Alternative paths to immune activation: the role of costimulatory risk genes for polygenic inflammatory disease in T helper cells

Reviewer #1 (Public Review):

Full activation of T cells requires not only antigen recognition through the T cell receptor, but also engagement of co-stimulation by the T cell. There are multiple co-stimulatory receptors that can be engaged by the T cell; yet, the downstream effects of signaling through these different receptors on T cell gene programs and function and are not yet fully understood. These questions are clinically important because genomic variants associated with immune and inflammatory disease map onto these different co-stimulatory receptors and, potentially, their downstream gene programs.

Based on these observations, the authors hypothesize that different modes of co-stimulation engage different genes and pathways that may be differentially associated with risk for inflammatory disease. To ask this question, the authors performs a comparative analysis of different co-stimulatory receptors, both CD28 - the most widely used form of co-stimulation for in vitro assays - as well as alternative modes of co-stimulation involving ICOS, CD6, CD27. They analyzing their effects on their T cell activation in vitro for human naive and memory CD4 cells, on gene expression using RNA-seq (at 24 hrs), on chromatin accessibility using ATAC-seq, and on specific proteins identified from transcriptomic data using flow cytometry.

From these experimental analysis, the authors conclude the following (1) alternative co-stimulation (ICOS, CD6, Cd27) can induce a *qualitatively* different gene and cellular program compared to canonical co-stim (CD28), resulting not only in less proliferation and cytokine production, as expected, but also in higher lysosome production and different metabolic programming. They also found that risk variants for inflammatory bowel disease mapped onto genes that were both shared across different modes of co-stimulation, as well as onto targets of specific co-stimulation.

This study and the authors' experimental system is well-designed to precisely identify genomic effects of co-stimulation, employing sorted subsets of human CD4 cells, as well as a in vitro setting that can effectively eliminate many confounding variables associated more complex scenarios. The transcriptome/chromatin accessibility measurements were also robustly analyzed and offer some support for the author's conclusion. However, there were two main weaknesses that limit that, if overcome, would enhance the authors' argument:

(1) It is not clear whether the qualitatively different effects of alternate co-stimulation compared to canonical CD28 co-stimulation, e.g. increased OXPHOS or lysosomal abundance for CD6, or heightened expression of genes or represent truly unique effects, or whether they simply represent effects of having quantitatively weaker strengths of CD28 co-stimulation. This concern would be addressed by an experiment doing a dose response curve for CD28 co-stimulation while measuring these variables (Fig. 6) or, more systematically, while performing RNA-seq. Also, to strengthen this argument, the authors would benefit from further in-depth literature discussion/analysis of the signaling pathways downstream of co-stimulation, to discuss molecular bases for different signaling, if any.

(2) There is no functional evidence to link differential activation of risk variant-associated genes by alternate co-stimulation with inflammatory disease. To show this, the authors can examine the activation of these genes (e.g. Bach2, Il18R1, from Table 2) using their assay, either using T cells from humans containing disease-associated variants at these gene loci, or by using T cells with a genetic disruption of the associated loci.

While providing insights for the pathogenesis of IBD, this study's main impact would be in the enhancing our understanding of how different modes of co-stimulation differ to activate T cells and prompt broader consideration of use of different co-stimulatory ligands in these in vitro assays and evaluation of their function in vivo.

Reviewer #2 (Public Review):

Voda et al examined the role of multiple co-stimulations on gene expression and chromatin accessibility of T cells. They further linked the roles of co-stimulatory proteins to genetic variants associated with IBD. They reported a shared effect of co-stimulatory proteins on gene expression and chromatin accessibility. In particular they reported the induction of genes associated with lysosome production with alternative co-stimulatory proteins. In linking human genetics to the effect of costimulation, they reported the largest enrichment of IBD risk variants in open chromatin regions shared by all costimulatory molecules.

The question that is being investigated in this manuscript is significant considering the requirement of costimulatory proteins in controlling T cell responses. However, the data presented and analyzes performed remain exploratory and it is not clear how it can advance our understanding of the link between IBD risk association and immune responses. At least one locus ( a target of shared/unique costimulatory molecules) should be selected and mechanistic investigation of the locus, transcription factors involved, and perturbation studies for understanding gene regulation should be performed.