Lymphocyte subsets in the peripheral blood are disturbed in systemic sclerosis patients and can be changed by immunosuppressive medication

Systemic sclerosis (SSc) is a severe chronic disease with a broad spectrum of clinical manifestations. SSc displays disturbed lymphocyte homeostasis. Immunosuppressive medications targeting T or B cells can improve disease manifestations. SSc clinical manifestations and immunosuppressive medication in itself can cause changes in lymphocyte subsets. The aim of this study was to investigate peripheral lymphocyte homeostasis in SSc with regards to the immunosuppression and to major organ involvement. 44 SSc patients and 19 healthy donors (HD) were included. Immunophenotyping of peripheral whole blood by fluorescence-activated cell sorting was performed. Cytokine secretions of stimulated B cell cultures were measured. SSc patients without immunosuppression compared to HD displayed lower γδ T cells, lower T helper cells (CD3+/CD4+), lower transitional B cells (CD19+/CD38++/CD10+/IgD+), lower pre-switched memory B cells (CD19+/CD27+/IgD+), and lower post-switched memory B cells (CD19+/CD27+/IgD−). There was no difference in the cytokine production of whole B cell cultures between SSc and HD. Within the SSc cohort, mycophenolate intake was associated with lower T helper cells and lower NK cells (CD56+/CD3−). The described differences in peripheral lymphocyte subsets between SSc and HD generate further insight in SSc pathogenesis. Lymphocyte changes under effective immunosuppression indicate how lymphocyte homeostasis in SSc might be restored.

The Intraflagellar Transport Protein IFT20 Recruits ATG16L1 to Early Endosomes to Promote Autophagosome Formation in T Cells

Lymphocyte homeostasis, activation and differentiation crucially rely on basal autophagy. The fine-tuning of this process depends on autophagy-related (ATG) proteins and their interaction with the trafficking machinery that orchestrates the membrane rearrangements leading to autophagosome biogenesis. The underlying mechanisms are as yet not fully understood. The intraflagellar transport (IFT) system, known for its role in cargo transport along the axonemal microtubules of the primary cilium, has emerged as a regulator of autophagy in ciliated cells. Growing evidence indicates that ciliogenesis proteins participate in cilia-independent processes, including autophagy, in the non-ciliated T cell. Here we investigate the mechanism by which IFT20, an integral component of the IFT system, regulates basal T cell autophagy. We show that IFT20 interacts with the core autophagy protein ATG16L1 and that its CC domain is essential for its pro-autophagic activity. We demonstrate that IFT20 is required for the association of ATG16L1 with the Golgi complex and early endosomes, both of which have been identified as membrane sources for phagophore elongation. This involves the ability of IFT20 to interact with proteins that are resident at these subcellular localizations, namely the golgin GMAP210 at the Golgi apparatus and Rab5 at early endosomes. GMAP210 depletion, while leading to a dispersion of ATG16L1 from the Golgi, did not affect basal autophagy. Conversely, IFT20 was found to recruit ATG16L1 to early endosomes tagged for autophagosome formation by the BECLIN 1/VPS34/Rab5 complex, which resulted in the local accumulation of LC3. Hence IFT20 participates in autophagosome biogenesis under basal conditions by regulating the localization of ATG16L1 at early endosomes to promote autophagosome biogenesis. These data identify IFT20 as a new regulator of an early step of basal autophagy in T cells.

The intraflagellar transport protein IFT20 recruits ATG16L1 to early endosomes to promote autophagosome formation in T cells

ABSTRACTLymphocyte homeostasis, activation and differentiation crucially rely on basal autophagy. The fine-tuning of this process depends on autophagy-related (ATG) proteins and their interaction with the trafficking machinery that orchestrates the membrane rearrangements leading to autophagosome biogenesis. The underlying mechanisms are as yet not fully understood. The intraflagellar transport (IFT) system, known for its role in cargo transport along the axonemal microtubules of the primary cilium, has emerged as a regulator of autophagy in ciliated cells. Growing evidence indicates that ciliogenesis proteins participate in cilia-independent processes, including autophagy, in the non-ciliated T cell. Here we investigate the mechanism by which IFT20, an integral component of the IFT system, regulates basal T cell autophagy. We show that IFT20 interacts with the core autophagy protein ATG16L1 through its CC domain, which is also essential for its pro-autophagic activity. We demonstrate that IFT20 is required for the association of ATG16L1 with the Golgi complex and early endosomes, both of which have been identified as membrane sources for phagophore elongation. This involves the ability of IFT20 to interact with proteins that are resident at these subcellular localizations, namely the golgin GMAP210 at the Golgi apparatus and Rab5 at early endosomes. GMAP210 depletion, while leading to a dispersion of ATG16L1 from the Golgi, did not affect basal autophagy. Conversely, IFT20 was found to recruit ATG16L1 to early endosomes tagged for autophagosome formation by the BECLIN 1/VPS34/Rab5 complex, which resulted in the local accumulation of LC3. Hence IFT20 participates in autophagosome biogenesis under basal conditions by regulating the localization of ATG16L1 at early endosomes to promote autophagosome biogenesis. These data identify IFT20 as a new regulator of an early step of basal autophagy in T cells.

Cathepsin K maintains the number of lymphocytes in vivo

In this study, we investigated the influence of the loss of Cathepsin K (Ctsk) gene on the hematopoietic system in vitro and in vivo. We found that cultures with Lineage- SCA1+ KIT+ (LSK) cells on Ctsk deficient stromal cells display reduced colony formation and proliferation, with increased differentiation, giving rise to repopulating cells with reduced ability to repopulate the donor LSK and T cell compartments in the bone marrow. Subsequent in vivo experiments showed impairment of lymphocyte numbers, but, gross effects on early hematopoiesis or myelopoiesis were not found. Most consistently in in vivo experimental settings, we found a significant reduction of (donor) T cell numbers in the bone marrow. Lymphocyte deregulation is also found in transplantation experiments, which revealed that Ctsk is required for optimal regeneration not only of T cells, but also of B cells. Interestingly, cell non-autonomous Ctsk regulates both B- and T cell numbers, but T cell numbers in the bone marrow require an additional autonomous Ctsk-dependent process. Thus, we show that Ctsk is required for the maintenance of hematopoietic stem cells in vitro, but in vivo, Ctsk deficiency most strongly affects lymphocyte homeostasis, particularly of T cells in the bone marrow.

YAP and TAZ in Fibroblastic Reticular Cells Support Hematopoiesis and Retention of Lymphocytes in Lymph Nodes

INTRODUCTION: Fibroblastic reticular cells (FRCs) are essential for adaptive immune response and maintaining lymph node (LN) homeostasis. FRCs regulate immune cell entry into the LN and confer compartmentalization of lymphocytes by secretion of essential chemokines. Indeed, FRCs are activated and display an undifferentiated phenotype in LN of patients with follicular lymphoma, but their meaning and mode action are unclear. The mammalian Hippo signaling pathway is an essential regulator of cell fate, differentiation, and homeostasis. Its final downstream effectors YAP and TAZ (YAP/TAZ) are inhibited by LATS1 and LATS2 (LATS1/2) in a phosphorylation-dependent manner. The roles of YAP/TAZ in hematopoiesis are considered to be dispensable under healthy physiological condition, but their significance appears to be context dependent in pathologies including hematologic malignancies. OBJECTIVES: We hypothesized that the Hippo pathway plays important roles in regulating FRCs by interacting with lymphocytes, with relevance to structural and functional properties of LNs. Using FRC-specific YAP/TAZ depletion or hyperactivation mouse models, we sought to investigate the roles of YAP/TAZ in orchestrating lymphocyte homeostasis by FRCs. METHODS: Animal care and experimental procedures were performed under the approval of the Institutional Animal Care and Use Committee (KA2016-12) of KAIST. Yap/Taz∆FRC or Lats1/2∆FRC mouse were generated by crossing FRC-specific Ccl19-Cre or inducible Pdgfrb-Cre-ERT2 mouse with Yapfl/fl/Tazfl/fl or Lats1fl/fl/Lats2fl/fl mouse, respectively. Cre-ERT2-negative but flox/flox-positive littermates were defined as wild-type mice (WT). In order to induce Cre activity, 2 mg of tamoxifen (Sigma-Aldrich) was dissolved in corn oil and intra-peritoneally injected for four consecutive days. Skin-draining LNs were analyzed with immunofluorescence staining, flow cytometry, or fluorescence activated cell sorting. Chemokine expression was measured from sorted FRCs with qRT-PCR, immunoblotting, or ELISA. APC BrdU flow kit (BD Biosciences) was used for cell cycle analysis. Blood count in mice was measured with hemocytometer (pocH-100iV DiFF, Sysmex). RESULTS: Both YAP/TAZ were highly and equally distributed in nucleus and cytoplasm of mouse LN FRCs, in addition to blood and lymphatic endothelial cells. LNs of Yap/Taz∆FRC mice compared with WT revealed a slight reduction in LN weight and reduced total cell number (~64.9%), decreased proportion (~42.9%), and number (~61.1 %) of FRCs among CD45- stromal cells, which were less proliferative (~65.2%) but had no difference in apoptosis. However, neither proliferation nor apoptosis of immune cells was altered. Analysis of isolated FRCs revealed that mRNA levels of lymphoid chemokines for immune cell trafficking and function were significantly attenuated in Yap/TazΔFRC mice compared with WT. Intriguingly, Lats1/2∆FRC mice looked pale from 1 week after birth. We found lower red blood cell (RBC) count (~16.1%) with decreased concentration of hemoglobin (~25.8%) in Lats1/2∆FRC mice compared with WT, suggesting hypochromic anemia. Moreover, although whole body length and overall organ size were small in Lats1/2∆FRC mice compared with WT at 2 weeks after birth, we could not observe any gross or microscopic abnormalities in major organs including brain, heart, lung, liver, and kidney in Lats1/2∆FRC mice. We further analyzed the primary lymphoid organ thymus and the secondary lymphoid organ spleen, as they are both supported by CCL19+ FRCs. Similar to major organs, the overall size of thymus and spleen was smaller in Lats1/2∆FRC mice compared with WT at 2 weeks after birth. However, microscopic analysis of thymus revealed that the border between the cortex and medulla was disrupted by the expanded medulla at the periphery that blends into the surrounding cortex in Lats1/2∆FRC mice, and analysis of spleen also revealed unclear lymphoid follicles with significantly reduced number of immune cells within the expanded white pulp in Lats1/2∆FRC mice compared with WT. CONCLUSIONS: Our findings present YAP/TAZ as essential governors of FRCs by supporting hematopoiesis and lymphocyte homeostasis. Considering that YAP/TAZ are enriched in bone marrow stromal cells, additional work to elucidate their role in promoting bone marrow microenvironment will improve our understanding on hematopoietic stem cell niche. Disclosures No relevant conflicts of interest to declare.