Imaging ocular immune responses by intravital microscopy

The eye offers excellent opportunities to observe cellular interactions in vivo. This applies especially to the immune response in which discrete events can be studied, including cell trafficking, transendothelial migration, adhesion, antigen presentation, and T cell activation. Intravital microscopy has allowed study of immune cell interactions in tissues such as the conjunctiva, the inflamed cornea, and the iris. Thus the realtime observation of presentation of antigen injected into the anterior chamber of the eye can be imaged using fluorescently labelled antigen and cells. Application of the scanning laser ophthalmoscope to the rat and mouse eye allows analysis of leukocyte-endothelial interactions in the retinal and choroidal circulations. These studies have provided important information on rolling and adhesion of leukocytes in real time in different microvascular beds that have not been manipulated in any way and has, for instance, provided quantitation to the effects of shear stress on leukocyte-endothelial adhesion. In addition, the model permits an accurate analysis of the timing of trafficking of T cells into the eye and the possibility of determining which cells, if any, may be responsible for antigen presentation in the tissues as opposed to the secondary lymphoid organs. Finally, these experimental methods are now being applied to the human eye and should prove valuable in determining the nature of tissue damage events in the eye as well as evaluating the response to treatments.     

Properties of glycolipid-enriched membrane rafts in antigen presentation

Presentation of antigen to T cells represents one of the central events in the engagement of the immune system toward the defense of the host against pathogens. Accordingly, understanding the mechanisms by which antigen presentation occurs is critical toward our understanding the properties of host defense against foreign antigen, as well as insight into other features of the immune system, such as autoimmune disease. The entire antigen-presentation event is complex, and many features of it remain poorly understood. However, recent studies have provided evidence showing that glycolipid-enriched membrane rafts are important for efficient antigen presentation; the studies suggest that one such function of rafts is trafficking of antigen-MHC II complexes to the presentation site on the surface of the antigen-presenting cell. Here, we present a critical discussion of rafts and their proposed functions in antigen presentation. Emerging topics of rafts and antigen presentation that warrant further investigation are also highlighted.     

Trypanosoma cruzi-infected macrophages are defective in major histocompatibility complex class II antigen presentation

Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagas' disease, interferes with the host immune response to establish a persistent infection. In this report, we demonstrate that macrophages infected with T. cruzi are unable to effectively present antigens to CD4 T cells. The interference is due to defective antigen-presenting cell (APC) function, as antigen-independent stimulation of the T cell in the presence of infected macrophages is not affected. The defect is distal to antigen processing and is not due to decreased major histocompatibility complex (MHC) class II expression, decreased viability, defective peptide loading in the infected macrophages, nor absence of CD28 co-stimulation. There was a role for gp39:CD40 co-stimulation during antigen presentation to the T cells we studied, but the expression of CD40 on T. cruzi-infected macrophages was not decreased. Antigen-specific adhesion between macrophages and T cells was reduced by infection. Equivalent levels of the adhesion molecules lymphocyte function-associated antigen-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or very late antigen-4 are found on infected and uninfected APC, suggesting that reduced expression of these adhesion molecules was not responsible for the defect in antigen-specific adhesion. The defective T cell:macrophage adhesion may be due to the reduced expression of other adhesion molecules or other changes in the cell induced by infection. Interfering with MHC class II antigen presentation in infected macrophages may help T. cruzi to blunt the immune response by the host.     

Regulation of lymphocyte adhesion and migration by the small GTPase Rap1 and its effector molecule, RAPL

Dynamic regulation of integrin-mediated adhesion is central to lymphocyte trafficking and antigen recognition. The small GTPase Rap1 is a potent stimulator of leukocyte integrins through modulation of affinity and avidity. In addition, lymphocyte Rap1 has unique abilities to trigger cell polarization and enhance cell motility. These characteristics of Rap1 contribute to adhesive interactions with antigen-presenting cells (APC) and the vascular endothelium. In the process of elucidating the molecular mechanisms of Rap1-mediated integrin activation, we have identified a novel Rap1-binding molecule, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL). RAPL is predominantly expressed in immune cells, and mediates Rap1-triggered integrin activation upon TCR engagement and chemokine stimulation. Importantly, Rap1/RAPL signaling cooperatively regulates cell polarization and integrin activation. The linkage between cell polarization and integrin activation through Rap1/RAPL signaling likely provides immune cells with their dynamic trafficking capability.     

The microstructure of secondary lymphoid organs that support immune cell trafficking

Immune cell trafficking in the secondary lymphoid organs is crucial for an effective immune response. Recirculating T cells constantly patrol not only secondary lymphoid organs but also the whole peripheral organs. Thoracic duct lymphocytes represent an ideal cell source for analyzing T cell trafficking: high endothelial venules (HEVs) allow recirculating lymphocytes to transmigrate from the blood directly, and recirculating T cells form a cluster with dendritic cells (DCs) to survey antigen invasions even in a steady state. This cluster becomes an actual site for the antigen presentation when DCs have captured antigens. On activation, effector and memory T cells differentiate into several subsets that have different trafficking molecules and patterns. DCs also migrate actively in a manner depending upon their maturational stages. Danger signals induce the recruitment of several DC precursor subsets with different trafficking patterns and functions. In this review, we describe general and specialized structures of the secondary lymphoid organs for the trafficking of T cells and DCs by a multicolor immunoenzyme staining technique. The lymph nodes, spleen, and Peyer's patches of rats were selected as the major representatives. In vivo trafficking of subsets of T cells and DCs within these organs under steady or emergency states are shown and discussed, and unsolved questions and future prospects are also considered.     

Dynamic programming of CD8+ T cell trafficking after live viral immunization

After viral infection, activated T cells are present in multiple tissues regardless of the infection route. How these cells acquire pleiotropic homing ability is unclear. By using a cutaneous vaccinia virus infection model, we demonstrate that regulation of T cell trafficking is multiphasic. Upon completion of three cell divisions, CD8+ T cells upregulated specific skin-homing molecules within draining lymph nodes (LN). By 60 hr after infection, some activated T cells reached the infected tissue, while others entered distant antigen-free LN. These latter cells continued to divide and acquire additional tissue-homing molecules in this new setting, independent of antigen presentation. After viral clearance, the initial skin-homing imprint became the predominant homing phenotype on memory cells and provided superior protection against secondary cutaneous challenge. These observations demonstrate a mechanism by which T cells provide both immediate tissue-specific immune control at the pathogen entry site and a more flexible systemic protection against pathogen dissemination.     

Antigen archiving by lymph node stroma: A novel function for the lymphatic endothelium

Secondary lymphoid stroma performs far more functions than simple structural support for lymphoid tissues, providing a host of soluble and membrane‐bound cues to trafficking leukocytes during inflammation and homeostasis. More recently it has become clear that stromal cells can manipulate T‐cell responses, either through direct antigen‐mediated stimulation of T cells or more indirectly through the retention and management of antigen after viral infection or vaccination. In light of recent data, this review provides an overview of stromal cell subsets and functions during the progression of an adaptive immune response with particular emphasis on antigen capture and retention by follicular dendritic cells as well as the recently described “antigen archiving” function of lymphatic endothelial cells (LECs). Given its impact on the maintenance of protective immune memory, we conclude by discussing the most pressing questions pertaining to LEC antigen capture, archiving and exchange with hematopoetically derived antigen‐presenting cells.     

Immune cell trafficking to the islets during type 1 diabetes

Inhibition of immune cell trafficking to the pancreatic islets during type 1 diabetes (T1D) has therapeutic potential, since targeting of T cell and B cell trafficking has been clinically effective in other autoimmune diseases. Trafficking to the islets is characterized by redundancy in adhesion molecule and chemokine usage, which has not enabled effective targeting to date. Additionally, cognate antigen is not consistently required for T cell entry into the islets throughout the progression of disease. However, myeloid cells are required to enable T cell and B cell entry into the islets, and may serve as a convergence point in the pathways controlling this process. In this review we describe current knowledge of the factors that mediate immune cell trafficking to pancreatic islets during T1D progression.     

T cell anergy and costimulation

Summary: T lymphocytes play a key role in immunity by distinguishing self from nonself peptide antigens and regulating both the cellular and humoral arms of the immune system. Acquired, antigen‐specific unresponsiveness is an important mechanism by which T cell responses to antigen are regulated in vivo. Clonal anergy is the term that describes T cell unresponsiveness at the cellular level. Anergic T cells do not proliferate or secrete interleukin (IL)‐2 in response to appropriate antigenic stimulation. However, anergic T cells express the IL‐2 receptor, and anergy can be broken by exogenous IL‐2. Anergy can be induced by submitogenic exposure to peptide antigen in the absence of a costimulatory signal provided by soluble cytokines or by interactions between costimulatory receptors on T cells and counter‐receptors on antigen‐presenting cells. The molecular events that mediate the induction and maintenance of T cell anergy are the focus of this review. The molecular consequences of CD28–B7 interaction are discussed as a model for the costimulatory signal that leads to T cell activation rather than the induction of anergy.     

Dendritic cell-based cancer immunotherapies

Because of their unique role in linking the innate and adaptive immune systems, dendritic cells (DCs) have been a logical focus for novel immunotherapies. However, strategies employing active immunization with ex vivo generated and antigen–pulsed DCs have shown limited efficacy in clinical trials. These past approaches did not take into account the complex interactions between cells of the innate immune system and DCs during DC maturation, antigen processing, and presentation to naïve T cells. By better understanding the natural sequence of events occurring in vivo during an effective immune response, we can tailor antitumor immunotherapeutic strategies to augment aspects of this response from the activation of innate immune cells to antigen uptake and DC maturation to priming of naïve T cells and, ultimately, to the establishment of antitumor immunity. Current DC vaccination strategies utilize a number of methods to recapitulate the cascade of events that culminate in a protective antitumor immune response.     

Antigen presentation by the endothelium: a green light for antigen-specific T cell trafficking?

The functional consequences of recognition of antigen displayed by the endothelium during T cell extravasation in the development of an immune response have been a matter of debate for a long time. Most investigations have focused on the induction of proliferative responses and cytokine production by T cells. In parallel, endothelial cells have been shown to express costimulatory molecules with positive and negative regulatory effects on T cell responses. Recent studies have provided an alternative view of the antigen presenting cell function of endothelial cells, suggesting that cognate recognition of the endothelium by trafficking T cells is a key event in selecting the migration of antigen-specific lymphocytes into the site of inflammation.     

Mechanisms of immunity to infection with typhus rickettsiae: infected fibroblasts bear rickettsial antigens on their surfaces.

As with any immune response to infectious organisms, both antibody and T cell-mediated immune responses to infection with Rickettsia typhi require the appropriate presentation of rickettsial antigens to immunocompetent cells. Considering the obligate intracellular nature of rickettsiae, the exact mechanisms by which lymphocytes and macrophages encounter and respond to rickettsial antigens may depend on certain aspects of pathogenesis and on the availability of organisms or their antigens to cells of the immune system. One potential mode of rickettsial antigen presentation, not previously identified, is the appearance in vitro of rickettsial antigens on the cell membrane of R. typhi-infected L-929 fibroblasts. Polyvalent fluoresceinated rabbit antisera directed against whole R. typhi cells used in flow cytometric analysis of infected fibroblasts showed an increasing presence of R. typhi antigen on the host cell membrane 1 to 3 days postinfection. The significance of this finding in the pathophysiology of rickettsia-host interactions and the generation of cytotoxic T cell-mediated immunity and antibody immunity is discussed.     

Fibronectin in immune responses in organ transplant recipients

The immune response to an organ allograft involves perpetuation of T cell infiltration and activation. Advances in understanding the mechanisms of T cell activation have placed particular emphasis on the interactions between the T-cell receptor and antigen presenting cells, with little reference to the fact that in vivo activation occurs in the physical context of extracellular matrix proteins (ECM). Indeed, the possibility that ECM proteins may have a determining role in lymphocyte adhesion and tissue localization and function is now becoming more appreciated in view of growing evidence indicating that integrins and other T cell antigens bind ECM components, with some of these components exerting synergistic effects on T-cell activation. This review focuses on the importance of interactions between lymphocytes and fibronectin, a prominent ECM component, for cell migration and function in organ allograft recipients. It explores novel therapeutic approaches based on the assumption that fibronectin represents an active element in the process of T cell activation in the immune cascade triggered by organ transplantation.     

Migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive CD4 T cells

To initiate an adaptive immune response, rare antigen-specific naive CD4(+) T cells must interact with equally rare dendritic cells (DCs) bearing cognate peptide-major histocompatibility complex (MHC) complexes. Lymph nodes (LNs) draining the site of antigen entry are populated by lymphoid-resident DCs as well as DCs that have immigrated from tissues, although the requirement for each population in initiating the T cell response remains unclear. Here, we show that antigen processing and presentation by both lymphoid-resident and migratory DCs was required for clonal selection and expansion of CD4(+) T cells after subcutaneous immunization. Early antigen presentation by lymphoid-resident DCs initiated activation and trapping of antigen-specific T cells in the draining LN, without sufficing for clonal expansion. Migratory DCs, however, interacted with the CD4(+) T cells retained in the LN to induce proliferation. Therefore, distinct DC subsets cooperate to alert and trap the appropriate cell and then license its expansion and differentiation.     

Immune recognition,response, and regulation

Effective immunity to infectious agents requires the initial recognition of antigen by specific receptors, which leads to the activation of immunocytes and the elicitation of an immune response. Because T cell antigen recognition and directed responses are complex processes, they are initiated and quelled in a highly regulated manner. Our laboratory has focused on defining the molecular processes that control T cell antigen presentation and recognition. Research in this area is focused on determining the chemical nature of antigens displayed by Major histocompatibility complex (MHC)-encoded class I molecules and the nonclassical class I-like prote in CD1d. Quantitative aspects of antigen presentation and recognition are also being studied to determine how these factors control the initiation of a T cell response. Our studies provide insights into the biochemical basis of T cell antigen recognition and response as well as the molecular processes underlying the initiation and regulation of immune responses by CD1d-restricted natural T lymphocytes.     

Antigen-presenting cells in the human female reproductive tract: analysis of antigen presentation in pre- and post-menopausal women.

PROBLEM: To determine whether cells in the female reproductive tract (FRT) are functionally capable of presenting antigen to T cells. METHOD OF STUDY: Analysis was done by determining the proliferation of purified autologous T cells to antigen, following co-incubation with non-proliferating cell suspensions isolated from the uterus and prepared by enzymatic digestion of reproductive tract tissues from hysterectomy patients with benign disease. RESULTS: All uterine preparations analyzed were functionally capable of presenting antigen; the ability to present antigen was independent of pre- and post-menopausal status. In contrast, some, but not all, tissues from the ovary, Fallopian tube, cervix, and vagina were capable of presenting antigen. CONCLUSION: These results suggest that the human FRT is an inductive site for immune responses. Regulation of antigen presentation in the reproductive tract may be important for protection against sexually transmitted diseases.     

Calcium responses elicited in human T cells and dendritic cells by cell-cell interaction and soluble ligands.

The interactions between a human CD4+ T cell clone and monocyte-derived human dendritic cells (DC) were analyzed with an imaging system. The first question addressed was the relationship between the formation of a contact zone and the triggering of a Ca2+ response in the T cells, in the presence or absence of antigen. Interaction of T cells with DC pulsed with the antigen led to the formation of a stable contact zone, followed by the appearance in the T cells of large and sustained Ca2+ oscillations. In the absence of antigen, contact zones formed normally and, surprisingly, Ca2+ responses were also observed, characterized by rare and small transients. Antigen-independent Ca2+ responses were not MHC restricted. The possible influence of Ca2+ responses in the DC on the efficiency of antigen presentation was then Investigated. In DC, Ca2+ responses can be elicited by a variety of stimuli: cell adhesion, platelet-activating factor, UTP and chemotactic molecules (formyl-Met-Leu-Pro, RANTES, MIP-1beta and SDF-1alpha). Importantly, Ca2+ responses were also induced in approximately 30% of DC as a result of their interaction with T cells. However, the efficiency of antigen presentation (as judged by the percentage of T cells presenting a Ca2+ response) was independent of the Ca2+ level in DC. Thus, imaging the interactions between human T cells and DC led us to observe two novel phenomena: DC-induced but antigen-independent Ca2+ responses in T cells and T cell-induced Ca2+ responses in DC.     

Expression of heat-stable antigen on tumor cells provides co-stimulation for tumor-specific T cell proliferation and cytotoxicity in mice

Heat-stable antigen (HSA/J11d/possibly homologous to CD24), a cell adhesion molecule capable of providing a co-stimulatory signal for T cell proliferation, is expressed on B cells, activated T cells, monocytes, granulocytes, Langerhans cells and thymocytes. Recent studies have demonstrated that co-stimulatory signals provided by cell adhesion molecules such as B7-1 play an essential role in generation of an anti-tumor immune response. To examine whether the co-stimulatory signal provided by HSA can induce an anti-tumor immune response, we have transfected HSA cDNA into the murine melanoma cell line K1735M2, and examined the ability of this transfected cell line to induce tumor-specific T cell responses. The results demonstrate that spleen cells from mice immunized with HSA-transfected K1735M2 cells showed enhanced T cell proliferation in a mixed lymphocyte tumor reaction (MLTR) assay and also demonstrated a significant anti-tumor cytotoxicity to the parent tumor cell (K1735M2). This anti-tumor cytolytic activity could be abrogated by pretreatment of effector cells with anti-mouse CD8 monoclonal antibody and complement. Under similar conditions, spleen cells from C3H mice immunized with vector-transfected K1735M2 cells neither actively proliferate in an MLTR assay, nor did they exert significant cytolytic activity against the respective tumor cells. In summary, our study demonstrated that HSA can provide a co-stimulatory signal for the T cell immune response against tumor cells in a murine model.     

Tracking lymphocytes in vivo

The ability to track antigen (Ag)-specific lymphocyte populations in vivo has greatly increased our understanding of the location and functional status of these cells throughout the course of an immune response. Recent technical advances have enhanced researchers' capability to follow migration, activation and cellular interactions of Ag-specific lymphocytes in situ. It is now possible to monitor changes in T cell subsets, co-stimulatory molecules, and chemokine expression within the physiological context of secondary lymphoid organs. Furthermore, the Ag-presenting cell-T cell interaction can be studied,thus dissecting the role and timing of Ag presentation of particular dendritic cell subsets in the initiation of the immune response. The capacity to adoptively transfer small populations of Ag-specific T lymphocytes has also increased our knowledge of the physiologically important role of regulatory T cells in autoimmunity and immunosuppression. New fluorescence imaging techniques such as multicolor video microscopy, laser scanning cytometry, and multiphoton tissue imaging have provided new ways in which researchers can track cellular changes within Ag-specific lymphocytes in vivo. This review summarizes some of the ways in which these techniques have led to discoveries in the role of signaling cascades, cell cycle progression, and apoptosis in maintaining an Ag-specific immune response.     

Antigen presentation of myoglobin: profiles of T cell proliferative responses following priming with synthetic overlapping peptides encompassing the entire molecule

Recently, the regions of myoglobin, which are recognized by T cells (T sites), were localized by a comprehensive synthetic strategy in which uniform synthetic overlapping peptides encompassing the entire protein chain were examined for stimulation of T cell proliferative activity. In this study, we report about the proliferative response to these peptides, as well as to the native protein, of lymph node cells from mice primed with the overlapping peptides either individually or in a mixture. Some, but not all, of the T site-containing peptides were effective in priming for an anti-myoglobin T cell response. Further, several peptides, which were highly immunogenic as free synthetic peptides, were not associated with any of the known T sites in this protein. Thus, the pattern of T cell recognition following priming with the overlapping peptides differs from the pattern observed when the native protein is the priming antigen. If antigen processing proceeds via fragmentation, then only those regions containing T sites would be expected to be effective in priming for a T cell response to the intact protein and, conversely, highly immunogenic peptides would correspond to T sites of the protein. Therefore, these findings indicate that the current concept of antigen fragmentation as a prerequisite for its presentation must be reappraised. We suggest that, in the presentation of a protein antigen, the protein is recognized predominantly intact and that the crucial aspects of presentation are determined by interaction with the cell membrane which trigger cellular activating events.