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 average % 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, all individual mutant cell line values were normalized to their paired control values. Average percent responders are presented. Bars represent the mean +/- SEM. One-way ANOVA with paired comparisons was performed with a Sidak’s posttest for specific comparisons of normalized values using GraphPad Prism9. All generated cells lines were considered for the multiple comparisons.

(C) Phosphorylation intensity of pCD3ζ for T1 and T1Δbind (left), normalized pCD3ζ intensity for T1 and T1Δbind (center), and % 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) compared to the average of their paired T1 control lines.

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) are shown compared to the average of their paired T1 control. Analysis was performed as in Figure 4C.