Complex regulation of CCR9 at multiple discrete stages of T cell development

We have conducted a comprehensive assessment of CCR9 expression and function at the important milestone stages of murine thymocyte development. We reveal an unusually complex regulatory pattern, in which CCR9 influences T cell development at several widely dispersed stages. We find that CCR9 is not expressed within the thymus until the double-negative (DN)3 stage, although it appears to contribute to T cell precursor development prior to residence in the thymus. CCR9 expression is influenced by pre-T cell receptor signals, and is dramatically up-regulated in a population that appears to be transitional between the DN4 and double-positive stages. In the periphery, functional CCR9 is expressed by all naive CD8 T cells, but not by naive CD4 T cells. To our knowledge, this latter finding is the first difference observed in homing receptor expression between naive lymphocyte populations. This suggests that naive CD8 T cells might have access to lymphoid microenvironments from which naive CD4 T cells are excluded.     

CCRL1/ACKR4 is expressed in key thymic microenvironments but is dispensable for T lymphopoiesis at steady state in adult mice

Thymus colonisation and thymocyte positioning are regulated by interactions between CCR7 and CCR9, and their respective ligands, CCL19/CCL21 and CCL25. The ligands of CCR7 and CCR9 also interact with the atypical receptor CCRL1 (also known as ACKR4), which is expressed in the thymus and has recently been reported to play an important role in normal αβT‐cell development. Here, we show that CCRL1 is expressed within the thymic cortex, predominantly by MHC‐II^lowCD40^? cortical thymic epithelial cells and at the subcapsular zone by a population of podoplanin⁺ thymic epithelial cells in mice. Interestingly, CCRL1 is also expressed by stromal cells which surround the pericytes of vessels at the corticomedullary junction, the site for progenitor cell entry and mature thymocyte egress from the thymus. We show that CCRL1 suppresses thymocyte progenitor entry into the thymus, however, the thymus size and cellularity are the same in adult WT and CCRL1^?/? mice. Moreover, CCRL1^?/? mice have no major perturbations in T‐cell populations at different stages of thymic differentiation and development, and have a similar rate of thymocyte migration into the blood. Collectively, our findings argue against a major role for CCRL1 in normal thymus development and function.     

Ligand thresholds at different stages of T cell development

Certain T cell ligands can stimulate most or all T cells whose receptor is encoded by particular V beta genes. Exposure to these same ligands during intrathymic development leads to deletion of T cells bearing these same receptors. We have utilized one such ligand, staphylococcal enterotoxin B (SEB), to examine the quantitative differences in these two responses. By comparing the dose of SEB required to delete developing T cells in thymic organ culture with that required to stimulate spleen cells to expand clonally, we observe that clonal deletion is one to two orders of magnitude more sensitive to SEB. Because the T cell ligand involves not only SEB but also class II MHC molecules, and because the culture conditions are distinct, this system does not allow one to state that intrathymic T cells are intrinsically more sensitive to negative selection than peripheral T cells are to activation. However, these studies for the first time do establish a quantitative comparison of these two processes and suggest that there is a margin for error built into the clonal deletion process, such that ligands presented in the thymus are 30- to 100-fold more active in clonal deletion than is the same ligand in activation of peripheral T cells. This result agrees well with the observation that naturally occurring unknown ligands associated with I-E can clonally delete cells that are not activated by the same ligand in mixed lymphocyte culture.     

Involvement of CCR3-reactive chemokines in eosinophil survival

BACKGROUND: Although eosinophils undergo apoptosis and are thus eliminated from sites of inflammation, they survive longer if survival factors, such as IL-3, IL-5 and GM-CSF, are present. However, it is often observed that some eosinophils survive even when incubated without any survival factors (spontaneous survival). The aim of the present study was to investigate what kind of factor(s) is associated with this spontaneous survival of eosinophils. METHODS: Eosinophils were purified from bronchoalveolar lavage of antigen-exposed Balb/c mice and stained with propidium iodide to detect apoptotic ones. RESULTS: We found that the spontaneous survival of eosinophils was reduced by treatment of the cells with anti-CC chemokine receptor-3 (CCR3) antibody (Ab). Moreover, eotaxin-1, eotaxin-2, and eotaxin-3 all prolonged the survival of eosinophils in a dose-dependent fashion. The survival-prolonging effect of eotaxin-1 was enhanced in the presence of eotaxin-3, indicating that these chemokines might work synergistically. CONCLUSION: We speculate that eosinophils survive longer under the influence of CCR3-reactive chemokines, which aid the infiltration of these cells into the tissue and that eosinophils may survive even longer if they encounter survival factors at local inflammatory sites.     

CCR4 frameshift mutation identifies a distinct group of adult T cell leukaemia/lymphoma with poor prognosis

Adult T cell leukaemia/lymphoma (ATLL) is an intractable T cell neoplasm caused by human T cell leukaemia virus type 1. Next‐generation sequencing‐based comprehensive mutation studies have revealed recurrent somatic CCR4 mutations in ATLL, although clinicopathological findings associated with CCR4 mutations remain to be delineated. In the current study, 184 cases of peripheral T cell lymphoma, including 113 cases of ATLL, were subjected to CCR4 mutation analysis. This sequence analysis identified mutations in 27% (30/113) of cases of ATLL and 9% (4/44) of cases of peripheral T cell lymphoma not otherwise specified. Identified mutations included nonsense (NS) and frameshift (FS) mutations. No significant differences in clinicopathological findings were observed between ATLL cases stratified by presence of CCR4 mutation. All ATLL cases with CCR4 mutations exhibited cell‐surface CCR4 positivity. Semi‐quantitative CCR4 protein analysis of immunohistochemical sections revealed higher CCR4 expression in cases with NS mutations of CCR4 than in cases with wild‐type (WT) CCR4. Furthermore, among ATLL cases, FS mutation was significantly associated with a poor prognosis, compared with NS mutation and WT CCR4. These results suggest that CCR4 mutation is an important determinant of the clinical course in ATLL cases, and that NS and FS mutations of CCR4 behave differently with respect to ATLL pathophysiology. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Estren-mediated inhibition of T lymphopoiesis is estrogen receptor-independent whereas its suppression of T cell-mediated inflammation is estrogen receptor-dependent

Estrogen has extensive effects on the immune system. The aim of the present experiments was to compare the effects of 17beta-estradiol (E2) and 4-estren-3alpha,17beta-diol (estren) on T lymphopoiesis and T cell-dependent inflammation. In order to investigate the role of estrogen receptors (ER) in the effects of E2 and estren on the immune system, ER knock-out mice lacking both ERalpha and ERbeta (DERKO) were used. T lymphopoiesis and T cell-dependent inflammation were studied by investigating thymus cellularity, the delayed-type hypersensitivity (DTH) reaction, CD4(+) T cells in spleen and serum levels of interleukin (IL)-6. As expected, the presence of ERs was mandatory for all the effects of E2. In contrast, treatment with estren reduced thymus cellularity in ER knock-out mice, indicating an effect through ER-independent pathways. Interestingly, estren suppressed only DTH, the frequency of CD4(+) T cells in spleen and serum levels of IL-6 in wild-type (WT) mice, but not in mice lacking ERs. Thus, our study is the first to show that estren inhibits T lymphopoiesis via ER-independent pathways, whereas its suppressive effects on inflammation are ER-dependent.     

Involvement of CC chemokines in gammadelta T lymphocyte trafficking during allergic inflammation: the role of CCL2/CCR2 pathway

In the present study, we show that the intra-thoracic injection of ovalbumin (OVA, 12.5 microg per cavity) into C57BL/10 mice induced a significant increase in gammadelta T lymphocyte numbers in the pleural cavity, blood and thoracic lymph node of challenged mice. Such increase was significant within 12 h, peaked within 48 h and returned to basal counts within 120 h. Levels of CC chemokine ligand (CCL)-2/monocyte chemotactic protein-1, CCL5/regulated upon activation, normal T cell expressed and secreted, CCL3/macrophage inflammatory protein-1 alpha and CCL25/thymus-expressed chemokine were above control values in pleural washes recovered 24 h after OVA challenge (OPW) and were likely produced by pleural macrophages and mesothelial cells. Antigenic challenge also induced an up-regulation in CC chemokine receptor (CCR)-2, CCR5 and CCR9 on gammadelta T cells from pleural cavities, blood and lymph nodes, suggesting that cells found in mice pleural cavity migrate from secondary lymphoid organs into the inflammatory site via blood stream. The in vitro neutralization of CCL2 (but not of CCL3, CCL5 or CCL25) abrogated OPW-induced gammadelta T lymphocyte transmigration. Confirming such results, the in vivo administration of alpha-CCL2 mAb inhibited gammadelta T lymphocyte accumulation in the pleural cavity of challenged mice, whereas the blockade of CCL3, CCL5 or CCL25 showed no effect on gammadelta T cell mobilization. In addition, OVA challenge failed to induce gammadelta T lymphocyte accumulation in the pleural cavity of C57BL/6 CCR2 knockout mice, which also showed decreased numbers of these cells in blood and lymph nodes when compared with wild-type mice. Overall, such results demonstrate that CCR2/CCL2 pathway is crucial for gammadelta T lymphocyte mobilization during the allergic response.     

Regulation of T lymphopoiesis by Notch1 and Lunatic fringe-mediated competition for intrathymic niches

Notch1 activation regulates T lineage commitment and early T cell development. Fringe glycosyltransferases alter the sensitivity of Notch receptors to Delta-like versus Jagged Notch ligands, but their functions in T lymphopoiesis have not been defined. Here we show that developmental stage-specific expression of the glycosyltransferase lunatic fringe (Lfng) is required for coordination of the access of T cell progenitors to intrathymic niches that support Notch1-dependent phases of T cell development. Lfng-null progenitors generated few thymocytes in competitive assays, whereas Lfng overexpression converted thymocytes into 'supercompetitors' with enhanced binding of Delta-like ligands and blocked T lymphopoiesis from normal progenitors. We suggest that the ability of Lfng and Notch1 to control progenitor competition for limiting cortical niches is an important mechanism for the homeostatic regulation of thymus size.     

CCR9+ T cells contribute to the resolution of the inflammatory response in a mouse model of intestinal amoebiasis

Analysis of the mechanisms underlying the inflammatory response in amoebiasis is important to understand the immunopathology of the disease. Mucosal associated effector and regulatory T cells may play a role in regulating the inflammatory immune response associated to Entamoeba histolytica infection in the colon. A subpopulation of regulatory T cells has recently been identified and is characterized by the expression of the chemokine receptor CCR9. In this report, we used CCR9 deficient (CCR9(-/-)) mice to investigate the role of the CCR9(+) T cells in a murine model of E. histolytica intestinal infection. Intracecal infection of CCR9(+/+), CCR9(+/-) and CCR9(-/-) mice with E. histolytica trophozoites, revealed striking differences in the development and nature of the intestinal inflammatory response observed between these strains. While CCR9(+/+) and CCR9(+/-) mice were resistant to the infection and resolved the pathogen-induced inflammatory response, CCR9(-/-) mice developed a chronic inflammatory response, which was associated with over-expression of the cytokines IFN-γ, TNF-α, IL-4, IL-6 and IL-17, while IL-10 was not present. In addition, increased levels of CCL11, CCL20 and CCL28 chemokines were detected by qRT-PCR in CCR9(-/-) mice. E. histolytica trophozoites were identified in the lumen of the cecum of CCR9(-/-) mice at seven days post infection (pi), whereas in CCR9(+/+) mice trophozoites disappeared by day 1 pi. Interestingly, the inflammation observed in CCR9(-/-) mice, was associated with a delayed recruitment of CD4(+)CD25(+)FoxP3(+) T cells to the cecal epithelium and lamina propria, suggesting that this population may play a role in the early regulation of the inflammatory response against E. histolytica, likely through IL-10 production. In support of these data, CCR9(+) T cells were also identified in colon tissue sections obtained from patients with amoebic colitis. Our data suggest that a population of CCR9(+)CD4(+)CD25(+)FoxP3(+) T cells may participate in the control and resolution of the inflammatory immune response to E. histolytica infection.     

Requirement for Notch1 signals at sequential early stages of intrathymic T cell development

Signaling through the transmembrane Notch1 receptor directs thymus-seeding progenitors (TSPs) to suppress their B cell potential and 'choose' the T cell fate. Present paradigms suggest that TSPs are contained in the multipotent early T lineage precursor (ETP) subset of thymocytes. However, we show here that the B cell potential of ETPs was not augmented in microenvironments that limited Notch1 activation. Furthermore, low-threshold Notch1 signals suppressed B cell production by TSPs before they reached the ETP stage. Notch1 signals of a higher threshold were needed to drive proliferation of ETPs and development into CD4(+)CD8(+) double-positive thymocytes. Thus, TSPs can be differentiated from all previously identified early T cell progenitors by their robust B cell potential and exquisite sensitivity to Notch1 signals.     

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.     

B lymphopoiesis on stromal cell clone: stromal cell clones acting on different stages of B cell differentiation

B lymphopoiesis supporting activities of two stromal cell clones, MC3T3-G2/PA6 (PA6) and ST2, were compared. When normal bone marrow cells were cultured in these clones under Whitlock-Witte-type condition, mature B cells were generated only in the culture with the ST2 layer. The cells maintained on the PA6 layer, however, contained the precursor cells giving rise to mature B cells when transferred to the ST2 layer. Thus, PA6 is a stromal cell clone capable of supporting the early B progenitors but cannot support a further maturation step into pre-B cells. The immunoglobulin heavy chain gene configuration of B progenitors maintained on the PA6 layer diversified after their transfer onto ST2 layer. This suggests that they are actually the earliest progenitors. This marked difference in the stromal cell activities between PA6 and ST2 could also be distinguished by stromal cell-dependent pre-B cell lines. Among four ST2-dependent pre-B cell lines tested, two grew only on the ST2 layer, which is capable of supporting B lymphopoiesis, while the others grew both on the ST2 and PA6 layers. These results strongly suggest that the process of intra-marrow B cell development is controlled by more than one signal acting on different stages of B cell differentiation.     

Involvement of 9p in metastatic ovarian adenocarcinomas

By a direct method and after in vitro culture, we cytogenetically analyzed five ovarian adenocarcinomas, using six samples of ascitic fluid. The structural aberrations included rearrangements of chromosomes 1 and 3, in agreement with the results of other researchers. The long arm of chromosome 6 was rearranged in three samples, but no translocation t(6;14) was found. Rearrangements involving 9p were present in all cases, with breakpoints at p13 or p22-23.