Juan Moyron-Quiroz

Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity

Bronchus-associated lymphoid tissue (BALT) is occasionally found in the lungs of mice and humans; however, its role in respiratory immunity is unknown. Here we show that mice lacking spleen, lymph nodes and Peyers patches generate unexpectedly robust primary B- and T-cell responses to influenza, which seem to be initiated at sites of induced BALT (iBALT). Areas of iBALT have distinct B-cell follicles and T-cell areas, and support T and B-cell proliferation. The homeostatic chemokines CXCL13 and CCL21 are expressed independently of TNFα and lymphotoxin at sites of iBALT formation. In addition, mice with iBALT, but lacking peripheral lymphoid organs, clear influenza infection and survive higher doses of virus than do normal mice, indicating that immune responses generated in iBALT are not only protective, but potentially less pathologic, than systemic immune responses. Thus, iBALT functions as an inducible secondary lymphoid tissue for respiratory immune responses.

Omental Milky Spots Develop in the Absence of Lymphoid Tissue-Inducer Cells and Support B and T Cell Responses to Peritoneal Antigens

The omentum is a site of B1 cell lymphopoiesis and immune responsiveness to T cell-independent antigens. However, it is unknown whether it supports immune responses independently of conventional lymphoid organs. We showed that the omentum collected antigens and cells from the peritoneal cavity and supported T cell-dependent B cell responses, including isotype switching, somatic hypermutation, and limited affinity maturation, despite the lack of identifiable follicular dendritic cells. The omentum also supported CD4+ and CD8+ T cell responses to peritoneal antigens and recruited effector T cells primed in other locations. Unlike conventional lymphoid organs, milky spots in the omentum developed in the absence of lymphoid tissue-inducer cells, but required the chemokine CXCL13. Although the lymphoid architecture of milky spots was disrupted in lymphotoxin-deficient mice, normal architecture was restored by reconstitution with lymphotoxin-sufficient hematopoietic cells. These results indicate that the milky spots of the omentum function as unique secondary lymphoid organs that promote immunity to peritoneal antigens.

Pulmonary expression of CXC chemokine ligand 13, CC chemokine ligand 19, and CC chemokine ligand 21 is essential for local immunity to influenza

CXC chemokine ligand 13 (CXCL13), CC chemokine ligand 21 (CCL21), and CCL19 are constitutively expressed in secondary lymphoid organs, where they control the placement of lymphocytes and dendritic cells. However, these chemokines are also inducibly expressed in the lung after influenza infection. Here we show that, in the absence of spleen and lymph nodes, the expression of homeostatic chemokines in the lung is essential for local B and T cell responses to influenza and for the development and organization of inducible bronchus-associated lymphoid tissue (iBALT). Surprisingly, despite the association between local CXCL13 expression and the formation of ectopic lymphoid tissues, the loss of CXCL13 in the lung had minimal impact on either the development or function of iBALT. In contrast, the loss of CCL19 and CCL21 impaired iBALT formation as well as B and T cell responses. These results demonstrate that the local expression of homeostatic chemokines in nonlymphoid organs, such as the lung, plays an important role in protective immune responses.

CD4 T Cell-Independent Antibody Response Promotes Resolution of Primary Influenza Infection and Helps to Prevent Reinfection

It is generally believed that the production of influenza-specific IgG in response to viral infection is dependent on CD4 T cells. However, we previously observed that CD40-deficient mice generate influenza-specific IgG during a primary infection, suggesting that influenza infection may elicit IgG responses independently of CD4 T cell help. In the present study, we tested this hypothesis and show that mice lacking CD40 or CD4 T cells produce detectable titers of influenza-specific IgG and recover from influenza infection in a manner similar to that of normal mice. In contrast, mice completely lacking B cells succumb to influenza infection, despite the presence of large numbers of functional influenza-specific CD8 effector cells in the lungs. Consistent with the characteristics of a T-independent Ab response, long-lived influenza-specific plasma cells are not found in the bone marrow of CD40−/− and class II−/− mice, and influenza-specific IgG titers wane within 60 days postinfection. However, despite the short-lived IgG response, CD40−/− and class II−/− mice are completely protected from challenge infection with the same virus administered within 30 days. This protection is mediated primarily by B cells and Ab, as influenza-immune CD40−/− and class II−/− mice were still resistant to challenge infection when T cells were depleted. These data demonstrate that T cell-independent influenza-specific Ab promotes the resolution of primary influenza infection and helps to prevent reinfection.

Role of CXC Chemokine Ligand 13, CC Chemokine Ligand (CCL) 19, and CCL21 in the Organization and Function of Nasal-Associated Lymphoid Tissue

Nasal-associated lymphoid tissue (NALT) orchestrates immune responses to Ags in the upper respiratory tract. Unlike other lymphoid organs, NALT develops independently of lymphotoxin-α (LTα). However, the structure and function of NALT are impaired in Ltα−/− mice, suggesting a link between LTα and chemokine expression. In this study we show that the expression of CXCL13, CCL19, CCL21, and CCL20 is impaired in the NALT of Ltα−/− mice. We also show that the NALT of Cxcl13−/− and plt/plt mice exhibits some, but not all, of the structural and functional defects observed in the NALT of Ltα−/− mice. Like the NALT of Ltα−/− mice, the NALT in Cxcl13−/− mice lacks follicular dendritic cells, BP3+ stromal cells, and ERTR7+ lymphoreticular cells. However, unlike the NALT of Ltα−/− mice, the NALT of Cxcl13−/− mice has peripheral node addressin+ high endothelial venules (HEVs). In contrast, the NALT of plt/plt mice is nearly normal, with follicular dendritic cells, BP3+ stromal cells, ERTR7+ lymphoreticular cells, and peripheral node addressin+ HEVs. Functionally, germinal center formation and switching to IgA are defective in the NALT of Ltα−/− and Cxcl13−/− mice. In contrast, CD8 T cell responses to influenza are impaired in Ltα−/− mice and plt/plt mice. Finally, the B and T cell defects in the NALT of Ltα−/− mice lead to delayed clearance of influenza from the nasal mucosa. Thus, the B and T cell defects in the NALT of Ltα−/− mice can be attributed to the impaired expression of CXCL13 and CCL19/CCL21, respectively, whereas impaired HEV development is directly due to the loss of LTα.

CD40, but Not CD154, Expression on B Cells Is Necessary for Optimal Primary B Cell Responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40−/− and CD154−/− mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.

The Function of Local Lymphoid Tissues in Pulmonary Immune Responses

Primary adaptive immune responses are initiated in secondary lymphoid organs, such as spleen, lymph nodes, and Peyer’s patches. These lymphoid organs recruit naive lymphocytes1 as well as activated antigen-presenting cells (APCs)2, and facilitate lymphocyte activation, expansion, and differentiation. For example, infection of the lung with influenza virus leads to activation of pulmonary dendritic cells, which engulf local antigens and traffic to the draining mediastinal lymph node (MLN)3, where they home to the T cell area surrounding the high endothelial venules (HEVs) (Figure 1). Naive B and T cells are constantly recruited into the lymph node via these HEVs and rapidly become activated as they encounter cognate antigen on APCs. Activated lymphocytes subsequently expand and differentiate into effector cells. For T cells, this differentiation primarily occurs in the T cell zone. In contrast, B cells rapidly expand and are selected for high-affinity variants in the germinal centers (GCs) that develop on the border between the T cell area and the B cell follicle. As the immune response progresses, effector B and T cells leave the lymph node via the efferent lymphatics, which drain into the blood via the thoracic duct. Once in the blood, activated effector cells recirculate to sites of inflammation, including the original site of infection in the lung, and use their effector functions to combat infection. An important point of this model is that, while infection occurs locally in non-lymphoid organs, primary immune responses are initiated centrally in secondary lymphoid organs. This scheme is outlined in Figure 1.