Lymphostromal interactions in thymic development and function

The generation of a peripheral T-cell pool is essential for normal immune system function. CD4+ and CD8+ T cells are produced most efficiently in the thymus, which provides a complexity of discrete cellular microenvironments. Specialized stromal cells, that make up such microenvironments, influence each stage in the maturation programme of immature T-cell precursors. Progress has recently been made in elucidating events that regulate the development of intrathymic microenvironments, as well as mechanisms of thymocyte differentiation. It is becoming increasingly clear that the generation and maintenance of thymic environments that are capable of supporting efficient T-cell development, requires complex interplay between lymphoid and stromal compartments of the thymus.     

De novo production of antigen-specific suppressor cells in vivo

Foxp3-expressing regulatory T cells (Treg) play an essential role in maintaining tolerance to self antigens and are generated under physiological conditions when developing T cells encounter antigens expressed by thymic epithelial cells. We have addressed the possibility that Treg can be exploited to prevent or even suppress ongoing immune responses to foreign antigens. To this end, one must develop methods that permit the de novo generation of Treg specific for foreign antigens in peripheral lymphoid tissue. This report describes the methodology of generating Treg by delivering minute doses of peptide contained in fusion Abs directed against the DEC-205 endocytic receptor on steady-state dendritic cells. The process, from cloning and production of fusion Abs to antigen-specific Treg induction in vivo, takes approximately 2 months. The results show that delivery of T-cell receptor agonist ligands under subimmunogenic conditions represents a suitable approach for converting naive T cells into Treg.     

Anatomical and cellular requirements for the activation and migration of virus-specific CD8+ T cells to the brain during Theiler's virus infection

Theiler's murine encephalomyelitis virus (TMEV) infection of the brain induces a virus-specific CD8(+) T-cell response in genetically resistant mice. The peak of the immune response to the virus occurs 7 days after infection, with an immunodominant CD8(+) T-cell response against a VP2-derived capsid peptide in the context of the D(b) molecule. The process of activation of antigen-specific T cells that migrate to the brain in the TMEV model has not been defined. The site of antigenic challenge in the TMEV model is directly into the brain parenchyma, a site that is considered immune privileged. We investigated the hypothesis that antiviral CD8(+) T-cell responses are initiated in situ upon intracranial inoculation with TMEV. To determine whether a brain parenchymal antigen-presenting cell is responsible for the activation of virus-specific CD8(+) T cells, we evaluated the CD8(+) T-cell response to the VP2 peptide in bone marrow chimeras and mutant mice lacking peripheral lymphoid organs. The generation of the anti-TMEV CD8(+) T-cell response in the brain requires priming by a bone marrow-derived antigen-presenting cell and the presence of peripheral lymphoid organs. Although our results show that activation of TMEV-specific CD8(+) T cells occurs in the peripheral lymphoid compartment, they do not exclude the possibility that the immune response to TMEV is initiated by a brain-resident, bone marrow-derived, antigen-presenting cell.     

Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T-cell transfer was more obligatory than exogenous IL-2 treatment to sustain adoptively transferred T cells in vivo. Ag-specific T-cell responses were weak in mice that either received IL-2 alone or did not receive the cognate peptide boost after T-cell transfer. The T-cell clones were also monitored by flow cytometry or RT-PCR based on expression of the T-cell receptor Vbeta-chain, which was previously characterized. Ag-specific T cells were recovered from both spleens and lungs of recipient mice, demonstrating that the T-cell clones could localize to both lymphoid and nonlymphoid tissues. This study demonstrates that both uncultured and in vitro-cloned T lymphocytes can migrate to lymphoid tissues and nonlymphoid (e.g., lung) tissues in recipient hosts and that their functional activities can be maintained at these sites after transfer, if they are exposed to peptide Ag in vivo.     

Thymocyte antigens do not induce tolerance in the CD4+ T cell compartment.

Thymocytes fail to tolerize the developing T cell repertoire to self MHC class I (MHC I) Ags because transgenic (CD2Kb) mice expressing H-2Kb solely in lymphoid cell lineages reject skin grafts mismatched only for H-2Kb. In this study, we examined why thymocytes fail to tolerize the T cell repertoire to self MHC I Ags. The ability of CD2Kb mice to reject H-2Kb skin grafts was age dependent because CD2Kb mice older than 20 wk accepted skin grafts. T cells from younger CD2Kb mice proliferated, but did not develop cytotoxic functions in vitro in response to H-2Kb. Proliferative responses were dominated by H-2Kb-specific, CD4+ T cells rather than CD8+ T cells. Representative CD4+ T cell clones from CD2Kb mice were MHC II restricted and recognized processed H-2Kb. TCR transgenic mice were generated from one CD4+ T cell clone (361) to monitor development of H-2Kb-specific immature thymocytes when all thymic cells or lymphoid cell lineages only expressed H-2Kb. Thymocyte precursors were not eliminated and mice were not tolerant to H-2Kb when Tg361 TCR transgenic mice were intercrossed with CD2Kb mice. In contrast, all thymocyte precursors were eliminated efficiently in thymic microenvironments in which all cells expressed H-2Kb. We conclude that self MHC I Ags expressed exclusively in thymocytes do not induce T cell tolerance because presentation of processed self MHC I Ags on self MHC II molecules fails to induce negative selection of CD4+ T cell precursors. This suggests that some self Ags are effectively compartmentalized and cannot induce self-tolerance in the T cell repertoire.     

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell-T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell-mediated immunity.     

Selection and fine-tuning of the autoimmune T-cell repertoire

The immune system must avoid aggressive T-cell responses against self-antigens. But, paradoxically, exposure to self-peptides seems to have an important role in positive selection in the thymus and the maintenance of a broad T-cell repertoire in the periphery. Recent experiments have highlighted situations that allow high-avidity self-reactive T cells to avoid negative selection in the thymus. Accumulating evidence indicates that other, non-deleting mechanisms control the avidity with which T cells recognize self-antigens--a phenomenon that is known as 'tuning'. This might maximize the peripheral T-cell repertoire by allowing the survival of T cells that can respond to self, but only at concentrations that are not normally reached in vivo.     

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.     

Neutrophils sustain effective CD8(+) T-cell responses in the respiratory tract following influenza infection

Neutrophils have an important role in early host protection during influenza A virus infection. Their ability to modulate the virus-specific adaptive immune response is less clear. Here, we have used a mouse model to examine the impact of neutrophils on CD8(+) T-cell responses during influenza virus infection. CD8(+) T-cell priming, expansion, migration, cytokine secretion and cytotoxic capacity were investigated in the virus-infected airways and secondary lymphoid organs. To do this, we utilised a Ly6G-specific monoclonal antibody (mAb; 1A8) that specifically depletes neutrophils in vivo. Neutrophil depletion early after infection with influenza virus strain HKx31 (H3N2) did not alter influenza virus-derived antigen presentation or na?ve CD8(+) T-cell expansion in the secondary lymphoid organs. Trafficking of virus-specific CD8(+) T cells into the infected pulmonary airways was also unaltered. Instead, early neutropenia reduced both the overall magnitude of influenza virus-specific CD8(+) T cells, together with impaired cytokine production and cytotoxic effector function. Therefore, neutrophils are important participants in anti-viral mechanisms that sustain effective CD8(+) T-cell responses in the respiratory tract of influenza virus-infected mice.     

Thymic function and allogeneic T-cell responses in stem-cell transplantation

The thymus is the primary site of T-cell production early in life, and has now been shown to continue to function in both healthy and immunocompromised individuals late into life. Positive and negative selection occurring in the thymus are two of the most important processes that govern the development and specificity of peripheral T cells, including their restriction to self HLA and their ability to respond in an alloreactive manner. In the chimeric state that follows successful allogeneic stem-cell transplants, the specificity of alloreactive cells may be governed by either host- or recipient-derived cellular elements, as well as maturing lymphoid cells, which are, in turn, derived from donor stem cells or host cells surviving transplant conditioning. The ability to measure recent thymic emigrants via the detection of T-cell receptor excision circles has facilitated studies of thymic function in immunodeficient individuals, including HIV-1 infected subjects and recipients of autologous or allogeneic stem-cell transplant (SCT). These studies have now demonstrated that thymic function is likely to play a beneficial role in immune reconstitution in these settings, but have yet to clearly demonstrate what clinical variables are the most important determinants of thymic persistence. It is also not yet clear how much the degree of thymic function following allogeneic SCT influences the alloreactive T-cell repertoire, although studies in animal models and early clinical studies suggest that GvHD results in thymic injury and dysfunction. Future studies will further clarify how thymic function shapes the repertoire of T cells that mediate alloreactivity, as well as protective pathogen-specific immune responses, following SCT. Finally, these studies will also demonstrate whether endogenous mediators of thymic function could be selectively applied to regulate post-SCT thymic function and alloreactivity.     

Antiviral therapy during primary simian immunodeficiency virus infection fails to prevent acute loss of CD4+ T cells in gut mucosa but enhances their rapid restoration through central memory T cells

Gut-associated lymphoid tissue (GALT) is an early target of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) and a site for severe CD4+ T-cell depletion. Although antiretroviral therapy (ART) is effective in suppressing HIV replication and restoring CD4+ T cells in peripheral blood, restoration in GALT is delayed. The role of restored CD4+ T-cell help in GALT during ART and its impact on antiviral CD8+ T-cell responses have not been investigated. Using the SIV model, we investigated gut CD4+ T-cell restoration in infected macaques, initiating ART during either the primary stage (1 week postinfection), prior to acute CD4+ cell loss (PSI), or during the chronic stage at 10 weeks postinfection (CSI). ART led to viral suppression in GALT and peripheral blood mononuclear cells of PSI and CSI animals at comparable levels. CSI animals had incomplete CD4+ T-cell restoration in GALT. In PSI animals, ART did not prevent acute CD4+ T-cell loss by 2 weeks postinfection in GALT but supported rapid and complete CD4+ T-cell restoration thereafter. This correlated with an accumulation of central memory CD4+ T cells and better suppression of inflammation. Restoration of CD4+ T cells in GALT correlated with qualitative changes in SIV gag-specific CD8+ T-cell responses, with a dominance of interleukin-2-producing responses in PSI animals, while both CSI macaques and untreated SIV-infected controls were dominated by gamma interferon responses. Thus, central memory CD4+ T-cell levels and qualitative antiviral CD8+ T-cell responses, independent of viral suppression, were the immune correlates of gut mucosal immune restoration during ART.     

Acute hepatotoxin exposure effects lymphoid and accessory cell types in inbred mice

The effect of acute hepatotoxin exposure on in vivo and in vitro immune responses were investigated in inbred mice. Splenic anti-SRBC PFC responses were slightly enhanced by carbon tetrachloride or galactosamine administration 5 hr prior to immunization. Whereas splenic anti-SRBC PFC responses were slightly enhanced in euthymic mice exposed to carbon tetrachloride 5 hr prior to immunization, immune responses to the TI antigens, Fl-LPS, Fl-Ficoll, and TNP-LPS, were significantly suppressed. Athymic mice receiving similar hepatotoxin exposure elicited enhanced immune responses to the TI immunogens, thereby suggesting that the activities of B cells and macrophages are enhanced in treated animals and in euthymic mice, T suppressor cells are also activated. By admixture of purified B- and T-cell and macrophage populations from either carbon tetrachloride-treated or control animals, it was demonstrated that hepatotoxin exposure also induces suppressor T cells regulating immune responses to the T-dependent antigen, SRBC, and that macrophages from treated animals are more functional. Further, B-cell responsiveness is enhanced. In addition to these observations, an active factor could be demonstrated in sera from hepatotoxin-treated animals which augments immune responses to SRBC in normal mice and promotes immune responses to this antigen in athymic mice. These findings indicate that the effects of acute hepatotoxin exposure are multifocal, influencing the activity of lymphoid and accessory cells.     

The complexity of the CD4 T-cell responses: old and new T-cell subsets

The T-cell compartment of the immune system reacts to an enormous variety of antigens, including self antigens, due to its a wide repertoire of T-cell clones. Self-reactive T cells undergo a negative selection process resulting in apoptosis of T cells with high affinity for self-peptides. Self-reactive T cells escaped to negative selection are then controlled by natural T regulatory (Treg) cells. Regulation also controls excessive effector T-cell responses. Three types of effector T cells are recognized: T helper 1 (Th1) cells, which protect against intracellular bacteria; Th2 cells, which play a role against parasites; Th17 cells, which would face extracellular bacteria, but also are involved in autoimmunity. Effector T-cell polarization is determined by the complex interaction of antigen-presenting cells with naive T cells and involves a multitude of factors, including the dominant cytokine environment, costimulatory molecules, type and load of antigen presented and signaling cascades. The decision for the immune response to go in a certain direction is based not onto one signal alone, rather onto many different elements acting synergistically, antagonistically and through feedback loops leading to activation of Th1, Th2, or Th17 responses. Both Th1 and Th2 can be suppressed by adaptive Treg cells through contact-dependent mechanisms and/or cytokines.     

Inhibition of adaptive Vgamma2Vdelta2+ T-cell responses during active mycobacterial coinfection of simian immunodeficiency virus SIVmac-infected monkeys

Adaptive immune responses of gammadelta T cells during active mycobacterial coinfection of human immunodeficiency virus-infected humans have not been studied. Macaques infected with the simian immunodeficiency virus (SIV) SIVmac were employed to determine the extent to which a coincident AIDS virus infection might compromise immune responses of mycobacterium-specific Vgamma2Vdelta2(+) T cells during active mycobacterial infection. Control SIVmac-negative macaques developed primary and recall expansions of phosphoantigen-specific Vgamma2Vdelta2(+) T cells after Mycobacterium bovis BCG infection and BCG reinfection, respectively. In contrast, SIVmac-infected macaques did not exhibit sound primary and recall expansions of Vgamma2Vdelta2(+) T cells in the blood and pulmonary alveoli following BCG infection and reinfection. The absence of adaptive Vgamma2Vdelta2(+) T-cell responses was associated with profound CD4(+) T-cell deficiency and subsequent development of SIVmac-related tuberculosis-like disease in the coinfected monkeys. Consistently, Vgamma2Vdelta2(+) T cells from coinfected monkeys displayed a reduced capacity to expand in vitro following stimulation with phosphoantigen. The reduced ability of Vgamma2Vdelta2(+) peripheral blood lymphocytes (PBL) to expand could be restored to some extent by coculture of these cells with CD4(+) T cells purified from PBL of SIV-negative monkeys. Furthermore, na?ve monkeys inoculated simultaneously with SIVmac and BCG were unable to sustain expansion of Vgamma2Vdelta2(+) T cells at the time that the coinfected monkeys developed lymphoid depletion and a fatal tuberculosis-like disease. Nevertheless, no deletion in Vdelta2 T-cell receptor repertoire was identified in SIVmac-BCG-coinfected macaques, implicating an SIVmac-induced down-regulation rather than a clonal exhaustion of these cells. Thus, an SIVmac-induced compromise of the adaptive Vgamma2Vdelta2(+) T-cell responses may contribute to the immunopathogenesis of the SIV-related tuberculosis-like disease in macaques.     

Heterogeneity and function of human B lymphocytes

B lymphocytes represent an important arm of the immune system. Besides their main function of providing antibodies protecting against pathogens, they also exert some regulatory functions, in particular for secondary lymphoid tissue differentiation. Human B cells can be divided in various subsets representing different maturation stages and different pathways of humoral immune responses. Na?ve IgMlow IgDhigh CD27- B cells can participate in T-cell dependent immune responses leading to germinal center formation in follicles of secondary lymphoid organs. Interactions with follicular helper T cells, a recently identified CD185+ T cell population providing help to follicular B cell, involve costimulatory molecules including CD40, CD27, CD278 and SAP-recruiting receptors. B cell interaction with follicular helper T cells represents a critical step controlling the generation of plama cells that ultimately produce high affinity, somatically mutated, class-switched antibodies or of their memory B cell counterpart (identified as CD27+ Ig switched or IgMonly B cells). IgMhigh IgDlow CD27+ B cells are a puzzling population apparently specialized in T-independent responses to bacterial capsular polysaccharides. The extra-follicular, probably antigen-independent, differentiation pathway of these cells, allowing pre-immune repertoire diversification by somatic hypermutation, is not yet characterized. However, circulating IgMhigh IgDlow CD27+ B cells are similar to splenic marginal zone B cells. In addition to these subsets, minor populations can also be identified in peripheral blood, such as transitional B cells and plasma blasts. All together, deciphering human B cell heterogeneity provides tools for investigations of humoral immunodeficiencies and auto-immune diseases, that will in return shed more light on B cell biology.     

Lymphotoxin/light,lymphoid microenvironments and autoimmune disease

Much of the efficiency of the immune system is attributed to the high degree of spatial and temporal organization in the secondary lymphoid organs. Signalling through the lymphotoxin (LT) pathway is a crucial element in the maintenance of this organized microenvironment. The effect of altering lymphoid microenvironments on immune responses remains relatively unexplored. Inhibitors of the LT and LIGHT pathways have been shown to reduce disease in a wide range of autoimmune models. This approach has provided a tool to probe the effect of manipulation of the microenvironment on both normal and pathological immune responses.     

B cells as antigen presenting cells

Several characteristics confer on B cells the ability to present antigen efficiently: (1) they can find T cells in secondary lymphoid organs shortly after antigen entrance, (2) BCR-mediated endocytosis allows them to concentrate small amounts of specific antigen, and (3) BCR signaling and HLA-DO expression direct their antigen processing machinery to favor presentation of antigens internalized through the BCR. When presenting antigen in a resting state, B cells can induce T cell tolerance. On the other hand, activation by antigen and T cell help converts them into APC capable of promoting immune responses. Presentation of self antigens by B cells is important in the development of autoimmune diseases, while presentation of tumor antigens is being used in vaccine strategies to generate immunity. Thus, detailed understanding of the antigen presenting function of B cells can lead to their use for the generation or inhibition of immune responses.     

Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells

T cell tolerance to self Ags is in part established in the thymus by induction of apoptosis or anergy of potentially autoreactive thymocytes. Some autospecific T cells nevertheless migrate to peripheral lymphoid organs but are kept under control by the recently identified CD4(+)CD25(+) regulatory T cell subset. Because these cells inhibit autoimmunity more efficiently than useful non-self Ag-specific immune responses, they are probably autospecific, posing important questions as to how they develop in the thymus. In this study we show that significantly more peripheral CD4(+)CD25(+) regulatory T cells recognize self than non-self Ags. However, we also show for a large panel of endogenous superantigens as well as for self peptide/MHC complexes that autospecific CD4(+)CD25(+) thymocyte precursors are normally deleted during ontogeny. Combined, our data firmly establish that the repertoire of regulatory T cells is specifically enriched in autospecific cells despite the fact that their precursors are normally susceptible to thymic deletion.     

Intraspleen DNA inoculation elicits protective cellular immune responses

DNA immunization or inoculation is a recent vaccination method that induces both humoral and cellular immune responses in a range of hosts. Independent of the route or site of vaccination, the transfer of antigen-presenting cells (APC) or antigens into lymphoid organs is necessary. The aim of this investigation was to test whether intraspleen (i.s.) DNA inoculation is capable of inducing a protective immune response. We immunized mice by a single i.s. injection of a DNA construct expressing the immunoglobulin (Ig) heavy-chain variable domain (VH) in which the complementarity-determining regions (CDR) had been replaced by a Taenia crassiceps T-cell epitope. In these mice, immune responses and protective effects elicited by the vaccine were measured. We have shown here for the first time that i.s. DNA inoculation can induce protective cellular immune responses and activate CD8(+) T cells. Also, Ig V(H) appeared to be the minimal delivery unit of "antigenized" Ig capable of inducing T-cell activation in a lymphoid organ. The strategy of introducing T-cell epitopes into the molecular context of the V(H) domain in combination with i.s. DNA immunization could have important implications and applications for human immunotherapy.     

Dermal fibroblasts induce maturation of dendritic cells

To trigger an effective T cell-mediated immune response in the skin, cutaneous dendritic cells (DC) migrate into locally draining lymph nodes, where they present Ag to naive T cells. Little is known about the interaction of DC with the various cellular microenvironments they encounter during their migration from the skin to lymphoid tissues. In this study, we show that human DC generated from peripheral blood monocytes specifically interact with human dermal fibroblasts via the interaction of beta(2) integrins on DC with Thy-1 (CD90) and ICAM-1 on fibroblasts. This induced the phenotypic maturation of DC reflected by expression of CD83, CD86, CD80, and HLA-DR in a TNF-alpha- and ICAM-1-dependent manner. Moreover, fibroblast-matured DC potently induced T cell activation reflected by CD25 expression and enhanced T cell proliferation. Together these data demonstrate that dermal fibroblasts that DC can encounter during their trafficking from skin to lymph node can act as potent regulators of DC differentiation and function, and thus may actively participate in the regulation and outcome of DC-driven cutaneous immune responses.