cell-T Cell Interactions Research Articles

Abstract A4: Hodgkin and Reed-Sternberg cells secrete soluble factors to modulate endothelial cell-T cell interactions in classical Hodgkin lymphoma

Abstract Introduction: Classical Hodgkin lymphoma (CHL) is characterized by the presence of a minority of malignant Hodgkin and Reed-Sternberg cells (H-RS cells) surrounded by an abundant mixed inflammatory infiltrate that includes CD4+ T helper 2 cells, regulatory T cells and CD8+ cytotoxic T cells. The mechanisms exploited by HRS cells to influence T cell recruitment into the involved lymph nodes are still unknown. Objective: The aim of this study is to elucidate whether H-RS cells can induce endothelial cell activation to modulate T cell recruitment in CHL. Procedures: Fresh cell culture supernatant (C/S) from the H-RS cell line, KM-H2, was used to stimulate endothelial cells (EC) in-vitro. Controls were EC incubated in medium alone; or with 10ng/ml TNF-α Up-regulation of adhesion molecule expression was assessed by ELISA and FACS. Using the transwell migration system and the parallel plate flow chamber apparatus respectivel, the ability of these stimulated EC to support T cell interactions in-vitro was assessed under static and defined flow conditions. Signaling pathways involved in C/S induced EC activation were examined by western blotting. Results: KM-H2 C/S-stimulated EC up-regulate ICAM-1, VCAM-1 and E-selectin to levels comparable to those of TNF-α-stimulated EC. Both C/S-stimulated EC and TNF-α stimulated-EC support naïve and memory T-cell interactions under defined flow conditions. Furthermore, both CD4+ naïve and memory T cell demonstrated enhanced transmigration across C/S-stimulated EC monolayers in response to SDF-1α, as compared to TNF-α-stimulated or unstimulated EC. The activation of EC by KM-H2 C/S is dependent on the NFkB pathway. Blocking of p65 nuclear translocation by the specific IκBα phosphorylation inhibitor (BAY11-8075) abrogated the stimulatory effect of C/S on EC. Phosphorylation of ERK, p38 and JNK were also detected in the KM-H2 C/S stimulated EC. Intracytoplasmic expression of TNF-α was detected in KM-H2 cells. The bioactivity assay showed that the TNF-α sensitive cell line, L929 is sensitive to KM-H2 C/S. However, treatment of cells with TNF-α neutralizing antibody did not effectively prevent L929 cell death, nor inhibit the stimulatory effect of KM-H2 C/S on EC. Conclusion: Our data suggest that the malignant Hodgkin and Reed-Sternberg cells secrete soluble mediators that modulate endothelial cell function and influence recruitment of T cells into the cHL lesion. While the C/S induced EC activation is dependent on the NFkB pathway, our data suggest that the dominant mediator involved is not TNF-α. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A4.

CD70 Is Selectively Expressed on Th1 but Not on Th2 Cells and Is Required for Th1-Type Immune Responses

The interaction between CD27 and CD70 provides a costimulatory signal for T-cell survival. Although the role of CD27 signaling in CD8(+) T cells has been well defined, its role in CD4(+) T cells is relatively unknown. Here, we report that CD70 is specifically expressed on differentiated T-helper (Th)1 cells, but not on Th2 cells. Upon activation, CD70 expression increased markedly on Th1 cells, but remained undetectable on Th2 cells. We demonstrate that CD27 is involved in naive T-cell expansion in Th1-type, but not in Th2-type, immune responses as in vivo treatment with anti-CD70 monoclonal antibody at induction resulted in a significant reduction of delayed-type and contact hypersensitivity responses, but not asthmatic responses. In both Th1-type responses, during the priming phase, CD70 was detected at earlier time points on dendritic cells (DCs) and at later time points on CD4(+) T cells. Our results indicate that CD70 may be useful as a marker to distinguish Th1 from Th2 cells. More importantly, CD27 function may be controlled by the differentially regulated kinetics of CD70 expression on DCs and CD4(+) T cells, and Th1 cell-specific CD70 expression may be involved in an amplification loop for polarized Th1-type immune responses through T cell-T cell interactions.

Decoy Strategies: The Structure of TL1A:DcR3 Complex

Decoy Receptor 3 (DcR3), a secreted member of the Tumor Necrosis Factor (TNF) receptor superfamily, neutralizes three different TNF ligands: FasL, LIGHT, and TL1A. Each of these ligands engages unique signaling receptors which direct distinct and critical immune responses. We report the crystal structures of the unliganded DcR3 ectodomain and its complex with TL1A, as well as complementary mutagenesis and biochemical studies. These analyses demonstrate that DcR3 interacts with invariant backbone and side-chain atoms in the membrane-proximal half of TL1A which supports recognition of its three distinct TNF ligands. Additional features serve as antideterminants that preclude interaction with other members of the TNF superfamily. This mode of interaction is unique among characterized TNF:TNFR family members and provides a mechanistic basis for the broadened specificity required to support the decoy function of DcR3, as well as for the rational manipulation of specificity and affinity of DcR3 and its ligands.

Blocking the NOTCH Pathway Inhibits Vascular Inflammation in Large-Vessel Vasculitis

Giant cell arteritis is a granulomatous vasculitis of the aorta and its branches that causes blindness, stroke, and aortic aneurysm. CD4 T cells are key pathogenic regulators, instructed by vessel wall dendritic cells to differentiate into vasculitic T cells. The unique pathways driving this dendritic cell-T-cell interaction are incompletely understood, but may provide novel therapeutic targets for a disease in which the only established therapy is long-term treatment with high doses of corticosteroids. Immunohistochemical and gene expression analyses of giant cell arteritis-affected temporal arteries revealed abundant expression of the NOTCH receptor and its ligands, Jagged1 and Delta1. Cleavage of the NOTCH intracellular domain in wall-infiltrating T cells indicated ongoing NOTCH pathway activation in large-vessel vasculitis. NOTCH activation did not occur in small-vessel vasculitis affecting branches of the vasa vasorum tree. We devised 2 strategies to block NOTCH pathway activation: γ-secretase inhibitor treatment, preventing nuclear translocation of the NOTCH intracellular domain, and competing for receptor-ligand interactions through excess soluble ligand, Jagged1-Fc. In a humanized mouse model, NOTCH pathway disruption had strong immunosuppressive effects, inhibiting T-cell activation in the early and established phases of vascular inflammation. NOTCH inhibition was particularly effective in downregulating Th17 responses, but also markedly suppressed Th1 responses. Blocking NOTCH signaling depleted T cells from the vascular infiltrates, implicating NOTCH- NOTCH ligand interactions in regulating T-cell retention and survival in vessel wall inflammation. Modulating the NOTCH signaling cascade emerges as a promising new strategy for immunosuppressive therapy of large-vessel vasculitis.

Dissection of the reciprocal interaction between T helper cells and B cells during an immune response in vivo (90.4)

Abstract Cognate interaction of B cells with antigen-specific T helper cells in secondary lymphoid organs is required for production of high-affinity antibodies and for generation of memory B cells and long-lived plasma cells. To gain insight into the nature of signals regulating these different aspects of B cell responses, we set up an experimental system in which CD3ε-/- mice - which lack T cells but have normal B cell development - are reconstituted with small numbers of TCR-transgenic T cells. Serum antibody levels, germinal center reaction, as well as generation of antibody-secreting cells and long-lived plasma cells were monitored at different time points after primary and secondary immunization. We found that adoptively transferred antigen-specific CD4+ T cells induced effective primary antibody responses; however, serum antibody levels were not sustained. Further analysis revealed that upon immunization T cells proliferated extensively but became exhausted, did not respond to secondary antigenic stimulation, and also failed to provide B cell help upon booster immunization. Although immunoglobulin class switch recombination, initiation of germinal center reaction, and differentiation of B cells to antibody-secreting short-lived plasma blasts were comparable in reconstituted CD3ε-/- mice, affinity-maturation was impaired and long-lived plasma cells were not generated or did not migrate to the bone marrow. Data will be presented on the mechanistic link between B cell-T cell interaction and the exhausted state of T helper cells in our experimental system. The results of the study suggest the existence of distinct early and late T cell-dependent events in initiation and maintenance of humoral immune responses. This work was supported by a Boehringer Ingelheim Fonds PhD Scholarship to D.B.

Dendritic Cell-Nerve Clusters Are Sites of T Cell Proliferation in Allergic Airway Inflammation

Interactions between T cells and dendritic cells in the airway mucosa precede secondary immune responses to inhaled antigen. The purpose of this study was to identify the anatomical locations where dendritic cell-T cell interactions occur, resulting in T cells activation by dendritic cells. In a mouse model of allergic airway inflammation, we applied whole-mount immunohistology and confocal microscopy to visualize dendritic cells and T cells together with nerves, epithelium, and smooth muscle in three dimensions. Proliferating T cells were identified by the detection of the incorporation of the nucleotide analogue 5-ethynyl-2'-deoxyuridine into the DNA. We developed a novel quantification method that enabled the accurate determination of cell-cell contacts in a semi-automated fashion. Dendritic cell-T cell interactions occurred beneath the smooth muscle layer, but not in the epithelium. Approximately 10% of the dendritic cells were contacted by nerves, and up to 4% of T cells formed clusters with these dendritic cells. T cells that were clustered with nerve-contacting dendritic cells proliferated only in the airways of mice with allergic inflammation but not in the airways of negative controls. Taken together, these results suggest that during the secondary immune response, sensory nerves influence dendritic cell-driven T cell activation in the airway mucosa.

Intracellular concentrations of Ca2+ modulate the strength of signal and alter the outcomes of cytotoxic T‐lymphocyte antigen‐4 (CD152)–CD80/CD86 interactions in CD4+ T lymphocytes

The costimulatory receptors CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 and their ligands, CD80 and CD86, are expressed on T lymphocytes; however, their functional roles during T cell-T cell interactions are not well known. The consequences of blocking CTLA-4-CD80/CD86 interactions on purified mouse CD4(+) T cells were studied in the context of the strength of signal (SOS). CD4(+) T cells were activated with phorbol 12-myristate 13-acetate (PMA) and different concentrations of a Ca(2+) ionophore, Ionomycin (I), or a sarcoplasmic Ca(2+) ATPase inhibitor, Thapsigargin (TG). Increasing concentrations of I or TG increased the amount of interleukin (IL)-2, reflecting the conversion of a low to a high SOS. During activation with PMA and low amounts of I, intracellular concentrations of calcium ([Ca(2+)](i)) were greatly reduced upon CTLA-4-CD80/CD86 blockade. Further experiments demonstrated that CTLA-4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of I or TG (high SOS) but the opposite occurred with PMA and low amounts of I or TG (low SOS). These results were confirmed by surface T-cell receptor (TCR)-CD3 signalling using a low SOS, for example soluble anti-CD3, or a high SOS, for example plate-bound anti-CD3. Also, CTLA-4-CD80/CD86 interactions enhanced the generation of reactive oxygen species (ROS). Studies with catalase revealed that H(2)O(2) was required for IL-2 production and cell cycle progression during activation with a low SOS. However, the high amounts of ROS produced during activation with a high SOS reduced cell cycle progression. Taken together, these results indicate that [Ca(2+)](i) and ROS play important roles in the modulation of T-cell responses by CTLA-4-CD80/CD86 interactions.

CD137-CD137 Ligand Interactions in Inflammation

The main stream of CD137 studies has been directed to the function of CD137 in CD8+ T-cell immunity, including its anti-tumor activity, and paradoxically the immunosuppressive activity of CD137, which proves to be of a great therapeutic potential for animal models of a variety of autoimmune and inflammatory diseases. Recent studies, however, add complexes to the biology of CD137. Accumulating is evidence supporting that there exists a bidirectional signal transduction pathway for the CD137 receptor and its ligand (CD137L). CD137/CD137L interactions are involved in the network of hematopoietic and nonhematopoietic cells in addition to the well characterized antigen-presenting cell-T cell interactions. Signaling through CD137L plays a critical role in the differentiation of myeloid cells and their cellular activities, suggesting that CD137L signals trigger and sustain inflammation. The overall consequence might be that the amplified inflammation by CD137L enhances the T-cell activity together with CD137 signals by upregulating costimulatory molecules, MHC molecules, cell adhesion molecules, cytokines, and chemokines. Solving this outstanding issue is urgent and will have an important clinical implication.

Receptor for Advanced Glycation End Products Expression on T Cells Contributes to Antigen-Specific Cellular Expansion In Vivo

Receptor for advanced glycation end products (RAGE) is an activation receptor triggered by inflammatory S100/calgranulins and high mobility group box-1 ligands. We have investigated the importance of RAGE on Ag priming of T cells in murine models in vivo. RAGE is inducibly up-regulated during T cell activation. Transfer of RAGE-deficient OT II T cells into OVA-immunized hosts resulted in reduced proliferative responses that were further diminished in RAGE-deficient recipients. Examination of RAGE-deficient dendritic cells did not reveal functional impairment in Ag presentation, maturation, or migratory capacities. However, RAGE-deficient T cells showed markedly impaired proliferative responses in vitro to nominal and alloantigens, in parallel with decreased production of IFN-gamma and IL-2. These data indicate that RAGE expressed on T cells is required for efficient priming of T cells and elucidate critical roles for RAGE engagement during cognate dendritic cell-T cell interactions.

Location, location, location: dendritic cell trafficking and transplant tolerance.

Use or targeting of dendritic cells for therapeutic manipulation of immune responses is being pursued in the areas of cancer, autoimmune disease, and allograft rejection. There is, however, a dearth of information regarding the optimal route of cell delivery or target location for maximal therapeutic effect, particularly in the field of transplantation. Further, little attention has been given to the roles that conventional experimental/immunosuppressive modalities have on the migratory capacity of these important antigen-presenting cells. Current understanding of the role of dendritic cells in immunologic ignorance, graft rejection, or tolerance to alloantigen suggests their function is influenced by subset, secondary lymphoid tissue location, and the type of organ transplanted. It also has been determined recently that dendritic cell subsets probably utilize distinct migratory routes to secondary lymphoid tissues, further underscoring the importance of understanding dendritic cell trafficking for optimization of dendritic cell therapy protocols. Increased comprehension of the requirements for dendritic cell-T cell interactions to take place in specific secondary lymphoid tissues for the induction of rejection versus tolerance, with and without antirejection therapy, will facilitate the ease with which cell-based therapy can be designed and implemented in transplant recipients.

Type I Interferons Attenuate T Cell Activating Functions of Human Mast Cells by Decreasing TNF-α Production and OX40 Ligand Expression While Increasing IL-10 Production

Recent studies have demonstrated that mast cells not only mediate inflammatory reactions in type I allergy but also play an important role in adaptive immunity. In the present study, we investigated the effects of interferon-alpha, which shares the same receptor as IFN-beta, on human cord blood-derived mast cells. Mast cells produced TNF-alpha, and IL-10, and expressed OX40 ligand upon activation by crosslinking of FcepsilonRI. When treated with interferon-alpha, TNF-alpha production was decreased while IL-10 and TGF-beta productions were increased. Furthermore, flow cytometric analysis revealed that interferon-alpha downregulated expression OX40 ligand on mast cells which is crucial for mast cell-T cell interaction. We confirmed that the viability of mast cells was not affected by interferon-alpha treatment. Accordingly, interferon-alpha-treated mast cells induced lower levels of CD4+ T cell proliferation compared with those without interferon-alpha treatment. These results suggest that type I interferons suppress T cell immune responses through their regulatory effects on mast cells.

OX40-OX40 Ligand Interaction through T Cell-T Cell Contact Contributes to CD4 T Cell Longevity

Signals through the OX40 costimulatory receptor on naive CD4 T cells are essential for full-fledged CD4 T cell activation and the generation of CD4 memory T cells. Because the ligand for OX40 is mainly expressed by APCs, including activated B cells, dendritic cells, and Langerhans cells, the OX40-OX40 ligand (OX40L) interaction has been thought to participate in T cell-APC interactions. Although several reports have revealed the expression of OX40L on T cells, the functional significance of its expression on them is still unclear. In this study, we demonstrate that Ag stimulation induced an increase in the surface expression and transcript levels of OX40L in CD4 T cells. Upon contact with OX40-expressing T cells, the cell surface expression of OX40L on CD4 T cells was markedly down-regulated, suggesting that OX40-OX40L binding occurs through a novel T cell-T cell interaction. To investigate the function of this phenomenon, we examined the proliferative response and survival of OX40L-deficient CD4 T cells when challenged with Ag. In vitro studies demonstrated markedly less CD3-induced proliferation of OX40L-deficient CD4 T cells compared with wild-type CD4 T cells. When using TCR transgenic CD4 T cells upon Ag stimulation, survival of OX40L-deficient T cells was impaired. Furthermore, we show that upon antigenic stimulation, fewer OX40L-deficient CD4 T cells than wild-type cells survived following transfer into wild-type and sublethally irradiated recipient mice. Taken together, our findings indicate that OX40L-expressing T cells have an autonomous machinery that provides OX40 signals through a T cell-T cell circuit, creating an additional mechanism for sustaining CD4 T cell longevity.

In Vitro Suppression of CD8+ T Cell Function by Friend Virus-Induced Regulatory T Cells

Regulatory T cell (Treg)-mediated suppression of CD8+ T cells has been implicated in the establishment and maintenance of chronic viral infections, but little is known about the mechanism of suppression. In this study an in vitro assay was developed to investigate the suppression of CD8+ T cells by Friend retrovirus (FV)-induced Tregs. CD4+CD25+ T cells isolated from mice chronically infected with the FV suppressed the development of effector function in naive CD8+ T cells without affecting their ability to proliferate or up-regulate activation markers. In vitro restimulation was not required for suppression by FV-induced Tregs, correlating with their high activation state in vivo. Suppression was mediated by direct T cell-T cell interactions and occurred in the absence of APCs. Furthermore, suppression occurred irrespective of the TCR specificity of the CD8+ T cells. Most interestingly, FV-induced Tregs were able to suppress the function of CD8+ effector T cells that had been physiologically activated during acute FV infection. The ability to suppress the effector function of activated CTLs is likely a requisite role for Tregs in limiting immunopathology by CD8+ T cells during antiviral immune responses. Such activity may also have adverse consequences by allowing viruses to establish and maintain chronic infections if suppression of antiviral immune responses occurs before virus eradication.

Cell Contact with CLL Cells Induces Defects in T Cell Differentiation and Effector Pathways: Impact of Silencing Specific Cytokine Production.

Previous studies have suggested that the development and progression of B cell CLL is dependent on interactions between malignant cells and normal components of the immune system. Although the T cell count may be normal or even increased, T cell dysfunction is a feature of CLL, with abnormal CD4/CD8 ratio, impaired mitogen response, and altered expression of surface antigens in response to antigen presentation. We are evaluating the impact of tumor cells on the immune system and have reported differences in gene expression profiles in T cells in previously untreated CLL patients. Specifically, in both CD4 and CD8 T cells we identified defects in genes regulating cytoskeleton formation, intracellular transportation and control of cytokines and chemokines. Analysis of the abnormal gene expression profiles suggested that many such abnormalities are induced by signaling through surface receptors on T cells by interaction with CLL cells or the microenvironment. To detemine the mechanism, we performed ex vivo tumor cell-T cell interaction assays using patient derived serum, transwell membrane and cell contact assays using CLL and CD4 or CD8 T cells from CLL patients and B cells and CD4 or CD8 T cells from healthy donors. Since the majority of the differentially expressed genes were involved in cell cytoskeleton formation and intracellular vesicle transportation pathways, we examined the impact on those specific pathways using proteomics. In keeping with the gene expression profiles in the T cells of CLL patients, we noted significant decreased expression of NFkB p65 and GDI1 and increase in Arp2/3 in healthy CD4 T cells and decreased expression of Rho-GAP and increased Arp2/3 in healthy CD8 T cells following CLL-T cell contact compared to healthy B cell-T cell contact. In contrast there was no change after exposure to patient sera or tumor cell derived soluble factors for 48h. To further analyze whether tumor cell derived cytokines have an impact on T cells in CLL, we inhibited IL-10 and IL-4 production in CLL cells and healthy B cells using siRNA targeting IL-10 and/or IL-4. The transfection efficiency monitored by flow cytometry with fluorescence labeled non-silencing RNA and observed 60–98 % transfection efficiency and at 48 h observed 40–85% inhibition in IL-10 protein expression. After 48 h incubation of autologous and allogeneic CD4 or CD8 T cells from CLL and healthy donors with non-transfected, mock transfected or IL-10 siRNA transfected CLL or healthy B cells for 48h, T cells were isolated and there was no significant difference in expression of cytoskeletal proteins in both CD4 or CD8 T cells. Addition of anti-IL-10 neutralizing monoclonal antibody also had no effect. Although no effect was noted on cytoskeletal proteins, after incubation with CLL but not healthy B cells, silencing or neutralization of IL-10 induced changes in expression of CXCR1, CXCR2, CXCR3, CXCR4 and CCR5 in CD4 and CCR5 and CCR4 in CD8 cells from healthy donors. We conclude that cell contact with CLL cells induces changes in expression of cytoskeletal proteins in healthy T cells similar to those observed in T cells from CLL patients and this is not induced by soluble factors including IL-10. However, IL-10 and potentially other soluble factors induce changes in chemokines and chemokine receptors on T cells, suggesting multiple mechanisms impair T cell function in CLL cells. Ongoing studies are assessing ways to repair the defects identified here to enhance immune responsiveness in this disease.

RANK and RANKL expression in RA synovium and lymph nodes as markers of dendritic cell-T cell interactions

The receptor activator of NF-κB/receptor activator of NF-κB ligand (RANK/RANKL) pathway is critical in osteoclastogenesis and bone erosion and has been implicated in the process of focal bone erosion in rheumatoid arthritis (RA). However, its involvement in the immune response during chronic inflammation remains to be clarified since deficient mice show major lymph node (LN) abnormalities. We investigated by immunohistochemistry the RANK and RANKL expression pattern of dendritic cell (DC) and T cell subsets in paired RA synovium-LN and also in normal peripheral blood mononuclear cells (PBMC) stimulated with phorbol miristate acetate/phytohemagglutin during 4, 24, and 48 hours. In RA synovium, RANKL+ cells were detected in the lining layer and the lymphocytic infiltrates, whereas RANK expression was restricted to perivascular infiltrates. In LN, RANK+ and RANKL+ cells were diffusely expressed in both the T-cell zone and germinal centers. Double staining with anti-RANK or anti-RANKL and anti-CD1a or anti-DC-LAMP antibodies showed that some immature CD1a+ DC expressed RANK and RANKL, whereas some mature DC-LAMP+ DC only expressed RANK. Double staining with the CD3, CD4 T-cell markers and the interferon gamma and IL-17 Th1 cell markers showed that some CD3+, CD4+, interferon gamma+, and IL-17+ cells produced RANKL whereas none of them express RANK. The same pattern was observed on activated PBMC. RANK+ cells were detected in unstimulated PBMC and identified as CD14+ monocytes. This study showed the involvement of RANK/RANKL in DC–T cell interactions as found during the inflammatory process, and makes RANK and RANKL potential therapeutic targets outside the bone field.

Impact of aging upon DBA/2J B cells

The influence of age on B lymphocyte phenotype and function in DBA/2J mice was examined. The B cells of this strain express the endogenous minor lymphocyte stimulatory (Mls) retroviral superantigen (SAg) Mls-1a permitting assessment of age-related changes in cognate B cell-T cell interaction. Relative to young DBA/2J mice (< 8 months), old mice (> 17 months) had greater numbers of B cells expressing high levels of IgM and low levels of the CD11b and CD5 antigens characteristic of B-1 B cells. As measured by the T cell proliferative response to Mls, the B cells from old DBA/2J mice had reduced ability to present SAg. Upon interaction with Mls-activated T cells, old B cells secreted more IgM while young B cells made more IgG1, IgG3, and IgG2a. DBA/2J BCL functioned poorly as Mls APCs and made considerably less serum Ig. T cells from old mice exhibited a lower response to SAg and were less capable of promoting B cell differentiation. These results indicate that aging influences the cellular collaboration necessary for humoral immunity.

Impaired Primary Immune Response in Type-1 Diabetes: Results from a Controlled Vaccination Study

Patients with diabetes have an increased risk for infections, but information on their adoptive immunity is incomplete and contradictory. Twenty patients with diabetes type-1 and 20 patients with type-2 diabetes were vaccinated with T-cell-dependent primary protein antigens (hepatitis A viral antigen, HAV; diphtheria toxoid) and a T-cell-independent polysaccharide antigen (pneumococcal polysaccharide). In parallel, the proliferative response of CD4+ T-cells to the primary protein antigens keyhole limpet hemocyanin (KLH) and sperm whale myoglobin (SWM) was measured in vitro using monocyte-derived dendritic cells (MDDC) as antigen-presenting cells. Compared to healthy controls, type-1 diabetes patients mounted a significantly impaired primary antibody response to hepatitis A vaccine (median HAV antibody titer after the first vaccination, 53 IU/L in diabetic patients vs 212 IU/L in the controls, P = 0.017) and diphtheria toxoid (median serum antibodies after vaccination, patients, 0.94 IU/ml, controls, 6.38 IU/ml, P = 0.004), while the response to pneumococcal polysaccharide was normal. Type-2 diabetes patients had a comparable metabolic dysregulation but showed a normal antibody response following vaccination, demonstrating that the effect was not due to hyperglycemia. Antigen-induced interferon-γ and interleukin-13 release was reduced in type-1 diabetes patients, localizing the impairment to the level of antigen-presenting cell–T-cell interaction. In addition, the proliferative response of CD4+ T-cells derived from type-1 diabetes patients to KLH and SWM was significantly reduced (P ≤ 0.01). FACS analysis of CD80 (B7.1), CD86 (B7.2), and HLA-DR expression on MDDC could not demonstrate significant differences in the expression of these molecules between type-1 and type-2 diabetes patients and healthy controls. An association of low HAV antibody response with HLA-DR3,4 expression in the patients was shown. Our results indicate that the primary antibody response to T-cell dependent antigens as well as the T-cell response to primary protein antigens is reduced in type-1 diabetes patients and that additional booster immunization can overcome the defect.

Autoreactive T cells revealed in the normal repertoire: escape from negative selection and peripheral tolerance.

Self-reactive T cells are known to be eliminated by negative selection in the thymus or by the induction of tolerance in the periphery. However, developmental pathways that allow self-reactive T cells to inhabit the normal repertoire are not well-characterized. In this investigation, we made use of anti-small nuclear ribonucleoprotein particle (snRNP) Ig transgenic (Tg) mice (2-12 Tg) to demonstrate that autoreactive T cells can be detected and activated in both normal naive mice and autoimmune-prone MRL lpr/lpr mice. In contrast, autoreactive T cells of nonautoimmune Tg mice are tolerized by Tg B cells in the periphery. In adoptive transfer studies, autoreactive T cells from MRL lpr/lpr mice can stimulate autoantibody synthesis in nonautoimmune anti-snRNP Tg mice. Transferred CD4 T cells migrate to regions of the spleen proximal to the B cell follicles, suggesting that cognate B cell-T cell interactions are critical to the autoimmune response. Taken together, our studies suggest that anti-snRNP B cells are important APCs for T cell activation in autoimmune-prone mice. Additionally, we have demonstrated that anti-snRNP B cell anergy in nonautoimmune mice may be reversed by appropriate T cell help.

The regulation of T cell homeostasis and autoimmunity by T cell–derived LIGHT

Costimulatory molecules on antigen-presenting cells (APCs) play an important role in T cell activation and expansion. However, little is known about the surface molecules involved in direct T-T cell interaction required for their activation and expansion. LIGHT, a newly discovered TNF superfamily member (TNFSF14), is expressed on activated T cells and immature dendritic cells. Here we demonstrate that blockade of LIGHT activity can reduce anti-CD3-mediated proliferation of purified T cells, suggesting that T cell-T cell interaction is essential for this proliferation. To test the in vivo activity of T cell-derived LIGHT in immune homeostasis and function, transgenic (Tg) mice expressing LIGHT in the T cell lineage were generated. LIGHT Tg mice have a significantly enlarged T cell compartment and a hyperactivated peripheral T cell population. LIGHT Tg mice spontaneously develop severe autoimmune disease manifested by splenomegaly, lymphadenopathy, glomerulonephritis, elevated autoantibodies, and severe infiltration of various peripheral tissues. Furthermore, the blockade of LIGHT activity ameliorates the severity of T cell-mediated diseases. Collectively, these findings establish a crucial role for this T cell-derived costimulatory ligand in T cell activation and expansion; moreover, the dysregulation of T cell-derived LIGHT leads to altered T cell homeostasis and autoimmune disease.

Multiparameter flow cytometric approach for simultaneous evaluation of T lymphocyte-endothelial cell interactions.

Since vascular endothelium is now recognized as an immunologically active tissue, a better understanding of the relationship between endothelial cells and T lymphocytes is critical to the field of solid organ transplantation. Investigations of endothelial cell-T cell interactions have been limited by methodology. We developed a flow cytometric method allowing for concurrent investigation of multiple cell populations within the same culture that can be applied to these complex interactions. Allogeneic CD8+ or CD4+ T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycle-blocking agent. At specific time points, the coculture was analyzed by flow cytometry. T-cell proliferation could be detected by gating on the T-cell subset and evaluating the CFSE fluorescence peaks. By directly analyzing cellular division, we minimized erroneous interpretation of the data encountered by previous studies, which utilized (3)H-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilitated the study of CD8+ lymphocyte activation, differentiation, and acquisition of effector function. By gating on the endothelial cell population, phenotypic changes such as upregulation of surface MHC molecules or immune-mediated apoptosis could be detected. In conclusion, we present a flow cytometric approach that could have important applications for clinical immunological monitoring in allogeneic or xenogeneic transplantation, and might provide the requisite information to better tailor immunotherapy to prevent chronic rejection.