Human Semaphorin-4A drives Th2 responses by binding to receptor ILT-4

Semaphorin-4A (Sema4A) has been implicated in the co-stimulation of T cells and drives Th1 immune responses by binding to the receptor T-cell immunoglobulin and mucin domain protein 2 (Tim-2) in mice. Here we show that human, but not murine, Sema4A is preferentially expressed on antigen-presenting cells, and co-stimulates CD4+ T-cell proliferation and drives Th2 responses. By employing two independent cloning strategies, we demonstrate that Immunoglobulin-like transcript 4 (ILT-4) is a receptor for human SEMA4A (hSEMA4A) on activated CD4+ T cells. We also find hSEMA4A to be highly expressed in human asthmatic lung tissue, implying its potential function in disease pathogenesis. Our study defines a different biological function of hSEMA4A from its murine homolog through its binding to the receptor of ILT-4 to co-stimulate CD4+T cells and regulate Th2 cells differentiation.

Semaphorin-4A (Sema4A)-Tim-2 axis has been implicated as a co-stimulation pathway in mouse T cells. Sema4A promotes Th1 immune responses by binding to Tim-2 in mice. However, it remains unclear if Sema4A acts as a co-stimulatory signal on human T cells as well, since Tim-2 is not conserved in the human genome. In this manuscript, the authors have shown that Sema4A is expressed in various human antigen presenting cells and co-stimulates human CD4+ T cell while promoting Th2 cytokine production. The co-stimulation effect of Sema4A is dependent on ILT-4 expressed on activated human T cells. The different signaling properties of Tim-2 and ILT-4 may explain the different co-stimulatory effect of Sema4A on human and mouse T cells-this making their report both novel and an important advance for the general immunology community.
Comments/concerns: 1. The authors have shown that Sema4A expression is elevated in asthma patient lung tissues. It would be more relevant to know if ILT-4 is expressed on T cells isolated from asthmatic lung tissues, or at least ILT-4 staining co-localizes in human asthmatic lung to CD4 T cells. 2. The authors imply in the abstract that asthma is an autoimmune disease-but there are no solid data suggesting that this is true and the reference should be deleted. 3. Fig. 2. Only representative data are shown. Aggregate data including statistical analyses are needed. 4. Fig. 3. Activation of STAT6-panel E. How do the investigators believe this is occurring-is this directly through activation of Sema4a or indirectly through the IL-4/13 that the T cells are producing as documented in panel C? If the latter, the result is trivial and can be excluded-this result is highly predictable and adds nothing to the study. A far more interesting result-and very important to clarify for this study-would be to show if Sema4a activation induces STAT6 activation independently IL-4/IL-13/IL-4R. This could be easily tested by culturing naïve T cells with anti-CD3 and Sema4a-expressing L cells while blocking IL-4R-then determine if you still get a Th2 phenotype and STAT6 activation. 5. Discussion. The discussion reasonably places prior work on Sem4A in the context of the current data. Please further discuss the current findings regarding prior work with ILT-4, including binding to fragments of C4(1). 6. General. The font size used in some of the flow plots is unreadable. Please increase font size to readable proportions. The manuscript further contains may grammatical mistakes and misspellings that should be corrected with an English editor. The issue of semaphorins being highly important in a wide spectrum of immune responses is a hot issue. Many semas such as sema3A and sema4A are inhibitory/stimulatory but do not have always a specific receptor or at least many of the receptors are still not well identified. This is why, the authors did a very nice step in elegantly showed the receptor ILT-4 on Th2 cells. The methodology and discussion are well designed and of high level, and therefore convincing. Some minor comments: 1. Why clean/ purified human sema4A was not applied and instead the authos chose a complicated sema4a-L expressing cells? A purified sema would alow us to see a calibration of doses and spectrum of responses!!

Reply:
Thank you for the comment.
In our study, we chose both purified Sema4A-Fc fusion protein and Sema4A-L expressing cells to show the same biological function of Sema4A as indicated in Fig 2 and

cells and enhance Th2 differentiation through its receptor ILT-4 on activated T cells.
We have added relevance of these findings in Discussion.

Reviewer #2 (Airway inflammation, Th function)(Remarks to the Author):
Semaphorin-4A (Sema4A)-Tim-2 axis has been implicated as a co-stimulation pathway in mouse T cells. Sema4A promotes Th1 immune responses by binding to Tim-2 in mice. However, it remains unclear if Sema4A acts as a co-stimulatory signal on human T cells as well, since Tim-2 is not conserved in the human genome. In this manuscript, the authors have shown that Sema4A is expressed in various human antigen presenting cells and co-stimulates human CD4+ T cell while promoting Th2 cytokine production. The co-stimulation effect of Sema4A is dependent on ILT-4 expressed on activated human T cells. The different signaling properties of Tim-2 and ILT-4 may explain the different co-stimulatory effect of Sema4A on human and mouse T cells-this making their report both novel and an important advance for the general immunology community.
Comments/concerns: 1. The authors have shown that Sema4A expression is elevated in asthma patient lung tissues. It would be more relevant to know if ILT-4 is expressed on T cells isolated from asthmatic lung tissues, or at least ILT-4 staining co-localizes in human asthmatic lung to CD4 T cells.

Reply:
Thank you for the comment. Figure 5 in the revised manuscript.

We have provided new data showing that ILT-4 colocalizes with CD4 T cells in asthmatic lung tissues as indicated in Supplementary
2. The authors imply in the abstract that asthma is an autoimmune disease-but there are no solid data suggesting that this is true and the reference should be deleted.

Reply:
Thank you for the comment.
We have removed this sentence and the reference.
3. Fig. 2. Only representative data are shown. Aggregate data including statistical analyses are needed.

Reply:
Thank you for the comment.
In our study, human CD4+ naïve T cells were purified from different donors.

The capacity of T cells proliferation in different donor is quite different. Even
the cells were harvested at the same day, as show in figure 2, the percentage of proliferated cells is quite different. However, the trend is always the same.

Fig. 3. Activation of STAT6-panel E. How do the investigators believe this is
occurring-is this directly through activation of Sema4a or indirectly through the IL-4/13 that the T cells are producing as documented in panel C? If the latter, the result is trivial and can be excluded-this result is highly predictable and adds nothing to the study. A far more interesting result-and very important to clarify for this study-would be to show if Sema4a activation induces STAT6 activation independently IL-4/IL-13/IL-4Rα. This could be easily tested by culturing naïve T cells with anti-CD3 and Sema4a-expressing L cells while blocking IL-4Rα-then determine if you still get a Th2 phenotype and STAT6 activation.

Reply:
Thank you for the comment.

Reply:
Thank you for the comment.
Yes, we added ILT-4 binding of fragments of C4 in discussion.
6. General. The font size used in some of the flow plots is unreadable. Please increase font size to readable proportions. The manuscript further contains may grammatical mistakes and misspellings that should be corrected with an English editor.

Reply:
Thank you for the comment.
We increased font size in the figures and we corrected mistakes carefully. It is critical to provide aggregate data including proper statistical analyses to show if Sema4A costimulates T cell proliferation. If "the trend is always the same" for most donor cells, aggregate data will provide a highly statistic significant result.

Reply:
Thank you for the comment.
In Fig 2, we only posted representative data. Actually, for each condition, we had more than 3 different donors' data. As analyzed the aggregate data as follows, we found that, there is significant difference between each treatment in each group.
Group 1 (Fig 2a left  Our data shows that human SEMA4A strongly co-stimulates T cells proliferation.