PD-1 negatively regulates helper T cell differentiation into Th2

Reviewer #1 (Public review):

Summary:

The authors analyze the roles of PD-1 in the early stages (pre-activation) of T cell differentiation and show that naïve CD4+ T cell differentiation is altered, especially Th2 differentiation is strongly impaired, upon early PD-1 stimulation. The results have important implications for the immunotherapy area, but I think the manuscript requires some revisions.

Strengths:

(1) Novel Insights into PD-1 in Early T Cell Differentiation:
The study provides new insights into the role of PD-1 during the pre-activation phase of T cell differentiation, particularly its impact on naïve CD4+ T cells and Th2 differentiation. This is a significant contribution to immunotherapy research.

(2) Relevance to Immunotherapy:
The findings have potential implications for the development of immunotherapies by demonstrating how PD-1 signaling affects specific T cell subsets early in differentiation.

Weaknesses:

(1) Inconsistent and Confusing Data:
There are contradictions between the figures and the conclusions, particularly regarding IL-4 and IFNgamma production in PD-1-expressing cells. This raises concerns about data interpretation and experimental accuracy.

(2) Unclear Experimental Rationale:
The reviewer questions the rationale behind key methodological choices, such as the high concentration of PDL-1 antibody and varying OVA peptide concentrations. These decisions need more justification.

Reviewer #2 (Public review):

Summary:

The authors try to demonstrate that PD-1 regulates not only the quantity but also the quality of the immune response determining the Th differentiation. The authors suggest that the ability of PD-1 agonists to dampen Th2 differentiation could be exploited in allergies or classical Th2-mediated disease as a therapeutical approach.

Strengths:
The authors performed a series of elegant experiments using OVA-specific CD4 T cells from mice, showing a strong reduction of Th2 differentiation in vitro. They also perform some experiments with a model of allergies, showing an amelioration of the phenotype after administration of PD-1 agonist with a reduction of Th2 cells.

Weaknesses:

The authors perform all the experiments using DO11.10 mouse cells. Such cells have a TCR with very high affinity, it would be relevant to repeat at least some of the in vitro assays in a more physiological setting (you can immunise mice with ova to increase the pool of OVA-specific T cells, and then repeat the restimulation experiment). Also, a longer kinetic would be of interest to see the effect of the agonist on Th1 cells.

Another drawback is the lack of experiments with human cells. It would be really important to repeat the experiments with CD4 T cells from healthy donors (the antibody that the authors use as PD-1 agonist is human, so it would not be a complicated experiment).

It would be also interesting to show in the allergic disease model the effect of the agonism on the T cell response in general.

Author Response:

We are grateful to the reviewers for their encouraging comments and constructive suggestions. These suggestions will be valuable to improve the revised manuscript.

Reviewer 1:

PD-1 signaling is suppressive to the establishment of cytokine-producing effector cells in general. However, as the reviewer pointed out, one of the results in Fig. 2H showing a decrease of IFN-gamma-producing cells is against this trend. The data indicate percentages of cytokine-producing cells, which are not always consistent with the absolute number of activated T cells. Nonetheless, we plan additional experiments in order to address the question.

For PD-1YFYF experiments in Figs. 3-5, there were moderate changes in cytokine production between wild-type and mutant PD-1. We conducted gene transduction to newly prepared T cells in each experiment. In addition, to monitor the immunosuppressive effect of PD-1 agonist antibodies, these T cells were stimulated using PD-L1-deficient APC. Therefore, we think these cytokine levels were most likely a technical variation, but not specific function of PD-1YFYF.

Anti-PD-L1 mAb was used for the optimal blockade of PD-1/PD-L1 blockade, and the concentration of antibody (5 microg/ml) is within a normal range for this purpose. We used variable concentrations of OVA peptide to set up experiments with different intensities of TCR stimulation. TCR signal intensity has been shown to affect CD4+ T cell differentiation into Th1 and Th2 cells. We lowered the peptide concentration to test the effect of PD-1 signals under the suboptimal TCR stimulation.

Reviewer 2:

Antigen-specific T cells from immunized mice are not ideal for Th differentiation studies because activated T cells in response to the antigen might have already undergone functional differentiation in vivo. Incorporating the reviewer’s suggestion, we will test alternative approach including human CD4+ T cells.

For the allergy model, we will expand the analysis for inflammatory effectors.