In the thymus, the development of T cell from CD4⁻CD8⁻ double-negative (DN) to CD4⁺CD8⁺ double-positive (DP) and then into CD8⁺ or CD4⁺ single-positive (SP) cells (a process known as positive selection) is regulated by multiple signalling pathways that induce cell-specific gene expression (1, 2). Failure of the appropriate signals occurring during T cell development can lead to immune disorders such as severe T cell immunodeficiency or autoimmune diseases. The mitogen-activated protein kinase (MAPK) signalling pathways, including extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, have been implicated in thymocyte differentiation (3–5). Of these three major MAPK pathways, the ERK pathway is known to be involved in T cell positive selection (6–8), since ERK1-deficient mice exhibit defects in T cell maturation (9). Results obtained with JNK1- and JNK2-deficient mice indicate that these kinases are not needed for the initial stages of T cell development (5, 10), although they are implicated in different aspects of T cell-mediated immune response, for example, in the subsequent differentiation of the activated CD4 T cells into Th1 or Th2 phenotype (3–5).

On the other hand, the p38 MAPK pathway has been suggested to participate in early thymocyte development. The mammalian p38 MAPK family is composed of four members, such as p38α, p38β, p38γ, and p38δ, encoded by distinct genes and all activated by phosphorylation mediated primarily by the MAPK kinases (MKK)3 and MKK6 (11, 12). p38 MAPK can be divided into two subsets, such as p38α/p38β and p38γ/p38δ, based on substrate specificities, protein similarity, expression patterns, and sensitivity to chemical inhibitors (11). Studies using a range of p38α inhibitors in vitro, dominant-negative or constitutively active transgenes, or the constitutive deletion of its activators, MKK6 and MKK3 to suppress p38 MAPK, have suggested a role for p38α in early T cell development and also in T cell function (5, 13–18). Thus, it has been shown that in mice that express dominant-negative forms of both MKK3 and MKK6 specifically in T cells, the positive selection of thymocytes is impaired (15). However, since MKK3 and MKK6 activate all p38 MAPK isoforms (12), some of the findings from experiments in which MKK3 and/or MKK6 have been overexpressed or constitutively deleted may be due to an effect on any of the four p38 MAPKs, and not necessarily on p38α specifically. Knockout mice for each p38 MAPK isoform have been generated and could be an adequate tool to elucidate the specific role of individual p38 MAPKs on T cell development. Gene targetting to delete p38α in the whole mouse yields an embryonic lethal phenotype (19–21); however, one study using p38α^−/−Rag^−/− chimaeras showed normal T cell development in these mice, with no alterations in mature T cell proliferation (22). Moreover, lymphocyte development or cytokine production in response to LPS is not affected in p38β-null mice (23). A recent study using knockout mice in which p38α has been specifically deleted in T cells (24, 25) shows that mice with p38α-deficient T cells express elevated amounts of p38β, and that mice with T cells simultaneously lacking p38α and p38β displayed a decreased size and cellularity of the thymus, lymph nodes (LNs), and spleen compared with those of wild-type (WT) animals (24). However, p38α and p38β deletion did not prevented the thymic development of CD4⁺ and CD8⁺ T cells or their homing and maintenance in the LNs and spleen (24) suggesting that p38α and p38β are dispensable for the development of thymocytes and that other p38 MAPK isoforms might be regulators of this process.

Using mice deficient in p38γ, p38δ, or both, it has been shown that these kinases are crucial for the inflammation and the innate immune response, by controlling immune cell response (11, 26–29). Particularly, they have recently been shown to play important roles in regulating: cytokine production by macrophages and dendritic cells; T cell activation and neutrophil migration; and also in modulating tumourigenesis associated with inflammation (11, 28, 30, 31). Despite the implication of the p38 MAPKs in the immune response, their role in the normal physiology of haematopoietic cells is largely unknown. No anomalies have thus far been reported in T cell development in p38γ-, p38δ-, or p38γ/δ-deficient mice. There are nonetheless indications that p38γ and p38δ are implicated in the development of some immune cells. Measurement of mRNA levels in inflammatory cell lineages showed that p38α and p38δ are the dominant p38 isoforms in monocyte/macrophages, neutrophils, CD4⁺ T cells, and endothelial cells, whereas p38β and p38γ mRNAs are poorly expressed (32). A possible role for p38γ in haematopoiesis has been proposed based on the mRNA expression pattern for each p38 isoform during differentiation of primary human erythroid progenitor. p38α and p38γ are expressed in early and late stages, whereas p38δ mRNA is expressed only at terminal stages of erythroid differentiation (33). Nonetheless, the role of p38γ and p38δ in the development and function of other haematopoietic cells remains unclear.

In this study, we investigate the role of p38γ and p38δ in T lymphocyte development. We performed a comparative analysis of both, the T cell differentiation in the thymus and the distribution of T cell populations in the LNs, in WT mice, and in mice lacking p38γ, p38δ, or both and in mice expressing kinase-inactive p38γ. We found that the combined action of p38γ and p38δ is necessary for thymocyte development and peripheral T cell homeostasis.

Here, we used mice lacking p38γ and/or p38δ, and mice expressing a kinase-inactive p38γ mutant to analyse the effect of these p38 isoforms on the development of T lymphocytes. Studies using p38α/p38β inhibitors and the expression of dominant-negative or active forms of p38α or MKK3 and MKK6 suggested that p38α is involved in T cell development by maintaining normal CD4⁻ CD8⁻ DN thymocyte numbers and by inhibiting formation of DP cells [reviewed in Ref. (18)]. However, neither the deletion of p38α or p38β alone nor the deletion of p38α and p38β in combination leads to thymocyte development defects (22, 23, 25). These observations do not rule out a potential role for p38α and p38β signals in T cell development, as some p38 isoforms could compensate for lack of others during this process. Here, we show that both single and the combined deletion of p38γ and p38δ led to specific thymocyte development defects. Total thymocyte cellularity and the absolute numbers of DN, DP, and SP CD4⁺ and CD8⁺ thymocytes were reduced in p38γ^−/−, p38δ^−/−, and p38γ/δ^−/− mice compared with WT mice; this reduction was more evident in p38γ/δ^−/− mice than in the other genotypes. In early thymocyte development, there is a reduction in the percentage of the CD25⁺CD44⁻ (DN3) subpopulation within the DN population, together with an accumulation of the CD25⁻CD44⁺ (DN1), CD25⁺CD44⁺ (DN2), and CD25⁻CD44⁻ (DN4) subpopulations in p38δ^−/− mice. These results suggest that p38δ regulates differentiation and expansion of DN3 thymocytes, and that has either opposite or different roles in the transition from DN2 to DN3 and from DN3 to DN4. In the regulation of DN2–DN3 transition, it is possible that p38δ controls signalling pathways, such the IL7 receptor (IL7R) or the Notch pathway, that are critical for the proliferative expansion of early pro-T cells within the thymus (2). Thus, p38δ could be mediating either the secretion of Notch ligands and/or of IL7 by stromal cells in the thymus or the signalling downstream of Notch and/or IL7R in pro-T cells, or both. In the DN3–DN4 transition, p38δ might negatively modulate proliferation or survival signalling since β-selection of DN3 thymocytes in p38δ-deficient mice is not affected compared with WT, and p38 MAPKs are downstream of the TCR receptor (42). However, further studies are required to determine the exact role p38δ in early thymocyte differentiation. p38δ^−/− and p38γ/pδ^−/− mouse phenotypes resembled those observed using the dominant-negative MKK3 and MKK6 [lck-MKK3(A)/MKK6b(A)] transgenic mice (15). These mice exhibit impaired DN thymocyte development and T-cell proliferation. Accordingly, MKK6(Glu) transgenic mice, which express a constitutively active form of MKK6, exhibit an arrest in T cell development at the DN3 stage (13). Because all p38 MAPKs are specifically activated by MKK3 and MKK6 (12), but neither p38α nor p38β deletion in mice results in thymocyte development defects (22, 23, 25, 43), our observations indicate that p38γ and p38δ signalling might account for the effects observed in T cell development using transgenic MKK3 and MKK6 mice. We show that p38γ and p38δ signalling control at least partially the correct development and maintenance of the different thymocyte subsets.

Also, our data suggest that these p38 MAPKs have a role in transmitting signals from the pre-TCR during DN to DP transition and αβ TCR-mediated negative selection signals during DP to SP transition. In late thymocyte development, signals mediated by the pre-TCR complex induce CD4⁻CD8⁻ (DN) thymocyte differentiation into CD4⁺CD8⁺ (DP). After TCR α-chain rearrangement and TCRαβ expression, DP thymocyte go through both positive and negative selection after the interaction of the TCR with the corresponding MHC, which leads to the downregulation of either CD8 or CD4, and to the differentiation of DP thymocytes into CD4⁺ or CD4⁺ SP cells (1). The lack of p38γ causes a moderate increase in DN and a decrease in DP thymocyte frequency, suggesting that p38γ modulate cell survival probably by modifying TCR signal strength and affecting the threshold between positive and negative selection. Also, there is an increase in CD4⁺ and in CD8⁺ populations in the thymus of p38γ^−/− mice, indicating a role of this kinase in cell survival. Interestingly, the effect of p38γ in late thymocyte development is not mediated by its kinase activity since the expression of inactive mutant p38γ (D171A) reverts the effect observed in the p38γ^−/− mice. p38γ is the only MAPK that has a C-terminal sequence that docks directly to PDZ domains of different proteins (11, 34, 35). This allows p38γ to regulate the composition of protein complexes independently of its kinase activity in different cellular compartments (11, 34, 35). For example, p38γ binds to the scaffold protein human discs large (hDlg, also known as Dlg1 and dlgh1) modulating the integrity and composition of hDlg complexes in the nucleus and in the cytoskeleton (11, 34, 35). In T cells, hDlg is implicated in TCR-mediated actin polymerisation, and it is essential for the preservation and regulation of cell polarity, which is important for T-cell development (44). Whether or not p38γ modulates hDlg complexes, TCR affinity and T cell proliferation in late thymocyte development are being investigated.

Analysis of secondary lymphoid organs showed that lack of p38γ decreased the frequency of CD4⁺ and CD8⁺ T cells in LNs and supports the role for p38γ in mature T cell differentiation. This reduction could be due to increased apoptosis or to a defect in cell proliferation. We have previously shown in in vitro T cell assays that LN cells from p38γ/δ^−/− mice showed reduced proliferation, and interferon γ and IL-17 production, compared with WT mice, in response to anti-CD3 (30).

An important observation in this study is that in some stages of thymocyte development the combined deletion of p38γ and p38δ do not causes the same effect that the individual deletion of these kinases. For example, in p38γ/δ^−/− mice, the percentage of DN2 thymocytes is similar to WT, whereas in the p38δ^−/− mice, DN2 subpopulation is increased; in late T cell development, changes in the percentages of DN, DP, and CD8⁺ SP caused by the lack of p38γ are not observed in p38γ/δ^−/− mice; and combined p38γ and p38δ deletion do not have any effect on CD4⁺ and CD8⁺ T cell frequency in LN, whereas these thymocyte populations are decreased in p38γ^−/− mice. It is likely that compensatory mechanisms are acting in p38γ/δ^−/− mice to overcome the loss of p38γ and p38δ. For example, we found that the protein expression level of p38β is increased in p38γ^−/− and p38γ/δ^−/− thymocytes compared with WT cells (Figure 1A). In future, studies will be crucial to determine changes in the levels of protein expression and activation of the different p38 MAPK isoforms or other MAPKs during T cell development. Further analyses are required to establish the exact molecular mechanism(s) by which p38γ and p38δ control some stages of T cell development, and conditional knockout mice in combination with different double, triple, or quadruple knockout mice for p38α, p38β, p38γ, and p38δ should provide important information for better understanding the physiological functions of these kinases.

This study was carried out in accordance with the recommendations of national and EU guidelines, with the approval of the Centro Nacional de Biotecnología Animal Ethics Committee (Reference: CAM PROEX 316/15).

AC, AR, MAMS and DFB designed experiments, performed experiments, and analysed the data. AC wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MT declared a shared affiliation, though no other collaboration, with the authors to the handling editor. The reviewer HH and handling Editor declared their shared affiliation.