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T lymphocytes (T cells) represent one of the major cell populations of the immune system. Named by the place of their development, the thymus, several types can be distinguished as the αβ T cells, the γδ T cells, the mucosa-associated invariant T cells (MAIT), and the natural killer T (NKT) cells. The αβ lineages of CD4+ THelper and the CD8+ T cytotoxic cells with the T cell receptor (TCR) composed of α- and β-chain are major players of the adaptive immune system. In the thymus, CD4+ and CD8+ single positive (SP) αβ cells represent the ultimate result of positive and negative selection of CD4+CD8+ double positive (DP) thymocytes. The DP population derives from the double negative (DN) thymocytes that develop from bone marrow-derived progenitors through different stages (DN1-DN4) that are characterized by CD25 and CD44 surface expression.
NFATc1, a member of the Nuclear Factor of Activated T cells (NFAT) transcription factors family, is critically involved in the differentiation and function of T cells. During thymocyte development, the nuclear expression of NFATc1 reaches the highest level at the DN3 (CD44-CD25+) stage. The hematopoietic cell-specific ablation of NFATc1 activity results in an arrest of thymocyte differentiation at the DN1 (CD44+CD25-) stage. On the other hand, over-expression of a constitutively active version of NFATc1 results in an impaired transition of DN3 cells to the DN4 (CD44-CD25-) stage, suggesting that a certain threshold level of NFATc1 activity is critical at this point.
ChIP-seq and RNA-seq analysis allowed us the identification of NFATc1/A target genes involved in lineage development as the Tcra and Tcrb gene loci. Furthermore, we identified multiple NFATc1-regulated genes that are involved in γδ T cell development. In the mouse models, Rag1Cre-Nfatc1fl/fl and Rag1Cre-E2fl/fl, in which the activity of NFATc1 or inducible NFATc1 in the latter is impaired during the early stages of thymocyte development, we observed increased numbers of γδ T cells. These γδ T cells showed an unusual overexpression of CD4, a lack of CD24 expression, and overexpression of the anti-apoptotic gene Bcl2a1a.
We hypothesize that during the DN stages NFATc1 plays an important role in regulating crucial steps of αβ thymocyte development and when NFATc1 activity is missing this may disturb αβ development resulting in alternative cell fates like γδ T cells.
The defense against invading pathogens is, amongst other things, mediated via the action of antibodies. Class-switched antibodies and antibodies of high affinity are produced by plasma cells descending from germinal center B (GCB) cells. GCB cells develop in the germinal center (GC), a specialized microstructure found in the B-cell follicle of secondary lymphoid organs. GCB-cell maturation and proliferation are supported by follicular T- helper (Tfh) cells. On the other hand, follicular regulatory T (Tfr) cells control this process in quantity and quality preventing, for instance, the formation of autoantibodies directed against endogenous structures. The development of GCB, Tfh and Tfr cells essentially depends on the migration into the GC, which is mediated via the expression of the chemokine receptor CXCR5.
One transcription factor highly expressed in follicular T cells, comprising Tfh and Tfr cells, is NFATc1. Tfr cells additionally express the transcriptional repressor Blimp-1, which is not expressed in Tfh cells. We found that NFATc1 is transactivating Cxcr5 via response elements in the promoter and enhancer in vitro. Blimp-1 binds to the same elements, transactivating Cxcr5 expression in cooperation with NFATc1, whilst mediating Cxcr5- repression on its own. In Tfr cells Blimp-1 suppresses CXCR5 expression in the absence of NFATc1. Blimp-1 itself is necessary to restrict Tfr-cell frequencies and to mediate Tfr- cell function as in mice with Blimp-1-ablated Tregs high frequencies of Tfr cells do not reduce GCB- or Tfh cell frequencies. NFATc1 and Blimp-1 double deficient Tfr cells show additional loss of function, which becomes visible in clearly expanded antibody titers.
To evaluate the function of NFATc1 in Tfr cells, we not only deleted it, but also overexpressed a constitutive active form of NFATc1/aA (caNFATc1/aA) in regulatory T cells (Tregs). The latter is leading to an upregulation of CXCR5 per cell, without changing Tfh or Tfr-cell frequencies. However, the high density of surface CXCR5 enhances the migration of Tfr cells deep into the GC, which results in a tighter control of the antigen- specific humoral immune response. Additionally, caNFATc1/aA increases the expression of genes coding for Tfr effector molecules like Il1rn, Il10, Tigit and Ctla4. Interestingly, this part of the transcriptional change is dependent on the presence of Blimp-1. Furthermore, Blimp-1 regulates the expression of multiple chemokine receptor genes on the background of caNFATc1/aA.
In contrast, when caNFATc1/aA is overexpressed in all T cells, the frequencies of Tfh- and GCB cells are dominantly reduced. This effect seems to stem from the conventional T- cell (Tcon) side, most probably originating from increased secretion of interleukin-2 (IL- 2) via the caNFATc1/aA overexpressing Tcons. IL-2 is known to hinder the germinal center reaction (GCR) and it might in its abundance not be neutralizable by Tfr cells.
Taken together, NFATc1 and Blimp-1 cooperate to control the migration of Tfr cells into the GC. Tfr cells in the GC depend on NFATc1 and Blimp-1 to perform their proper function. Overexpression of caNFATc1 in Tregs strengthens Tfr function in a Blimp-1-dependent manner, whilst overexpression of caNFATc1 in all T cells dominantly diminishes the GCR.