CD4+ T cell response phenotypes characterized by single cell analysis

CD4⁺ T cells are central players of the adaptive immune response. They mediate inflammation via clonal expansion, differentiation into specific response subtypes and production of inflammatory signals that promote cytotoxicity and antibody production by other immune cells. CD4⁺ T cells may also differentiate into regulatory T cells (Treg) that control autoimmune responses and promote tolerance. Loss of CD4⁺ T cell tolerance to autoantigen leads to unwanted T cell responses and autoimmunity.

Immunomodulation therapies that affect CD4⁺ T cells have become a paradigm to reverse T cell mediated autoimmunity, either by blocking T cell activation signals or by enhancing tolerogenic function. Some of these single drug therapies are now used in trials or in practice to treat patients with autoimmune diseases. However, their true immunomodulating capacity on CD4⁺ T cell phenotype is unclear.

The aim of my thesis is to identify immunomodulators that modify the transcriptional profile of antigen-responsive human CD4⁺ T cells toward anergy or tolerance. I selected compounds targeting critical pathways of T cell activation that have relevance to autoimmunity in type 1 diabetes. I examined the in vitro effect of immunomodulation on naïve and memory CD4⁺ T cell proliferation upon stimulation with model antigens. Responsiveness of immunomodulated cells to subsequent antigen stimulation was also investigated. Single cell transcriptomic analyses were performed on proliferating and non-proliferating CD4⁺ T cells stimulated with model antigens in the presence of immunomodulation to detect potential alterations in their profile in comparison to untreated cells.

CD4⁺ T cell responses to antigen stimulation were assessed using proliferation assays in the presence of absence of immunomodulating drugs. The drugs assessed were the JAK inhibitor Tofacitinib, the natural phenol Resveratrol, an anti-human IFN-αR monoclonal antibody, the anti-IL-6R monoclonal antibody Tocilizumab, the proteasome inhibitor Bortezomib, the IgG1-CTLA-4 fusion protein Abatacept, a DHODH inhibitor (Compound A) and a RORγT inverse agonist (Compound B). The effects on human peripheral blood memory CD4⁺ T cells were examined using the recall antigens Flu and Tetanus Toxoid and the superantigen SEB at a concentration that stimulated TCRV17⁺ cells. The effects on naïve CD4⁺ T cells were assessed using SEB. Responses were measured using flow cytometry. Re-stimulation of inhibited cells in the absence of immunomodulator was performed to examine the legacy of immunomodulated CD4⁺ T cells post-treatment. For the transcriptomic analyses, single cells were sorted with FACS and their transcriptome was sequenced with RNAseq. For memory CD4⁺ T cells stimulated with recall antigens, the proliferated responsive cells in cultures with and without immunomodulatory drug were examined and compared. For memory and naive CD4⁺ T cells stimulated with SEB, the non-proliferated TCRV17⁺ CD4⁺ T cells in cultures with and without immunomodulatory drug were examined and compared. The differentially expressed genes were examined and further processed using the Ingenuity Pathway Analysis.

All the selected compounds consistently reduced the frequencies of proliferating memory CD4⁺ T cells responding to recall antigens, with the exception of the anti-IFN-αR and Tocilizumab. Re-stimulation in the absence of inhibitor showed a persistent loss of responsive capacity to the recall antigen for cells that had been exposed to Resveratrol and to the RORγT inverse agonist. The transcriptome of isolated single CD4⁺ CD25⁺ T cells that proliferated in the presence of these compounds was comparable to that of not treated proliferative cells, showing minor and inconsistent across PBMC samples differences in gene expression. These data suggest that the immunomodulators induced little change in memory CD4⁺ T cell phenotype.

Resveratrol, the DHODH inhibitor and a RORγT inverse agonist, but none of the other compounds successfully inhibited proliferation of responsive to SEB naïve and memory TCRV17⁺ cells, without affecting their viability. Responsiveness of these inhibited cells to SEB was restored in the absence of immunomodulators. The transcriptome of isolated non-proliferating TCRV17⁺ cells exposed to SEB in the absence of immunomodulators was characteristic of stimulated T cells with upregulated pathways related to TCR signaling, anabolic biosynthetic processes and chromosomal replication. Non-proliferating cells that were stimulated but inhibited by the three compounds had a transcriptional profile consistent with downregulation of response to SEB.  Resveratrol had the strongest downregulating effect in both naïve and memory cells. The inhibited pathways that were commonly affected by all compounds were associated with cell cycle control of chromosomal replication, tRNA charging, spliceosomal cycle and necroptosis, demonstrating the role of these pathways in the CD4⁺ T cell response to SEB. The transcriptomic data of immunomodulated dividing or not dividing CD4⁺ T cells did not provide evidence of an induced Treg phenotype.

In conclusion the established in vitro models identified six inhibitors of memory antigen-specific CD4⁺ T cell response and three that were additionally able to inhibit memory and naïve response to SEB. The transcriptomic data suggest that the primary effect of the tested immunomodulators is on the T cell responsiveness to antigen and not on the T cell phenotype. I conclude that while the majority of immunomodulators can severely inhibit CD4⁺ T cell response, none of the tested immunomodulators have a combined capacity to inhibit and shift antigen-responsive CD4⁺ T cells toward tolerogenic phenotypes in vitro. I interpret these data as indicating that successful therapeutic application is likely to require chronic administration.

Publication:

Beneficial Effect of U-74389 G and Sildenafil in An Experimental Model of Flap Ischemia/Reperfusion Injury in Swine. Histological and Biochemical Evaluation of the Model.  S.L. Karamatsoukis, E.A. Trigka, M. Stasinopoulou, A. Stavridou, A. Zacharioudaki, K. Tsarea, M. Karamperi, T. Pittaras, O. Papadopoulos, E. Patsouris, N. Nikiteas, G.C. Zografos, A.E. Papalois. J Invest Surg. 2020;33:391-403.