ) for WT and CD4-T1 (T1) (left), normalized pCD3ζ intensity for WT and T1 (center), and normalized % responders of pCD3ζ for WT and T1 (right). Five independently generated cell lines (biological replicates) were tested for WT and T1. For phosphorylation intensity, each pair of lines (connected symbols) was tested in three independent experiments. Data were analyzed and collected as in Figure 2 – figure supplement 3. One-way ANOVA was performed with a Dunnett’s posttest using GraphPad Prism9. For normalized intensity and % responders, all individual mutant cell line values were normalized to their paired control values. Bars represent the mean +/− SEM. One-way ANOVA was performed with a Sidak’s posttest for specific comparisons of normalized values using GraphPad Prism9.

(C) Phosphorylation intensity of pCD3ζ for T1 and T1Δbind (left), normalized pCD3ζ intensity for T1 and T1Δbind (center), and normalized % responders of pCD3ζ for T1 and T1Δbind (right) were performed and analyzed as in B, with the exception that the open symbols represent data from a single experiment whereas the closed symbols represent aggregate data from three independent experiments. Two-tailed t tests were performed to compare the single T1 vs T1Δbind samples as no other samples were collected in parallel.

All values presented as percent of WT control (truncation average/WT average x 100) for normalized values

All values presented as percent of T1 control (mutant average/T1 average x 100) for normalized values

The GGXXG and CV+C motifs are key determinants of CD4 function

(A, B) Representative IL-2 production is shown in response to a titration of MCC peptide from one experiment (left). Experiments were performed in triplicate and each symbol equals the mean +/− SEM at that peptide concentration. AUC analysis for the dose response is shown as a measure of the response magnitude for the average of three independent experiments performed with one independently generated set of cell lines (center). The average response to a low dose (41nM) of peptide is shown as a measure of sensitivity for three independent experiments performed with one independently generated set of cell lines (right). Results are representative of those obtained with at least three independently generated sets of cell lines. One-way ANOVA was performed with a Dunnett’s posttest for comparisons with WT and T1 samples, and a Sidak’s posttest for comparisons between selected samples.

The GGXXG and CV+C motifs reduce pCD3ζ levels

(A) Phosphorylation intensity of CD3ζ for T1 and T1-TMD (left), T1 and T1-Palm (2C) (center), and T1 and T1-TP (2C) (right) are shown for independently generated pairs of (connecting line) T1 and T1-TMD (left), T1 and T1-Palm (2C) (center), and T1 and T1-TP (2C) (right) cell lines. Each pair of lines (connected closed symbols) was tested in three independent experiments (technical replicates). The data from those experiments was aggregated, and the symbols represent the mean intensity of the aggregated pCD3ζ intensity values. One-way ANOVA was performed with a Dunnett’s posttest using GraphPad Prims 9.

(B) Data for each cell line in A are shown as the average pCD3ζ intensity for all T1-TMD (left), T1-Palm (2C) (center), and T1-TP (2C) (right) cell lines normalized to their paired T1 control. Dotted line is the normalized pCD3ζ intensity for T1Δbind as a visual reference point for the contributions of CD4-pMHCII interactions. Bars represent the mean +/− SEM. One-way ANOVA was performed with a Sidak’s posttest for specific comparisons using GraphPad Prims 9.

(C) The average % responders for phosphorylation of CD3ζ is shown for T1-TMD (left), T1-Palm (2C) (center), and T1-TP (2C) (right) normalized to their paired T1 control line. Bars represent the mean +/− SEM. One-way ANOVA was performed with a Sidak’s posttest for specific comparisons using GraphPad Prims 9.

The (C/F)CVRC motifs reduces proximal TCR-CD3 signaling

(A) Phosphorylation intensity of CD3ζ for T1 and T1-Palm (3C) (left) and T1 and T1-TP (3C) (right) are shown for independently generated pairs of (connecting lines) T1 and T1-Palm (3C) (left) and T1 and T1-TP (3C) (right) cell lines. Analysis was performed as in Figure 4A.

(B) Data for each mutant cell line in A are shown as the average pCD3ζ intensity values for T1-Palm (3C) (left) and T1-TP (3C) (right) normalized to their paired T1 controls. The dotted line represents the normalized phosphorylation CD3ζ intensity for T1Δbind as a visual reference for the contributions of CD4-pMHCII interactions. Analysis was performed as in Figure 4C.

(C) Average % responders for phosphorylation of CD3ζ is shown for T1-Palm (3C) (left) and T1-TP (3C) (right) normalized to their paired T1 control. Analysis was performed as in Figure 4C.

Flow cytometry analysis of CD4 (left) and TCR (right) expression on 58α-β- hybridoma cells. Parental 58α-β- hybridoma cells served as negative control for surface expression (open black histogram trace).

TCR (left) and CD4 (right) endocytosis after pMHCII engagement is shown for the indicated cell lines after 16 hours coculture with APCs in the presence of 10μM MCC peptide. The change in TCR and CD4 gMFI, as measured by flow cytometry, is shown for each cell line relative to an equivalent sample cultured with APCs in the absence of MCC peptide. Each data point represents the mean ± SEM for three independent experiments. For endocytosis measurements were performed in triplicate for each experiment. One-way ANOVA was performed with a Dunnett’s posttest.

Example of intracellular signaling analysis workflow.

(A) Flow cytometry analysis of WT 58α-β- hybridoma:M12 cell couples. Representative dot plots are shown for TCRβGFP+ CD4+ 58α-β- hybridoma cells coupled to Tag-it Violet- labeled M12s expressing the indicated tethered pMCHII Hb:I-Ek (left) and MCC:I-Ek (center). Representative histograms of WT 58α-β- hybridoma cells coupled to M12 cells transduced to express the indicated tethered pMHCII are shown for pCD3ζ intensity (right). 10,000 coupled cells were collected per individual experiment.

(B) Flow cytometry analysis of T1 58α-β- hybridoma-M12 cell couples. Representative dot plots are shown for TCRβGFP+ CD4+ 58α-β- hybridoma cells coupled to Tag-it Violet- labeled Hb:I-Ek+ (left) and MCC:I-Ek+ (center) M12 cells. Representative histograms of T1 58α-β- hybridoma cells coupled to M12 cells expressing the indicated tethered pMHCII are shown for pCD3ζ intensity (right). 10,000 coupled cells were collected per individual experiment.

(C) A representative smoothed overlapping histogram of pCD3ζ intensity is shown for 58α-β- cells coupled to Hb:I-Ek+ (cyan) or MCC:I-Ek+ (black) M12 cells. Histogram of pCD3ζ intensity for 58α-β- cells coupled to MCC:I-Ek+ M12 cells subtracted from Hb:I- Ek+ M12 cell couples show the difference in pCD3ζ intensity on a bin-by-bin basis after stimulation with agonist MCC:I-Ek compared with null Hb:I-Ek for WT (left) and T1 (center) cells. Overlapping pCD3ζ histogram (right) of cells responding to MCC:I-Ek after Hb:I-Ek subtraction shows the responding populations for the WT and T1 cell lines. Data represent the aggregate from three individual experiments (30,000 couples analyzed). One-way ANOVA was performed with a Dunnett’s posttest for comparison with the WT sample because other mutants were simultaneously analyzed in this experiment (not shown).

(D) Concatenated pCD3ζ average intensity ± SEM of WT and T1 cells (left) and the percent of responding WT and T1 cells (right).

(A) Zap70 (left), and Plcγ1 (right) phosphorylation intensity for WT and T1 are shown for paired (connecting line) WT and T1 cell lines. Five independently generated cell lines were tested (biological replicates). For phosphorylation intensity, each pair of lines (connected symbols) was tested in three independent experiments (technical replicates). The data from those experiments was aggregated, and the symbols represent the mean intensity of the aggregated pCD3ζ intensity values. One-way ANOVA was performed with a Dunnett’s posttest.

(B) Average T1 phosphorylation for Zap70 (left) and Plcγ1 (right) from A are shown normalized to the paired WT controls. Bars represent the mean +/- SEM. One-way ANOVA was performed with a Sidak’s posttest for specific comparisons.

(C) Average T1 % responders for pZap70 (left), and pPlcγ1 (right) are shown normalized to the paired WT controls for the cell lines shown in A. Bars represent the mean +/- SEM. One-way ANOVA was performed with a Sidak’s posttest for specific comparisons.

(A) Phosphorylation intensity for T1 and T1Δbind Zap70 (left), and Plcγ1 (right) are shown for paired (connecting line) T1 and T1Δbind cell lines. Three independently generated cell lines were tested (biological replicates). Analysis was performed as in Figure 2— figure supplement 4 with the exception that the open symbol represent data from a single experiment whereas the closed symbols represent the average of aggregated data from three independent experiments. For the open symbol comparisons we performed a two- tailed t test as no other samples were collected in parallel.

(B) Average T1Δbind cell line phosphorylation intensity for Zap70 (left), and Plcγ1 (right) are shown normalized to the average intensity of the paired T1 control cells shown in A. Analysis was performed as in Figure 2— figure supplement 4.

(C) Average T1Δbind cell line % responders for pZap70 (left) and pPlcγ1 (right) are shown normalized to their paired T1 control. Analysis was performed as in Figure 2— figure supplement 4.

(A, B) Flow cytometry analysis of CD4 (left) and TCR (right) expression on 58α-β- hybridoma cells. Parental 58α-β- hybridoma cells served as negative control for surface expression (open black histogram trace).

(A, B) Lck signal is shown for each sucrose fraction normalized to the CD4 signal detected in the corresponding fraction (left). The AUC is shown for the normalized Lck signal in the DRM (center) and DSM (right) fractions. Each data point represents the mean +/- SEM for three independent experiments with the same cell line. Data are representative of experiments performed with three independently generated sets of lines for TMD, Palm(3C), and TP(3C) mutants. Analysis was performed with two set of lines for the Palm(2C) and TP(2C) mutants. One-way ANOVA was performed with a Dunnett’s posttest for comparisons with WT and T1 samples, and a Sidak’s posttest for comparisons between selected samples.

(A, B) Total Lck signal (total AUC for sucrose gradient) normalized to CD4 signal is shown for the indicated cell lines. Each data point represents the mean +/- SEM for three independent experiments with the same cell line (experimental replicates). Data are representative of experiments performed with three independently generated sets of lines for TMD, Palm(3C), and TP(3C) mutants (biological replicates). Analysis was performed with two set of lines for the Palm(2C) and TP(2C) mutants. One-way ANOVA was performed with a Dunnett’s posttest for comparisons with WT and T1 samples, and a Sidak’s posttest for comparisons between selected samples.

(A, B) TCR (left) and CD4 (right) endocytosis after pMHCII engagement is shown for the indicated cell lines after 16 hours coculture with APCs in the presence of 10μM MCC peptide. The change in TCR and CD4 gMFI, as measured by flow cytometry, is shown for each cell line relative to an equivalent sample cultured with APCs in the absence of MCC peptide. Each data point represents the mean ± SEM for three independent experiments (experimental replicates). Data are representative of those acquired with at least two independently generated sets of cell lines (biological replicates). Endocytosis measurements were performed in triplicate (technical replicates) for each experiment. One-way ANOVA was performed with a Dunnett’s posttest for comparisons with WT and T1 samples.

(A) Phosphorylation intensity of Zap70 for T1 and T1-TMD (left), T1 and T1-Palm (2C) (center), and T1 and T1-TP (2C) (right) are shown for paired (connecting line) cell lines. Analysis was performed as in Figure 4A.

(B) Normalized phosphorylation intensity of Zap70 for T1-TMD (left), T1-Palm (2C) (center), and T1-TP (2C) (right) are shown as bars. Analysis was performed as in Figure 4B.

(C) Normalized % responders of the phosphorylation Zap70 for T1-TMD (left), T1-Palm (2C) (center), and T1-TP (2C) (right) are shown as bars for T1 and T1Δbind cell lines. Analysis was performed as in Figure 4C.

(A) Phosphorylation intensity of Plcγ1 for T1 and T1-TMD (left), T1 and T1-Palm (2C) (center), and T1 and T1-TP (2C) (right) are shown for paired (connecting line) cell lines. Analysis was performed as in Figure 4A.

(B) Normalized phosphorylation intensity of Plcγ1 for T1-TMD (left), T1-Palm (2C) (center), and T1-TP (2C) (right) are shown as bars. Analysis was performed as in Figure 4B.

(C) Normalized % responders of phosphorylated pPlcγ1 for T1-TMD (left), T1-Palm (2C) (center), and T1-TP (2C) (right) are shown as bars for T1 and T1Δbind cell lines. Analysis was performed as in Figure 4C.

Representative IL-2 production is shown in response to M12 cells transduced to express tethered MCC:I-Ek constructs as used in phosphorylation analysis. Experiments were performed in triplicate and each bar equals the mean +/- SEM at that peptide concentration. Results are representative of those obtained with two independently generated matched sets of cell lines. One-way ANOVA was performed with a Dunnett’s posttest for comparisons with WT and T1 samples, and a Sidak’s posttest for comparisons between selected samples.

(A) Phosphorylation intensity of Zap70 for T1 and T1-Palm (3C) (left) and T1 and T1-TP (3C) (right) are shown for paired (connecting line) cell lines. Analysis was performed as in Figure 5A.

(B) Normalized phosphorylation intensity of Zap70 for T1-Palm (3C) (left) and T1-TP (3C) (right) are shown as bars. Analysis was performed as in Figure 5B.

(C) Normalized % responders of phosphorylated Zap70 for T1-Palm (3C) (left) and T1- TP (3C) (right) are shown as bars. Analysis was performed as in Figure 5C.

(A) Phosphorylation intensity of Plcγ1 for T1 and T1-Palm (3C) (left) and T1 and T1-TP (3C) (right) are shown for paired (connecting line) cell lines. Analysis was performed as in Figure 5A.

(B) Normalized phosphorylation intensity of Plcγ1 for T1-Palm (3C) (left) and T1-TP (3C) (right) are shown as bars. Analysis was performed as in Figure 5B.

(C) Normalized % responders of phosphorylated Plcγ1 for T1-Palm (3C) (left) and T1- TP (3C) (right) are shown as bars. Analysis was performed as in Figure 5C.