Cutting edge: Secondary lymphoid organs are essential for maintaining the CD4, but not CD8, naive T cell pool.

Despite declining thymic output with age, the peripheral naive T cell pool of an adult animal remains remarkably stable. Therefore, a central question in immunology is how the naive T cell pool is maintained. Here we show that the maintenance of the naive CD4, but not CD8, T cell population in the thymectomized adult mouse is dependent on the presence of secondary lymphoid tissues. This finding is explained by the inability of naive CD4 T cells to sustain normal levels of the survival molecule Bcl-2 or to undergo homeostatic proliferation in the absence of secondary lymphoid organs. Thus, naive CD4 T cells must traffic through secondary lymphoid organs to maintain a stable CD4 pool while naive CD8 T cells encounter their survival and proliferation signals outside the organized structures of secondary lymphoid tissues.     

Cellular interactions in lymph node development

The organized accumulation of lymphocytes is a biological phenomenon used to optimize both homeostatic immune surveillance, as well as chronic responses to pathogenic stimuli. During embryonic development, circulating hemopoietic cells gather at predestined sites throughout the body, where they are subsequently arranged in T and B cell-specific areas characteristic of secondary lymphoid organs. In contrast, the body seems to harbor a limited second set of selected sites that support formation of organized lymphoid aggregates. However, these are only revealed at times of local, chronic inflammation, when so-called tertiary lymphoid structures appear. Once thought of as two distinct phenomena, recent insights suggest that highly similar networks of paracrine interactions regulate the formation of both secondary and tertiary lymphoid structures. This review will focus on these cellular interactions between organizing and inducing cell populations leading to the formation of lymph nodes or organized inflammatory infiltrates.     

The role of lymphotoxin in development and maintenance of secondary lymphoid tissues

Secondary lymphoid organs provide the necessary microenvironment for the cooperation of antigen-specific T- and B-lymphocytes and antigen-presenting cells in order to initiate an efficient immune response. Remarkable progress in understanding of the mechanisms of lymphoid organogenesis was achieved due to the analysis of various gene-targeted mice. This review primarily focuses on the role of lymphotoxin (LT) in development, maturation and maintenance of secondary lymphoid organs.     

CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.

The proper function of immune surveillance requires well-coordinated mechanisms in order to guide the patrolling immune cells through peripheral tissues and into secondary lymphoid organs. Analyzing gene-targeted mice, we identified the chemokine receptor CCR7 as an important organizer of the primary immune response. CCR7-deficient mice show severely delayed kinetics regarding the antibody response and lack contact sensitivity and delayed type hypersensitivity reactions. Due to the impaired migration of lymphocytes, these animals reveal profound morphological alterations in all secondary lymphoid organs. Upon activation, mature skin dendritic cells fail to migrate into the draining lymph nodes. Thus, in order to bring together lymphocytes and dendritic cells to form the characteristic microarchitecture of secondary lymphoid organs, CCR7 is required to rapidly initiate an adoptive immune response.     

Thymic and extrathymic T cell development pathways follow different rules

Separation between primary and secondary lymphoid organs is a universal feature in jawed vertebrates. Strikingly, oncostatin M (OM)-transgenic mice present massive extrathymic T cell development, localized exclusively in the lymph nodes (LN). According to the prevailing paradigm, the thymus is the main source of T lymphocytes in gnathostomes mainly because thymic epithelial cells have a unique ability to support early steps in T cell development. It is therefore remarkable that productive T cell development occurs in the OM(+) LN, despite the absence of epithelial cells. The present study shows that in the OM(+) LN: 1) MHC class I expression strictly on hemopoietic cells is sufficient to support the development of a diversified repertoire of CD8 T cells; 2) the efficiency of positive selection of specific TCR-transgenic T cells is not the same as in the thymus; 3) negative selection is very effective, despite the lack of an organized thymic-like medulla. Furthermore, our data suggest that extrathymic T lymphocytes developing in the OM(+) LN undergo extensive postselection expansion because they live in the microenvironment in which they were positively selected. This work illustrates how the division of labor between primary and secondary lymphoid organs influences the repertoire and homeostasis of T lymphocytes.     

Impact of CCR7 on priming and distribution of antiviral effector and memory CTL

The chemokine receptor CCR7 is a key factor in the coordinate migration of T cells and dendritic cells (DC) into and their localization within secondary lymphoid organs. In this study we investigated the impact of CCR7 on CD8(+) T cell responses by infecting CCR7(-/-) mice with lymphocytic choriomeningitis virus (LCMV). We found that the absence of CCR7 affects the magnitude of an antiviral CTL response during the acute phase, with reduced numbers of virus-specific CTL in all lymphoid and nonlymphoid organs tested. On the single cell level, CCR7-deficient CTL gained full effector function, such that antiviral protection in CCR7-deficient mice was complete, but delayed. Similarly, adoptive transfer experiments using DC from CCR7-deficient or competent mice for the priming of CCR7-positive or CCR7-negative CD8(+) T cells, respectively, revealed that ectopic positioning of DC and CTL outside organized T cell zones results in reduced priming efficacy. In the memory phase, CCR7-deficient mice maintained a stable LCMV-specific CTL population, predominantly in nonlymphoid organs, and rapidly mounted protective CTL responses against a challenge infection with a vaccinia virus recombinant for the gp33 epitope of LCMV. Taken together, the CCR7-dependent organization of the T cell zone does not appear to be a prerequisite for antiviral effector CTL differentiation and the sustenance of antiviral memory responses in lymphoid or peripheral tissues.     

The ontogeny and distribution of B cells in normal and mutant immune-defective CBA/N mice: two-parameter analysis of surface IgM and IgD

A dual-laser fluorescence-activated cell sorter was utilized to study the distribution of the surface IgM and IgD on individual B cells of normal and immune-defective CBA/N mice. Cells from different lymphoid organs and from developing mice were studied. Two major populations of cells were seen. Those with low densities of surface IgM and intermediate-high densities of surface IgD were relatively or totally absent from the bone marrow, spleens, and lymph nodes of adult, immune-defective (CBA/N x DBA/2)F1 male mice, and developed late in ontogeny in the lymphoid organs of normal F1 female mice. By contrast, the second major population, with intermediate-high surface IgM and low surface IgD, was found in highest frequency in the lymphoid organs of immature mice, the bone marrow of adult mice, and the lymphoid organs of F1 male mice compared to F1 female mice at any age. These two major populations of B cells were further subdivided into five groups of cells to better define the surface IgM and IgD characteristics of developing B cells of immune-defective and normal mice. The relationship of these groups of cells to populations defined by other criteria are discussed.     

Establishment of early lymphoid organ infrastructure in transplanted tumors mediated by local production of lymphotoxin alpha and in the combined absence of functional B and T cells

Lymphoid organogenesis is a highly coordinated process involving orchestrated expression of a number of genes. Although the essential role of lymphotoxin alpha (LTalpha) for the normal development of secondary lymphoid organs is well established, it is not clear to which extent it depends upon cooperation with T and B lymphocytes for lymphoid neo-organogenesis. To determine whether LTalpha is sufficient to mediate recruitment of basic elements needed for lymphoid organogenesis, we made use of a LTalpha-transfected cell line as an experimental tool and established tumors in nude and SCID mice. Our data showed that high endothelial venules formed and follicular dendritic cells accumulated and differentiated in response to LTalpha in the absence of lymphocytes. A CD4(+)CD3(-)CD11c(+) cell population that is found in the secondary lymphoid organ was also recruited into tumors expressing LTalpha. Furthermore, in nude mice, B cells migrated in response to LTalpha and formed intratumoral follicles. These B cell follicles were structurally well equipped with follicular dendritic cell networks and high endothelial venules; however, they were not functionally active; e.g., those B cells specific for a surrogate Ag expressed by the tumor were found in the spleen, but not in the tumor. We show that, even in the absence of functional T and B lymphocytes, local expression of LTalpha in transplanted tumors induced typical stromal characteristics of lymphoid tissue, emphasizing that LTalpha is a critically important cytokine for formation of lymphoid organ infrastructure.     

Direct antigen presentation by a xenograft induces immunity independently of secondary lymphoid organs

The location of immune activation is controversial during acute allograft rejection and unknown in xenotransplantation. To determine where immune activation to a xenograft occurs, we examined whether splenectomized alymphoplastic mice that possess no secondary lymphoid organs can reject porcine skin xenografts. Our results show that these mice rejected their xenografts, in a T cell-dependent fashion, at the same tempo as wild-type recipients, demonstrating that xenograft rejection is not critically dependent on secondary lymphoid organs. Furthermore, we provide evidence that immune activation in the bone marrow did not take place during xenograft rejection. Importantly, immunity to xenoantigens was only induced after xenotransplantation and not by immunization with porcine spleen cells, as xenografted mutant mice developed an effector response, whereas mutant mice immunized by porcine spleen cells via i.p. injection failed to do so. Moreover, we provide evidence that antixenograft immunity occurred via direct and indirect Ag presentation, as recipient T cells could be stimulated by either donor spleen cells or recipient APCs. Thus, our data provide evidence that direct and indirect Ag presentation by a xenograft induces immunity in the absence of secondary lymphoid organs. These results have important implications for developing relevant xenotransplantation protocols.     

The power and the promise of organoid models for cancer precision medicine with next-generation functional diagnostics and pharmaceutical exploitation

As organ-specific three-dimensional cell clusters derived from cancer tissue or cancer-specific stem cells, cancer-derived organoids are organized in the same manner of the cell sorting and spatial lineage restriction in vivo, making them ideal for simulating the characteristics of cancer and the heterogeneity of cancer cells in vivo. Besides the applications as a new in vitro model to study the physiological characteristics of normal tissues and organs, organoids are also used for in vivo cancer cell characterization, anti-cancer drug screening, and precision medicine. However, organoid cultures are not without limitations, i.e., the lack of nerves, blood vessels, and immune cells. As a result, organoids could not fully replicate the characteristics of organs but partially simulate the disease process. This review attempts to provide insights into the organoid models for cancer precision medicine.     

Cell-cell interactions in synovitis. Interactions between T cells and B cells in rheumatoid arthritis

In rheumatoid arthritis, T cells and B cells participate in the immune responses evolving in the synovial lesions. Interaction between T cells and B cells is probably antigen specific because complex microstructures typical of secondary lymphoid organs are generated. Differences between patients in forming follicles with germinal centers, T-cell-B-cell aggregates without germinal center reactions, or loosely organized T-cell-B-cell infiltrates might reflect the presence of different antigens or a heterogeneity in host response patterns to immune injury. Tertiary lymphoid microstructures in the rheumatoid lesions can enhance the sensitivity of antigen recognition, optimize the collaboration of immunoregulatory and effector cells, and support the interaction between the tissue site and the aberrant immune response. The molecular basis of lymphoid organogenesis studied in gene-targeted mice will provide clues to why the synovium is a preferred site for tertiary lymphoid tissue. B cells have a critical role in lymphoid organogenesis. Their contribution to synovial inflammation extends beyond antibody secretion and includes the activation and regulation of effector T cells.     

Cutting edge: ectopic expression of the chemokine TCA4/SLC is sufficient to trigger lymphoid neogenesis

To test whether accumulation of naive lymphocytes is sufficient to trigger lymphoid development, we generated mice with islet expression of the chemokine TCA4/SLC. This chemokine is specific for naive lymphocytes and mature dendritic cells (DC) which express the CCR7 receptor. Islets initially developed accumulations of T cells with DC, with scattered B cells at the perimeter. These infiltrates consolidated into organized lymphoid tissue, with high endothelial venules and stromal reticulum. Infiltrate lymphocytes showed a naive CD44low CD25- CD69- phenotype, though half were CD62L negative. When backcrossed to RAG-1 knockout, DC were not recruited. Interestingly, islet lymphoid tissue developed in backcrosses to Ikaros knockout mice despite the absence of normal peripheral nodes. Our results indicate that TCA4/SLC can induce the development and organization of lymphoid tissue through diffential recruitment of T and B lymphocytes and secondary effects on stromal cell development.     

Cell-cell interactions in synovitis: Interactions between T cells and B cells in rheumatoid arthritis

In rheumatoid arthritis, T cells and B cells participate in the immune responses evolving in the synovial lesions. Interaction between T cells and B cells is probably antigen specific because complex microstructures typical of secondary lymphoid organs are generated. Differences between patients in forming follicles with germinal centers, T-cell–B-cell aggregates without germinal center reactions, or loosely organized T-cell–B-cell infiltrates might reflect the presence of different antigens or a heterogeneity in host response patterns to immune injury. Tertiary lymphoid microstructures in the rheumatoid lesions can enhance the sensitivity of antigen recognition, optimize the collaboration of immunoregulatory and effector cells, and support the interaction between the tissue site and the aberrant immune response. The molecular basis of lymphoid organogenesis studied in gene-targeted mice will provide clues to why the synovium is a preferred site for tertiary lymphoid tissue. B cells have a critical role in lymphoid organogenesis. Their contribution to synovial inflammation extends beyond antibody secretion and includes the activation and regulation of effector T cells.     

Crk-associated substrate lymphocyte type is required for lymphocyte trafficking and marginal zone B cell maintenance

The lymphocyte-specific Cas family protein Cas-L (Crk-associated substrate lymphocyte type) has been implicated to function in lymphocyte movement, mediated mainly by integrin signaling. However, its physiological role is poorly understood. In this study we analyzed the function of Cas-L in lymphocytes using gene-targeted mice. The mutant mice showed a deficit of marginal zone B (MZB) cells and a decrease of cell number in secondary lymphoid organs. An insufficient chemotactic response and perturbed cell adhesion were observed in Cas-L-deficient lymphocytes, suggesting that the aberrant localization was responsible for the deficit of MZB cells. Moreover, we found that lymphocyte trafficking was altered in Cas-L-deficient mice, which gave a potential reason for contraction of secondary lymphoid tissues. Thus, Cas-L affects homeostasis of MZB cells and peripheral lymphoid organs, which is considered to be relevant to impaired lymphocyte migration and adhesion.     

Tumor necrosis factor and the p55TNF receptor are required for optimal development of the marginal sinus and for migration of follicular dendritic cell precursors into splenic follicles

The development and function of secondary lymphoid tissue require signaling by tumor necrosis factor and lymphotoxins. Mice deficient in LTbetaR show defective organogenesis of lymph nodes and Peyer's patches and a severely disturbed splenic architecture. In contrast, TNF or p55TNF-R deficiency does not affect the organogenesis of peripheral lymphoid organs but interferes with the formation of B cell follicles and the appearance of FDC networks and germinal centers in all secondary lymphoid organs. Based on these differences, we have previously hypothesized that the role of TNF in lymphoid structure is distinct from that of LT and restricted in regulating cellular interactions that allow the differentiation and/or correct positioning of FDCs. In the present study we show that, in addition to the defects in follicular structure, TNF or p55TNF-R knockout mice exhibit defects in the formation of the macrophage populations and of the sinus lining cells of the splenic marginal zone. Interestingly, a large number of dendritic-shaped cells stained with FDC-specific markers and able to trap immune complexes are retained within the defective marginal zone of TNF and p55TNF-R knockout spleens. We conclude that the primary defect in the lymphoid phenotype of TNF or p55TNF-R knockout mice is the failure of FDC precursors to migrate through the disorganized marginal sinus and to home properly into the splenic follicular areas where they would promote the formation of B cell follicles and germinal centers.     

TNF receptor-1 is required for the formation of splenic compartments during adult, but not embryonic life

Lymphotoxin β-receptor (LTβR) and TNF receptor-1 (TNFR1) are important for the development of secondary lymphoid organs during embryonic life. The significance of LTβR and TNFR1 for the formation of lymphoid tissue during adult life is not well understood. Immunohistochemistry, morphometry, flow cytometry, and laser microdissection were used to compare wild-type, LTβR(-/-), TNFR1(-/-) spleens with splenic tissue that has been newly formed 8 wk after avascular implantation into adult mice. During ontogeny, LTβR is sufficient to induce formation of the marginal zone, similar-sized T and B cell zones, and a mixed T/B cell zone that completely surrounded the T cell zone. Strikingly, in adult mice, the formation of splenic compartments required both LTβR and TNFR1 expression, demonstrating that the molecular requirements for lymphoid tissue formation are different during embryonic and adult life. Thus, interfering with the TNFR1 pathway offers the possibility to selectively block the formation of ectopic lymphoid tissue and at the same time to spare secondary lymphoid organs such as spleen and lymph nodes. This opens a new perspective for the treatment of autoimmune and inflammatory diseases.     

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 Peyer's 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 TNFalpha 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.     

Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis

Despite their widespread expression, the in vivo recruitment activities of CCL19 (EBV-induced molecule 1 ligand chemokine) and CXCL12 (stromal cell-derived factor 1) have not been established. Furthermore, although CXCL13 (B lymphocyte chemoattractant) has been shown to induce lymphoid neogenesis through induction of lymphotoxin (LT)alpha1beta2, it is unclear whether other homeostatic chemokines have this property. In this work we show that ectopic expression in pancreatic islets of CCL19 leads to small infiltrates composed of lymphocytes and dendritic cells and containing high endothelial venules and stromal cells. Ectopic CXCL12 induced small infiltrates containing few T cells but enriched in dendritic cells, B cells, and plasma cells. Comparison of CCL19 transgenic mice with mice expressing CCL21 (secondary lymphoid tissue chemokine) revealed that CCL21 induced larger and more organized infiltrates. A more significant role for CCL21 is also suggested in lymphoid tissues, as CCL21 protein was found to be present in lymph nodes and spleen at much higher concentrations than CCL19. CCL19 and CCL21 but not CXCL12 induced LTalpha1beta2 expression on naive CD4 T cells, and treatment of CCL21 transgenic mice with LTbetaR-Fc antagonized development of organized lymphoid structures. LTalpha1beta2 was also induced on naive T cells by the cytokines IL-4 and IL-7. These studies establish that CCL19 and CXCL12 are sufficient to mediate cell recruitment in vivo and they indicate that LTalpha1beta2 may function downstream of CCL21, CCL19, and IL-2 family cytokines in normal and pathological lymphoid tissue development.     

Purging metastases in lymphoid organs using a combination of antigen-nonspecific adoptive T cell therapy, oncolytic virotherapy and immunotherapy

In many common cancers, dissemination of secondary tumors via the lymph nodes poses the most significant threat to the affected individual. Metastatic cells often reach the lymph nodes by mimicking the molecular mechanisms used by hematopoietic cells to traffic to peripheral lymphoid organs. Therefore, we exploited naive T cell trafficking in order to chaperone an oncolytic virus to lymphoid organs harboring metastatic cells. Metastatic burden was initially reduced by viral oncolysis and was then eradicated, as tumor cell killing in the lymph node and spleen generated protective antitumor immunity. Lymph node purging of tumor cells was possible even in virus-immune mice. Adoptive transfer of normal T cells loaded with oncolytic virus into individuals with cancer would be technically easy to implement both to reduce the distribution of metastases and to vaccinate the affected individual in situ against micrometastatic disease. As such, this adoptive transfer could have a great therapeutic impact, in the adjuvant setting, on many different cancer types.