The CD4 transmembrane GGXXG and juxtamembrane (C/F)CV+C motifs mediate pMHCII-specific signaling independently of CD4-Lck interactions

Reviewer #1 (Public Review):

Summary:

This study by Lee et al. is a direct follow-up on their previous study that described an evolutionary conservancy among placental mammals of two motifs (a transmembrane motif and a juxtamembrane palmitoylation site) in CD4, an antigen co-receptor, and showed their relevance for T-cell antigen signaling. In this study, they describe the contribution of these two motifs to the CD4-mediated antigen signaling in the absence of CD4-LCK binding. Their approach was the comparison of antigen-induced proximal TCR signaling and distal IL-2 production in 58-/- T-cell hybridoma expressing exogenous truncated version of CD4 (without the interaction with LCK), called T1 with T1 version with the mutations in either or both of the conserved motifs. They show that the T1 CD4 can support signaling to the extend similar to WT CD4, but the mutation of the conserved motifs substantially reduced the signaling. The authors conclude that the role of these motifs is independent of the LCK-binding.

Strengths:
The authors convincingly show that T1 CD4, lacking the interaction with LCK supports the TCR signaling and also that the two studied motifs have a significant contribution to it.

Weaknesses:
The study has several weaknesses.

1. The whole study is based on a single experimental system, genetically modified 58-/- hybridoma. It is unclear at this moment, how the molecular motifs studied here contribute to the signaling in a real T cell. The evolutionary conservancy suggests that these motifs are important for T cell biology. However, the LCK-binding motif is conserved as well (perhaps even more) and it plays a very minor role in their model. Without verifying their results in primary cells, the quantitative, but even qualitative, importance of these motifs for T-cell signaling and biology is unclear. Although the authors discuss this issue in the Discussion, it should be noted in all important parts of the manuscript, where conclusions are made (abstract, end of introduction, perhaps also in the title) that the results are coming from the hybridoma cells.

2. Many of the experiments lack the negative control. I believe that two types of negative controls should be included in all experiments. First, hybridoma cells without CD4 (or with CD4 mutant unable to bind MHCII). Second, no peptide control, i.e., activation of the hybridoma cells with the APC not loaded with the cognate peptide. These controls are required to distinguish the basal levels of phoshorylation and CD4-independent antigen-induced phosphorylation to quantify, what is the contribution of the particular motifs to the CD4-mediated support. Although these controls are included in some of the experiments, they are missing in other ones. The binding mutant appears in some FC results as a horizontal bar (without any error bar/variability), showing that CD4 does not give a huge advantage in these readouts. Why don't the authors show no peptide controls here as well? Why the primary FC data (histograms) are not shown? Why neither of these two controls is shown for the % of responders plots? Although the IL-2 production is a very robust and convincing readout, the phosphoflow is much less sensitive. It seems that the signaling is elevated only marginally. Without the mentioned controls and showing the raw data, the precise interpretation is not possible.

3. The processing of the data is not clear. Some of the figures seem to be overprocessed. For instance, I am not sure what "Normalized % responders of pCD3zeta" means (e.g., Fig. 1C and elsewhere)? Why do not the authors show the actual % of pCD3zeta+ cells including the gating strategy? Why do the authors subtract the two histograms in Fig. 2- Fig.S3? It is very unusual.

4. The manuscript lacks Materials and Methods. It only refers to the previous paper, which is very unusual. Although most of the methods are the same, they still should be mentioned here. Moreover, some of the mutants presented here were not generated in the previous study, as far as I understand. Perhaps the authors plan to include Materials and Methods during the revision...

5. Membrane rafts are a very controversial topic. I recommend the authors stick to the more consensual term "detergent resistant microdomains" in all cases/occurances.

6. Last, but not least, the mechanistic explanation (beyond the independence of LCK binding) of the role of these motifs is very unclear at the moment.

Reviewer #2 (Public Review):

Summary:
The paper by Kuhn and colleagues follows upon a 2022 paper in which they identified residues in CD4 constrained by evolutionary purifying selection in placental mammals and then performed functional analyses of these conserved sequences. They showed that sequences distinct from the CXC "clamp" involved in recruitment of Lck have critical roles in TCR signaling, and these include a glycine-rich motif in the transmembrane (TM) domain and the cys-containing juxtamembrane (JM) motif that undergoes palmitoylation, both of which promote TCR signaling, and a cytoplasmic domain helical motif, also involved in Lck binding, that constrains signaling. Mutations in the transmembrane and juxtamembrane sequences led to reduced proximal signaling and IL-2 production in a hybridoma's response to antigen presentation, despite retention of abundant CD4 association with Lck in the detergent-soluble membrane fraction, presumably mislocalized outside of lipid rafts and distal to the TCR. A major conclusion of that study was that CD4 sequences required for Lck association, including the CXC "clasp" motif, are not as consequential for CD4 co-receptor function in TCR signaling as the conserved TM and JM motifs. However, the experiments did not determine whether the functions of the TM and JM motifs are dependent on the Lck-binding properties of CD4 - the mutations in those motifs could result in free Lck redistributing to associate with CD4 in signaling-incompetent membrane domains or could function independently of CD4-Lck association. The current study addresses this specific question.

Using the same model system as in the earlier paper (the entire methods section is a citation to the earlier paper), the authors show that truncation of the Lck-binding intracellular domain resulted in a moderate reduction in IL-2 response, as previously shown, but there was no apparent effect on proximal phosphorylation events (CD3z, Lck, ZAP70, PLCg1). They then evaluated a series of TM and JM motif mutations in the context of the truncated Lck-nonbinding molecule, and showed that these had substantially impaired co-receptor function in the IL-2 assay and reduced proximal signaling. The proximal signaling could be observed at high ligand density even with a MHC non-binding mutation in CD4, although there was still impaired IL-2 production. This result additionally illustrates that phosphorylation of the proximal signaling molecules is not sufficient to activate IL-2 expression in the context of antigen presentation.

Strengths:
The strength of the paper is the further clear demonstration that the classical model of CD4 co-receptor function (MHCII-binding CD4 bringing Lck to the TCR complex, for phosphorylation of the CD3 chain ITAMs and of the ZAP70 kinase) is not sufficient to explain TCR activation. The data, combined with the earlier paper, further implicate the gly-rich TM sequence and the palmitoylation targets in the JM region as having critical roles in productive co-receptor-dependent TCR activation.

Weaknesses:
The major weakness of the paper is the lack of mechanistic insight into how the TM and JM motifs function. The new results are largely incremental in light of the earlier paper from this group as well as other literature, cited by the authors, that implicates "free" Lck, not associated with co-receptors, as having the major role in TCR activation. It is clear that the two motifs are important for CD4 function at low pMHCII ligand density. The proposal that they modulate interactions of TCR complex with cholesterol or other membrane lipids is an interesting one, and it would be worth further exploring by employing approaches that alter membrane lipid composition. The JM sequence presumably dictates localization within the membrane, by way of palmitoylation, which may be critical to regulate avidity of the TCR:CD4 complex for pMHCII or TCR complex allosteric effects that influence the activation threshold. Experiments that explore the basis of the mutant phenotype could substantially enhance the impact of this study.

Author Response

We thank the Editors and the Reviewers for the time spent on our manuscript entitled “The CD4 transmembrane GGXXG and juxtamembrane (C/F)CV+C motifs mediate pMHCII-specific signaling independently of CD4-Lck interactions”. We appreciate the helpful feedback and the opportunity to participate in eLife’s new model for publishing.

We are writing to provide the following provisional author responses for posting with the first version of the reviewed preprint:

1. To address comments about the limited scope of this study and referencing of the Methods section to our prior study, we would like to note that we submitted the current study via the Research Advance mechanism. Our goal was to build upon the conclusions of our 2022 eLife publication (PMID: 35861317) and address an unresolved question from that study (as nicely summarized by Reviewer #2). In the current manuscript we present data from reductionist experiments that were designed specifically for this purpose and, as noted by the reviewers, we provide answers to the question being asked. We think that the Research Advance mechanism is an ideal opportunity to make these results available to the field given the stated purpose of such articles (for reference: “A Research Advance might use a new technique or a different experimental design to generate results that build upon the conclusions of the original research by, for example, providing new mechanistic insights or extend the pathway under investigation…”).

a. The Methods were not duplicated in this manuscript because we referenced our prior study as per instructions for the Research Advance mechanism.

1. The constituent residues of the motifs analyzed in this and our prior study were determined to be functionally significant in vivo through the computational reconstruction of CD4’s evolutionary history, which provided us with data from ~435 million years of natural experiments with CD4 in numerous jawed vertebrate species. We agree that having conditional knock-in mice of these CD4 mutants, and those characterized in our last study, would be useful for determining how these mutations impact T cell development, activation, differentiation, and effector function. Given the costs involved with making genetically engineered mouse model systems, the computational and experimental data we have generated in the current and prior study will help us prioritize next steps to dig deeper into the details of why the residues we are studying are under purifying selection (fail to propagate to progeny if mutated, meaning terminal). In short, only now, with the data in hand, can we prioritize mouse studies. We think it is important for the advancement of the field that we make these results available in a timely manner rather than waiting to report them together with the results of mouse models once generated and analyzed.

2. The reductionist experimental data presented here provide us with mechanistic insights into why the residues we are studying are functionally important. We therefore think it is of value to note that 58a-b- T cell hybridomas were used in seminal work that established a link between CD4Lck association, via motifs in the CD4 intracellular domain, and signaling output as measured by IL-2 production (Glaichenhaus, et al., 1991). Importantly, the impact of disrupting CD4-Lck interactions on proximal signaling were not interrogated until the work we describe here and in our preceding study, wherein we establish that CD4-Lck association does not regulate proximal signaling in 58a-b- T cell hybridomas. Given that this experimental system was used to help establish the dominant paradigm (i.e. the widely held view that CD4 recruits Lck to TCR-CD3 to initiate pMHCII-specific signaling), we think it is a legitimate system to directly test this model and further test core questions of CD4 function by employing more modern experimental techniques.