Thymocyte‐derived BDNF influences T‐cell maturation at the DN3/DN4 transition stage

Brain‐derived neurotrophic factor (BDNF) promotes neuronal survival, regeneration, and plasticity. Emerging evidence also indicates an essential role for BDNF outside the nervous system, for instance in immune cells. We therefore investigated the impact of BDNF on T cells using BDNF knockout (KO) mice and conditional KO mice lacking BDNF specifically in this lymphoid subset. In both settings, we observed diminished T‐cell cellularity in peripheral lymphoid organs and an increase in CD4⁺CD44⁺ memory T cells. Analysis of thymocyte development revealed diminished total thymocyte numbers, accompanied by a significant increase in CD4/CD8 double‐negative (DN) thymocytes due to a partial block in the transition from the DN3 to the DN4 stage. This was neither due to increased thymocyte apoptosis nor defects in the expression of the TCR‐β chain or the pre‐TCR. In contrast, pERK but not pAKT levels were diminished in DN3 BDNF‐deficient thymocytes. BDNF deficiency in T cells did not result in gross deficits in peripheral acute immune responses nor in changes of the homeostatic proliferation of peripheral T cells. Taken together, our data reveal a critical autocrine and/or paracrine role of T‐cell‐derived BDNF in thymocyte maturation involving ERK‐mediated TCR signaling pathways.     

Distinct IL‐7 signaling in recent thymic emigrants versus mature naïve T cells controls T‐cell homeostasis

IL‐7 is essential for T‐cell survival but its availability is limited in vivo. Consequently, all peripheral T cells, including recent thymic emigrants (RTEs) are constantly competing for IL‐7 to survive. RTEs are required to replenish TCR diversity and rejuvenate the peripheral T‐cell pool. However, it remains unknown how RTEs successfully compete with resident mature T cells for IL‐7. Moreover, RTEs express low levels of IL‐7 receptors, presumably rendering them even less competitive. Here, we show that, surprisingly, RTEs are more responsive to IL‐7 than mature naïve T cells as demonstrated by markedly increased STAT5 phosphorylation upon IL‐7 stimulation. Nonetheless, adoptive transfer of RTE cells into lymphopenic host mice resulted in slower IL‐7‐induced homeostatic proliferation and diminished expansion compared to naïve donor T cells. Mechanistically, we found that IL‐7 signaling in RTEs preferentially upregulated expression of Bcl‐2, which is anti‐apoptotic but also anti‐proliferative. In contrast, naïve T cells showed diminished Bcl‐2 induction but greater proliferative response to IL‐7. Collectively, these data indicate that IL‐7 responsiveness in RTE is designed to maximize survival at the expense of reduced proliferation, consistent with RTE serving as a subpopulation of T cells rich in diversity but not in frequency.     

Naive CD4(+) lymphocytes convert to anergic or memory-like cells in T cell-deprived recipients

Recent demonstrations that naive T cells proliferate after transfer to lymphopenic hosts have led to the theory that active homeostatic mechanisms fill the peripheral pool of naive T cells. To extend these data, we injected naive CD4(+) T cells from AND TCR transgenic mice (H-2(b/b) or H-2(k/k)) into CD3 epsilon-deficient mice, and studied the absolute number, phenotype and functional capacities of the transferred lymphocytes, from the first days to a few months after transfer. Proliferation of naive CD4(+) T cells did not fill the peripheral naive T cell pool. Injected naive T cells acquired a memory-like phenotype that was stable with time, despite the absence of foreign antigenic stimulation. Their functional capacities were modified, enhanced or abolished depending on the MHC haplotype. Thus, "homeostatic" proliferation of naive CD4(+) T cells in T cell-deprived recipients does not regenerate the naive CD4(+) T cell pool.     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

IL‐15 modulates the balance between Bcl‐2 and Bim via a Jak3/1‐PI3K‐Akt‐ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL‐15 is an essential survival factor for CD8αα⁺ intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL‐15‐induced survival signals in primary CD8αα⁺ iIELs remains elusive. Although Bcl‐2 level in CD8αα⁺ iIELs positively correlates with IL‐15Rα expression in the intestinal epithelial cells, overexpression of Bcl‐2 only moderately restores CD8αα⁺ γδ iIELs in Il15^?/? mice. Here, we found that IL‐15 promptly activated a Jak3‐Jak1‐PI3K‐Akt pathway that led to the upregulation of Bcl‐2 and Mcl‐1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl‐2 but also resulted in the phosphorylation of extra‐long Bim at Ser⁶⁵. The latter event facilitated the dissociation of Bim from Bcl‐2 without affecting Bim abundance in IL‐15‐treated CD8αα⁺ iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl‐2 or removal of Bim from CD8αα⁺ iIELs promoted their survival in Il15ra^?/? mice. Taken together, IL‐15 promotes CD8αα⁺ iIEL survival by both increasing Bcl‐2 levels and dissociating Bim from Bcl‐2 through activation of a Jak3‐Jak1‐PI3K‐Akt‐ERK1/2 pathway, which differs from a previously reported IL‐15‐induced survival signal.     

Intestinal helminth infection impacts the systemic distribution and function of the naive lymphocyte pool

Homeostasis is a fundamental principle of biological systems. A paradigm of immune homeostasis is the remarkably constant number of naive T and B lymphocytes in the body that continuously circulate through the secondary lymphoid organs to maximize immune surveillance. Whether the dynamics and distribution of the systemic naive lymphocyte pool is affected following organ-specific infection is not known. Here we show that, following infection of mice with an enteric helminth, naive T and B lymphocytes accumulate in the T helper type 2-reactive mesenteric lymph node while they are concurrently depleted from non-draining peripheral lymph nodes. This systemic redistribution of naive lymphocytes is sustained into the chronic phase of the infection, requires lymphotoxin beta receptor-dependent signals and is associated with a reduced ability of parasitized animals to mount antigen-specific cellular and humoral immune responses to heterologous immunization or infection at peripheral sites. Our data suggest that the function of the homeostatic naive lymphocyte pool can be modulated by its systemic distribution following infection and may provide a novel concept underlying compromised immune responsiveness at peripheral sites in helminth-infected individuals.     

Peripheral Thy1+ lymphocytes rearranging TCR‐γδ genes in LAT‐deficient mice

Linker for activation of T cells (LAT) is an adaptor molecule indispensable for development of αβ and γδ T lymphocytes. Surprisingly, using a new model of LAT‐deficient mice we found that despite arrested thymic development, a discrete population of cells with active Lat promoter, expressing Thy1 molecules, accumulated in peripheral lymphoid organs of homozygous (Lat^Inv/Inv) mutant mice. By measuring frequencies of TCR gene rearrangements in conjunction with a panel of cell surface Ag, we dissected two subsets of these Thy1⁺ cells. Thy1^dull cells expressed markers of NK lymphocytes and contained low frequency of TCR‐γ gene rearrangements without detectable TCR‐δ rearrangements. Thy1^high cells resembled immature CD44⁺CD25⁺ thymocytes and contained high frequency of non‐productive TCR‐γ and TCR‐δ rearrangements, indicating that cells displaying molecular signatures of commitment toward γδ T‐cell lineage can develop and populate lymphoid tissues of LAT‐deficient mice. Phenotypically similar Thy1^high cells were also found in lymph nodes of lymphocyte‐deficient (Rag2^?/?) mice but not in T lymphocyte proficient, heterozygous Lat^+/Inv mice suggesting that Thy1^high cells of LAT‐deficient mice identified in this study accumulate in peripheral lymphoid organs as a result of congenital lymphopenia.     

Correction of T cell deficiency in ZAP‐70 knock‐out mice by simple intraperitoneal adoptive transfer of thymocytes

The tyrosine kinase zeta chain‐associated protein of 70 kDa (ZAP‐70) plays a key role in T cell development and signalling. In the absence of ZAP‐70, T cell development is arrested in the CD4⁺CD8⁺ double‐positive stage, thus ZAP‐70 homozygous knockout (ZAP‐70^–/–) mice have no mature T cells in their peripheral lymphoid organs and blood, causing severe immunodeficiency. We investigated the early kinetics and long‐term effects of wild‐type thymocyte transfer on T cell repopulation in ZAP‐70^–/– mice. We used a single intraperitoneal (i.p.) injection to deliver donor thymocytes to the recipients. Here, we show that after i.p. injection donor thymocytes leave the peritoneum through milky spots in the omentum and home to the thymus, where donor‐originated CD4^–CD8^– double‐negative thymocytes most probably restore T cell development and the disrupted thymic architecture. Subsequently, newly developed, donor‐originated, single‐positive αβ T cells appear in peripheral lymphoid organs, where they form organized T cell zones. The established chimerism was found to be stable, as donor‐originated cells were present in transferred ZAP‐70^–/– mice as late as 8 months after i.p. injection. We demonstrate that a simple i.p. injection of ZAP‐70^+/+ thymocytes is a feasible method for the long‐term reconstitution of T cell development in ZAP‐70‐deficient mice.     

Foxp3+ regulatory T‐cell homeostasis quantitatively differs in murine peripheral lymph nodes and spleen

Regulatory T (Treg) cells are essential for maintaining self‐tolerance and modulating inflammatory immune responses. Treg cells either develop within the thymus or are converted from CD4⁺ naive T (Tnaive) cells in the periphery. The Treg‐cell population size is tightly controlled and Treg‐cell development and homeostasis have been intensively studied; however, quantitative information about mechanisms of peripheral Treg‐cell homeostasis is lacking. Here we developed the first mathematical model of peripheral Treg‐cell homeostasis, incorporating secondary lymphoid organs as separate entities and encompassing factors determining the size of the Treg‐cell population, namely thymic output, homeostatic proliferation, peripheral conversion, transorgan migration, apoptosis, and the Tnaive‐cell population. Quantitative data were collected by monitoring Tnaive‐cell homeostasis and Treg‐cell rebound after selective in vivo depletion of Treg cells. Our model predicted the previously unanticipated possibility that Treg cells regulate migration of Tnaive cells between spleen and peripheral lymph nodes (LNs), whereas migration of Treg cells between these organs can largely be neglected. Furthermore, our simulations suggested that peripheral conversion significantly contributed to the maintenance of the Treg‐cell population, especially in LNs. Hence, we provide the first estimation of the peripheral Treg‐cell conversion rate and propose additional facets of Treg‐cell‐mediated immune regulation that may previously have escaped attention.     

Expression of toll‐like receptors in T lymphocytes stimulated with N‐(3‐oxododecanoyl)‐L‐homoserine lactone from Pseudomonas aeruginosa

The establishment of chronic Pseudomonas aeruginosa infections is correlated with the disturbance of the host immune system. The P. aeruginosa quorum‐sensing molecule N‐3‐(oxododecanoyl)‐L‐homoserine lactone (3‐O‐C12‐HSL) has the potential to modulate the host immune system. The immune system recognizes pathogens via toll‐like receptors (TLRs). We found that 3‐O‐C12‐HSL induced TLR changes in monocytes. However, the role of T cells in P. aeruginosa infection has not been delineated. In order to understand this activity, we examined whether 3‐O‐C12‐HSL has an effect on the immune function and the expression of TLRs in T lymphocytes. Human peripheral blood mononuclear cells (PBMCs) cells were cultured with 0, 1, 10, 50, or 100 μM 3‐O‐C12‐HSL for 12 h. TLR2/TLR4 expression and T‐lymphocyte proliferation were increased in a dose‐dependent manner, and 100 μM 3‐O‐C12‐HSL significantly increased TLR2 expression. Moreover, tumor necrosis factor‐α production of these PBMCs was inhibited. To conclude, 3‐O‐C12‐HSL can induce lymphocyte cell proliferation. These findings provide a new perspective on our understanding of the persistence of the chronic inflammation that accompanies P. aeruginosa infection.     

Differences in Bcl‐2 expression by T‐cell subsets alter their balance after in vivo irradiation to favor CD4+Bcl‐2hi NKT cells

Although it is well known that in vivo radiation depletes immune cells via the Bcl‐2 apoptotic pathway, a more nuanced analysis of the changes in the balance of immune‐cell subsets is needed to understand the impact of radiation on immune function. We show the balance of T‐cell subsets changes after increasing single doses of total body irradiation (TBI) or after fractionated irradiation of the lymphoid tissues (TLI) of mice due to differences in radioresistance and Bcl‐2 expression of the NKT‐cell and non‐NKT subsets to favor CD4⁺Bcl‐2^hi NKT cells. Reduction of the Bcl‐2^lo mature T‐cell subsets was at least 100‐fold greater than that of the Bcl‐2^hi subsets. CD4⁺ NKT cells upregulated Bcl‐2 after TBI and TLI and developed a Th2 bias after TLI, whereas non‐NKT cells failed to do so. Our previous studies showed TLI protects against graft versus host disease in wild‐type, but not in NKT‐cell‐deficient mice. The present study shows that NKT cells have a protective function even after TBI, and these cells are tenfold more abundant after an equal dose of TLI. In conclusion, differential expression of Bcl‐2 contributes to the changes in T‐cell subsets and immune function after irradiation.     

Aire controls the recirculation of murine Foxp3+ regulatory T‐cells back to the thymus

In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4⁺ and CD8⁺ single positive (SP) thymocytes. For example, mTEC expression of Aire controls intrathymic self‐antigen availability for negative selection. Interestingly, alterations in both Foxp3⁺ Regulatory T‐cells (T‐Reg) and conventional SP thymocytes in Aire^?/? mice suggest additional, yet poorly understood, roles for Aire during intrathymic T‐cell development. To examine this, we analysed thymocytes from Aire^?/? mice using Rag2GFP and Foxp3 expression, and a recently described CD69/MHCI subset definition of post‐selection CD4⁺ conventional thymocytes. We show that while Aire is dispensable for de novo generation of conventional αβT‐cells, it plays a key role in controlling the intrathymic T‐Reg pool. Surprisingly, a decline in intrathymic T‐Reg in Aire^?/? mice maps to a reduction in mature recirculating Rag2GFP^? T‐Reg that express CCR6 and re‐enter the thymus from the periphery. Furthermore, we show mTEC expression of the CCR6 ligand CCL20 is reduced in Aire^?/? mice, and that CCR6 is required for T‐Reg recirculation back to the thymus. Collectively, our study re‐defines requirements for late stage intrathymic αβT‐cell development, and demonstrates that Aire controls a CCR6‐CCL20 axis that determines the developmental makeup of the intrathymic T‐Reg pool.     

Prolonged exposure of naïve CD8+ T cells to interleukin-7 or interleukin-15 stimulates proliferation without differentiation or loss of telomere length

Interleukin (IL)‐7 and IL‐15 are cytokines implicated in homeostatic control of the peripheral CD8 T‐cell pool. We compared the effects of IL‐7 and IL‐15 on survival and proliferation of purified human CD8⁺ T‐cell subsets. Low concentrations of either cytokine reduced the spontaneous apoptosis of all subsets, and enhancement of survival corresponded to the extent of Bcl‐2 up‐regulation. Surprisingly, although minimal proliferation of naïve CD8⁺ T cells was observed during the first week of culture with cytokines, a marked expansion of these cells occurred at later time points, particularly in response to IL‐15. This occurred largely without phenotypic change or acquisition of effector function, indicating a dissociation of differentiation from proliferation. Notably, progression of naïve CD8⁺ T cells through several cell divisions resulted in up‐regulation of telomerase and the maintenance of telomere length. These data show that IL‐7 and IL‐15 induce cell proliferation and rescue from apoptosis in a concentration, time and subset‐dependent manner, and have implications for the homeostatic expansion of the naïve CD8⁺ T‐cell pool.     

IL‐15 regulates Bcl‐2 family members Bim and Mcl‐1 through JAK/STAT and PI3K/AKT pathways in T cells

Maintenance of T cells is determined by their survival capacity, which is regulated by Bcl‐2 proteins. Cytokines signalling through the common gamma chains such as IL‐2, IL‐7 and IL‐15 are important for T‐cell survival but how these cytokines determine the expression of Bcl‐2‐family proteins is not clear. We report signalling events of cytokines that regulate expression of two key Bcl‐2 proteins, pro‐apoptotic Bim and anti‐apoptotic Mcl‐1, in resting C57BL/6 mouse T cells. IL‐2, IL‐7 and IL‐15 inhibited apoptosis but paradoxically induced the expression of Bim, countered by concomitant induction of Mcl‐1. Bim induction by IL‐15 was found at the mRNA and protein levels and depended on both JAK/STAT and PI3K signals. A new STAT5‐binding site was identified in the Bim promoter, which was occupied by STAT5 upon IL‐15 stimulation. Although it also depended on JAK/STAT‐ and PI3K signalling, Mcl‐1 regulation was independent of Mcl‐1 mRNA levels and of regulation of protein stability, suggesting translational regulation. Concurrent CD3 signals inhibited some of the IL‐7 effect but not the IL‐15 effect on Bcl‐2 proteins. The data suggest that cytokines induce Bim and prime T cells for apoptosis, but also inhibit apoptosis by stabilising Mcl‐1. Later downregulation of short‐lived Mcl‐1 may induce efficient, Bim‐dependent apoptosis.     

A polymorphism in a phosphotyrosine signalling motif of CD229 (Ly9, SLAMF3) alters SH2 domain binding and T‐cell activation

Signalling lymphocyte activation molecule (SLAM) family members regulate activation and inhibition in the innate and adaptive immune systems. Genome‐wide association studies identified their genetic locus (1q23) as highly polymorphic and associated with susceptibility to systemic lupus erythematosus (SLE). Here we show that the Val₆₀₂ variant of the non‐synonymous single nucleotide polymorphism (SNP) rs509749 in the SLAM family member CD229 (Ly9, SLAMF3) has a two‐fold lower affinity compared with the SLE‐associated Met₆₀₂ variant for the small adaptor protein SAP. Comparison of the two variants in T‐cell lines revealed the Val₆₀₂ variant to be significantly more highly expressed than CD229 Met₆₀₂. Activation was diminished in cells expressing CD229 Val₆₀₂ compared with CD229 Met₆₀₂ as measured by up‐regulation of CD69. There was no correlation between homozygosity at rs509749 and activation in peripheral blood mononuclear cells from healthy donors. These findings identify potential mechanisms by which a single SNP can perturb fine‐tuning in the immune system with significant functional consequences.