Cbfβ2 controls differentiation of and confers homing capacity to prethymic progenitors

Multipotent hematopoietic progenitors must acquire thymus-homing capacity to initiate T lymphocyte development. Despite its importance, the transcriptional program underlying this process remains elusive. Cbfβ forms transcription factor complexes with Runx proteins, and here we show that Cbfβ2, encoded by an RNA splice variant of the Cbfb gene, is essential for extrathymic differentiation of T cell progenitors. Furthermore, Cbfβ2 endows extrathymic progenitors with thymus-homing capacity by inducing expression of the principal thymus-homing receptor, Ccr9. This occurs via direct binding of Cbfβ2 to cell type–specific enhancers, as is observed in Rorγt induction during differentiation of lymphoid tissue inducer cells by activation of an intronic enhancer. As in mice, an alternative splicing event in zebrafish generates a Cbfβ2-specific mRNA, important for ccr9 expression. Thus, despite phylogenetically and ontogenetically variable sites of origin of T cell progenitors, their robust thymus-homing capacity is ensured by an evolutionarily conserved mechanism emerging from functional diversification of Runx transcription factor complexes by acquisition of a novel splice variant.

Increased prevalence of lymphoid tissue inducer cells in the cerebrospinal fluid of patients with early multiple sclerosis

Background: Inflammatory cytokines produced by cells of the immune system are believed to play a central role in the pathogenesis of multiple sclerosis (MS). Innate lymphoid cells (ILCs) have been shown to produce and secrete a wide range of the cytokines involved in MS pathogenesis; however, a possible implication of ILCs in MS development and disease progression has not been investigated. Objective: With this study, we aimed to clarify a potential role of ILCs in the early stages of MS. Methods and Results: Using flow cytometry, we analysed the prevalence and phenotype of ILCs in the cerebrospinal fluid (CSF) of patients experiencing their first or second demyelinating event. We found a substantial increase in both frequency and number of ILCs, in particular the LTi subset, as compared to healthy controls. We also found an association between CSF pleocytosis and an increased frequency of LTi cells in the CSF, suggesting a favoured recruitment of blood derived LTi cells. Conclusion: Our data suggests a role for ILCs, and in particular the LTi subset, in the early stages of MS. This finding represents an important contribution to the understanding of early inflammation in MS, and adds new knowledge beneficial for future MS therapies.

Human innate lymphoid cells

Innate lymphoid cells (ILCs) are lymphoid cells that do not express rearranged receptors and have important effector and regulatory functions in innate immunity and tissue remodeling. ILCs are categorized into 3 groups based on their distinct patterns of cytokine production and the requirement of particular transcription factors for their development and function. Group 1 ILCs (ILC1s) produce interferon γ and depend on Tbet, group 2 ILCs (ILC2s) produce type 2 cytokines like interleukin-5 (IL-5) and IL-13 and require GATA3, and group 3 ILCs (ILC3s) include lymphoid tissue inducer cells, produce IL-17 and/or IL-22, and are dependent on RORγt. Whereas ILCs play essential roles in the innate immune system, uncontrolled activation and proliferation of ILCs can contribute to inflammatory autoimmune diseases. In this review, we provide an overview of the characteristics of ILCs in the context of health and disease. We will focus on human ILCs but refer to mouse studies if needed to clarify aspects of ILC biology.

Endothelial cell–specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation

The development of lymph nodes (LNs) and formation of LN stromal cell microenvironments is dependent on lymphotoxin-β receptor (LTβR) signaling. In particular, the LTβR-dependent crosstalk between mesenchymal lymphoid tissue organizer and hematopoietic lymphoid tissue inducer cells has been regarded as critical for these processes. Here, we assessed whether endothelial cell (EC)–restricted LTβR signaling impacts on LN development and the vascular LN microenvironment. Using EC-specific ablation of LTβR in mice, we found that conditionally LTβR-deficient animals failed to develop a significant proportion of their peripheral LNs. However, remnant LNs showed impaired formation of high endothelial venules (HEVs). Venules had lost their cuboidal shape, showed reduced segment length and branching points, and reduced adhesion molecule and constitutive chemokine expression. Due to the altered EC–lymphocyte interaction, homing of lymphocytes to peripheral LNs was significantly impaired. Thus, this study identifies ECs as an important LTβR-dependent lymphoid tissue organizer cell population and indicates that continuous triggering of the LTβR on LN ECs is critical for lymphocyte homeostasis.

Restoration of CCL21 Accelerates Lymph Node (LN) and Spleen Regeneration Post-Transplant and Improves Anti-Pathogen and Anti-Tumor Effector Responses

Abstract 212 Introduction: BMT conditioning causes substantial stromal cell injury, which may hinder lymphopoiesis and immune recovery post-transplant. The C-C chemokine CCL21, produced by LN fibroblastic reticular cells (FRCs), is critical for the trafficking of CCR7 expressing T cells into secondary lymphoid organs (i.e. spleen, LN). We first reported that radiation conditioning markedly reduces FRCs, CCL21 expression, and lymphoid tissue inducer cells that are critical for lymphoid organogenesis. As a consequence of these events, mice have substantial T cell lymphopenia and poor responses to pathogens and neoantigens (Kelly et al, Blood, 2010). We hypothesized that CCL21 deficiency would preclude the recruitment of recent thymic emigrants to secondary lymphoid organs where they would receive necessary signals for their survival. Moreover, within the secondary lymphoid organs, T cells would be unable to properly migrate to B cell zones. Thus, we tested whether restoring CCL21 protein could improve LN stromal cell architecture, T cell trafficking and consequently, T cell effector responses. Methods/Results: C57BL/6 mice were lethally irradiated, rescued with congenic T cell depleted bone marrow, and given CCL21 cDNA containing or null adenovirus vector transduced dendritic cells (DCs) that were pulsed with lysates from either vesicular stomatitis or attenuated Listeria monocytogenes expressing the nominal antigen ovalbumin (VSV-OVA; Listeria-OVA). The DC vaccines were given intramuscularly beginning 21 days post-transplant and then weekly for three total doses. On day 42, mice were challenged with either VSV-OVA or Listeria-OVA and euthanized on day 43 or 50, respectively. Compared to DC/null, DC/CCL21 vaccine substantially improved spleen and LN architecture and markedly increased ER-TR7+ LN stromal cell density. These architectural changes were associated with significantly increased CCL21 expression (p<0.05 for both CCL21 density per B cell area and normalization to non-BMT controls determined by ELISA), resulting in a significant increase in total LN cells, CD8+ T cells and lymphoid tissue inducer cells (all p<0.05). Expression of the B cell chemoattractant, CXCL13, and B cell follicle number were significantly increased (p<0.05) in the spleen and LN of DC/CCL21 vaccinated mice and T cells were found juxtaposed to B cells within B cell zones. Compared to DC/null, DC/CCL21 LN CD8+ T cell number normalized vs non-BMT controls, and clearance of Listeria-OVA by the liver was significantly (p<0.05) improved and similar to non-BMT controls (3.5 vs 1.8 vs 1.2 × 105 colony-forming units, respectively). Similarly, VSV-OVA clearance was significantly improved in DC/CCL21 vs DC/null vaccines, both less than non-BMT controls (13.5 vs 26 vs 8 splenic plaque-forming units, respectively; all group-wise comparisons, p<0.05). In order to determine whether improved immune function could enhance the endogenous CD8+ T cell response to acute myeloid leukemia (AML) challenge post-BMT, congeneic BMT recipients were vaccinated with DC/CCL21 or DC/null pre-loaded with AML lysates. All mice received a lethal dose (106) of C57BL/6 AML cells expressing firefly luciferase (C1498) on d.42 post-BMT (see Figure). Compared to the uniform lethality in both DC/null vaccines and non-vaccinated, non-BMT controls, mice receiving DC/CCL21 had a significant increase (p=.0241 compared to DC/null) in survival with 50% of mice surviving long-term. Conclusions: Taken together, these data indicate that DC/CCL21 vaccines can potently restore LN and splenic architecture as well as improve endogenous T cell responses to pathogens and AML challenge. Because DC/CCL21 vaccines are in clinical trial for patients with lung cancer or melanoma, our studies may provide the foundation for future clinical trials of DC/CCL21 vaccination in patients receiving pre-transplant conditioning regimens. If our preclinical data prove translatable, DC/CCL21 vaccination could increase immune reconstitution in BMT recipients. This could lead to decreased relapse and increased pathogenic responses to life-threatening infections and decreasing morbidity and mortality post transplant. Disclosures: No relevant conflicts of interest to declare.