Ex vivo expansion of regulatory T cells from long-term Belatacept-treated kidney transplant patients restores their phenotype and suppressive function but not their FOXP3 TSDR demethylation status.

We recently reported that Tregs from long-term Belatacept-treated kidney transplant patients displayed an altered phenotype and impaired suppressive function compared to Tregs from healthy controls. However, it remains unknown whether ex vivo expansion of Tregs from patients who underwent long-term immunosuppression may be feasible to be used in their treatment. In this work, Tregs from Belatacept-treated patients were polyclonally expanded in vitro in the presence of rapamycin and IL-2. After four weeks of expansion, Tregs from patients expressed high levels of FOXP3, CD25, CTLA-4, Helios and CCR7, and showed strong suppressive activity, even in the presence of pro-inflammatory cytokines. However, FOXP3 TSDR demethylation remained lower in expanded Tregs from Belatacept-treated patients compared to healthy control Tregs. These data suggest that ex vivo expansion of Tregs from patients undergoing long-term immunosuppression may require the use of epigenetic modifying agents to stabilize FOXP3 expression to be considered as treatment in kidney transplant patients.

CCR9 Is a Key Regulator of Early Phases of Allergic Airway Inflammation.

Airway inflammation is the most common hallmark of allergic asthma. Chemokine receptors involved in leukocyte recruitment are closely related to the pathology in asthma. CCR9 has been described as a homeostatic and inflammatory chemokine receptor, but its role and that of its ligand CCL25 during lung inflammation remain unknown. To investigate the role of CCR9 as a modulator of airway inflammation, we established an OVA-induced allergic inflammation model in CCR9-deficient mice. Here, we report the expression of CCR9 and CCL25 as early as 6 hours post-OVA challenge in eosinophils and T-lymphocytes. Moreover, in challenged CCR9-deficient mice, cell recruitment was impaired at peribronchial and perivenular levels. OVA-administration in CCR9-deficient mice leads to a less inflammatory cell recruitment, which modifies the expression of IL-10, CCL11, and CCL25 at 24 hours after OVA challenge. In contrast, the secretion of IL-4 and IL-5 was not affected in CCR9-deficient mice compared to WT mice. These results demonstrate for the first time that CCR9 and CCL25 expressions are induced in the early stages of airway inflammation and they have an important role modulating eosinophils and lymphocytes recruitment at the first stages of inflammatory process, suggesting that they might be a potential target to regulate inflammation in asthma.

Neonatal respiratory syncytial virus infection has an effect on lung inflammation and the CD4(+) CD25(+) T cell subpopulation during ovalbumin sensitization in adult mice.

In BALB/c adult mice, respiratory syncytial virus (RSV) infection enhances the degree of lung inflammation before and/or after ovalbumin (OVA) respiratory sensitization. However, it is unclear whether RSV infection in newborn mice has an effect on the immune response to OVA respiratory sensitization in adult mice. The aim of this study was to determine if RSV neonatal infection alters T CD4(+) population and lung inflammation during OVA respiratory sensitization in adult mice. BALB/c mice were infected with RSV on the fourth day of life and challenged by OVA 4 weeks later. We found that in adult mice, RSV neonatal infection prior to OVA sensitization reduces the CD4(+) CD25(+) and CD4(+) CD25(+) forkhead protein 3 (FoxP3)(+) cell populations in the lungs and bronchoalveolar lavage. Furthermore, it also attenuates the inflammatory infiltrate and cytokine/chemokine expression levels in the mouse airways. In conclusion, the magnitude of the immune response to a non-viral respiratory perturbation in adult mice is not enhanced by a neonatal RSV infection.

A CCL chemokine-derived peptide (CDIP-2) exerts anti-inflammatory activity via CCR1, CCR2 and CCR3 chemokine receptors: Implications as a potential therapeutic treatment of asthma.

Allergic asthma is a chronic inflammatory disease characterized by the accumulation of eosinophils, Th2 cells and mononuclear cells in the airways, leading to changes in lung architecture and subsequently reduced respiratory function. We have previously demonstrated that CDIP-2, a chemokine derived peptide, reduced in vitro chemotaxis and decreased cellular infiltration in a murine model of allergic airway inflammation. However, the mechanisms involved in this process have not been identified yet. Now, we found that CDIP-2 reduces chemokine-mediated functions via interactions with CCR1, CCR2 and CCR3. Moreover, using bone marrow-derived eosinophils, we demonstrated that CDIP-2 modifies the calcium fluxes induced by CCL11 and down-modulated CCR3 expression. Finally, CDIP-2 treatment in a murine model of OVA-induced allergic airway inflammation reduced leukocyte recruitment and decreases production of cytokines. These data suggest that chemokine-derived peptides represent new therapeutic tools to generate more effective antiinflammatory drugs.

CCR9+ T cells contribute to the resolution of the inflammatory response in a mouse model of intestinal amoebiasis.

Analysis of the mechanisms underlying the inflammatory response in amoebiasis is important to understand the immunopathology of the disease. Mucosal associated effector and regulatory T cells may play a role in regulating the inflammatory immune response associated to Entamoeba histolytica infection in the colon. A subpopulation of regulatory T cells has recently been identified and is characterized by the expression of the chemokine receptor CCR9. In this report, we used CCR9 deficient (CCR9(-/-)) mice to investigate the role of the CCR9(+) T cells in a murine model of E. histolytica intestinal infection. Intracecal infection of CCR9(+/+), CCR9(+/-) and CCR9(-/-) mice with E. histolytica trophozoites, revealed striking differences in the development and nature of the intestinal inflammatory response observed between these strains. While CCR9(+/+) and CCR9(+/-) mice were resistant to the infection and resolved the pathogen-induced inflammatory response, CCR9(-/-) mice developed a chronic inflammatory response, which was associated with over-expression of the cytokines IFN-γ, TNF-α, IL-4, IL-6 and IL-17, while IL-10 was not present. In addition, increased levels of CCL11, CCL20 and CCL28 chemokines were detected by qRT-PCR in CCR9(-/-) mice. E. histolytica trophozoites were identified in the lumen of the cecum of CCR9(-/-) mice at seven days post infection (pi), whereas in CCR9(+/+) mice trophozoites disappeared by day 1 pi. Interestingly, the inflammation observed in CCR9(-/-) mice, was associated with a delayed recruitment of CD4(+)CD25(+)FoxP3(+) T cells to the cecal epithelium and lamina propria, suggesting that this population may play a role in the early regulation of the inflammatory response against E. histolytica, likely through IL-10 production. In support of these data, CCR9(+) T cells were also identified in colon tissue sections obtained from patients with amoebic colitis. Our data suggest that a population of CCR9(+)CD4(+)CD25(+)FoxP3(+) T cells may participate in the control and resolution of the inflammatory immune response to E. histolytica infection.

CD11c decrease in mouse thymic dendritic cells after vanadium inhalation.

Vanadium (V) is a transition metal found in air adsorbed onto suspended particles. As a result, urban populations are often exposed to this element as a constituent of particulate matter (PM). One aspect of the myriad toxicities that might arise from these exposures is altered immune responses. Previous reports from the laboratory reported modifications in splenic architecture - with germinal center hyperplasia and a suppressed humoral immune response - in mice that had been exposed to vanadium agents via inhalation. This paper reports a decrease in the presence of the CD11c surface marker on mouse thymic dendritic cells (DC) as a result of host exposure to vanadium (here, in the form of vanadium pentoxide; V(2)O(5)) over a period of 4 weeks. All results were obtained using immunohistochemistry and flow cytometry. It is surmised that this decrease might induce a dysfunction, including possible negative selection of T-cells, which could increase the presence of autoreactive clones in the exposed host. Such an outcome could, in turn, increase the risk for development of autoimmune reactions in different organs specifically, and of autoimmune diseases in general in these V-exposed hosts.

When versatility matters: activins/inhibins as key regulators of immunity.

Activins and inhibins are members of the transforming growth factor-ß superfamily that have been considered crucial regulators of cell processes, such as differentiation, proliferation and apoptosis, in different cell types. Initial studies about the function of activin A in the immune system focused on the regulation of hematopoiesis in the bone marrow under homeostatic and inflammatory conditions. Recent data provide a more comprehensive understanding about the role of activins/inhibins in the immune system. Novel findings included in this review point out the important requirement of activin/inhibin signaling to maintain the balance between homeostatic and inflammatory signals that are required for the optimal development and function of immune cells. The purpose of this review is to highlight the versatile nature of activins/inhibins as key regulators of both the innate and adaptive immune responses.

Proteolytic cleavage of chemokines by Trypanosoma cruzi's cruzipain inhibits chemokine functions by promoting the generation of antagonists.

Chagas disease is a chronic inflammatory disease caused by infection with Trypanosoma cruzi. Although it had a decline in recent years, it still affects millions of people in Latin America. The host immune response against this parasite is complex and relies on the development of an efficient T cell-mediated response; however, T. cruzi displays a number of evasion mechanisms allowing it to remain undetected even for years. One of these is the secretion of anti-inflammatory molecules such as proteases and the modulation of biological functions of chemokines. Our objective was to analyze the effect of a major cysteine protease, cruzipain, on a number of critical functions of several CC chemokines, both in vitro and in vivo. Initially, using a murine model of T. cruzi infection, we demonstrated that CCL-2 and CCL-12 chemokines are highly expressed at different stages and correlated with an increase in the expression of cruzipain. In addition, we demonstrated that cruzipain is capable of differentially cleaving CCL-2 and CCL-12 chemokines, as well as CCL-13. Analysis of the proteolysed products identified unique cleavage sites in these chemokines. These cruzipain-modified chemokine products were tested in chemotaxis assays using monocytic cells. We found that cruzipain treated-CCL-2 maintained its biological activity, in contrast to the closely related CCL-12 and CCL-13 chemokines, which showed little or null agonist activity after treatment. Furthermore, based on this analysis, a 14-mer cruzipain-derived chemokine peptide (CDCP-1) was chemically synthesized and tested for agonist activity using in vitro chemotaxis assays. Interestingly, CDCP-1 showed antagonist activity affecting in vitro migration of monocytic cells and calcium flux release. In conclusion, our results demonstrate that cruzipain modulates biological functions of chemokines through proteolytic cleavage, by generating chemokine-derived peptides with antagonist activities. This event could play a role during the latest phases of Chagas disease, when the parasite may differentially modulate chemokine-mediated inflammatory responses.

CDIP-2, a synthetic peptide derived from chemokine (C-C motif) ligand 13 (CCL13), ameliorates allergic airway inflammation.

Airway inflammation is characterized by selective recruitment of mononuclear and granulocytic cells. This recruitment is mediated by the action of chemotactic cytokines, such as chemokines. A number of chemokines and their receptors have been identified and proposed as potential therapeutic agents in allergic airway inflammation. One of these chemokines is chemokine (C-C motif) ligand 13 (CCL13), a CC chemokine that has been associated with allergic inflammatory diseases such as asthma and allergic rhinitis. To investigate alternative therapeutic agents to alleviate allergic inflammatory diseases, a number of chemokine-derived synthetic peptides were designed and tested for their ability to modulate in vitro and in vivo chemokine-mediated functions. Our results show that one of these peptides, CDIP-2, displayed antagonist functions in in vitro chemotaxis assays using monocytic cell lines. In addition, we found that CDIP-2 significantly reduced peribronchial, perivascular infiltrate and mucus overproduction in an ovalbumin-induced allergic lung inflammation murine model. Thus, CDIP-2 may be considered as part of a novel group of anti-inflammatory agents based on chemokine-derived synthetic peptides.

The role of TGF-beta superfamily during T cell development: new insights.

Members of the transforming growth factor beta (TGF-beta) superfamily are soluble factors that regulate a variety of functional responses including proliferation, differentiation, apoptosis and cell cycle, among others, depending not only on the cell type and its differentiation state, but also on the milieu of cytokines present. All three members of this superfamily: TGF-betas, bone morphogenetic proteins (BMPs) and Activins, have been shown to be expressed in the thymus suggesting their potential role as regulators of the T lymphocyte differentiation process. Although initial reports described the role of TGF-beta in controlling specific checkpoints during thymocyte development, recent data has provided new evidence on the role of BMPs and Activins in this process. This review provides new insights on the function of members of the TGF-beta superfamily at different stages of thymocyte development.

Molecular signals involved in CD4 versus CD8 T cell commitment / Señales moleculares implicadas en la diferenciación cd4 versus CD8

During T cell development in the thymus, differentiation and selection processes take place to ensure the generation of a restricted self-tolerant T cell repertoire with the ability to recognize and eliminate foreign pathogens. Thymocytes that recognise self peptides on self MHC molecules with low avidity are rescued from programmed cell death and are positively selected, while those thymocytes recognising self peptide-MHC complexes with high avidity are deleted by negative selection. It is known that signals transduced through the T Cell Receptor are crucial for the survival and differentiation of immature thymocytes, during positive selection. One of the most important events during this selection process is the choice of a specific T cell lineage, to become a helper CD4 T cell or a cytotoxic CD8 T cell. The molecular events involved in this choice are still on debate. The initial models described to explain this selection process were known as the «instructive» and the «stochastic» models, both of which have found experimental evidence in favour and against. However, a «strength of signal» model, based on the intensity and/or duration of the TCR-MHC/peptide interaction has recently acquired more support. Finally we also describe a new model called “kinetic signalling” model which offers a new perspective of the mechanisms by which T cell commitment takes place in the thymus. In this review, we will also discuss the current knowledge about the molecular mechanisms involved in CD4 versus CD8 lineage commitment, including signalling and transcriptional pathways, which have been postulated to participate in this differentiation process (AU)

Durante el desarrollo de los linfocitos T se llevan a cabo distintos procesos de selección y diferenciación que aseguran la generación de un repertorio de linfocitos T restringido por MHC propio y auto-tolerante, capaz de reconocer y eliminar patógenos extraños. Los timocitos que reconocen con baja avidez péptidos propios son rescatados de la muerte celular programada y seleccionados positivamente, mientras que los timocitos que reconocen complejos péptido-MHC con alta avidez son eliminados mediante selección negativa. Se sabe que las señales moleculares transducidas a través del receptor para el antígeno de los linfocitos T (TCR), son cruciales para la supervivencia y diferenciación de los timocitos inmaduros durante la selección positiva. Uno de los eventos más importantes durante este proceso de selección es la elección de un linaje T específico, que diferenciará el timocito en un linfocito T CD4 cooperador, o bien CD8 citotóxico. Las señales moleculares implicadas en esta elección están todavía en debate. Inicialmente los modelos propuestos para explicar la elección de linaje, conocidos como los modelos «instructivo» y estocástico, encontraron evidencia experimental a favor y en contra. Recientemente ha adquirido mayor apoyo el modelo de la «fuerza de la señal» basado en la intensidad y/o duración de la interacción TCR-MHC/péptido. Por último, describimos un nuevo modelo «de la cinética de señalización» que aporta una nueva perspectiva sobre los mecanismos implicados en el proceso de elección de linaje que tiene lugar durante el desarrollo de los timocitos. Esta revisión discute el conocimiento actual sobre los mecanismos moleculares implicados en el compromiso hacia linaje CD4 versus CD8, incluyendo las vías de señalización y los factores transcripcionales que se ha postulado participan en dicho proceso de diferenciación (AU)

Entamoeba histolytica cysteine protease 2 (EhCP2) modulates leucocyte migration by proteolytic cleavage of chemokines.

Human amoebiasis is a disease produced by infection with the protozoan Entamoeba histolytica currently affecting many millions of people worldwide. Amoebic colitis is the most common clinical manifestation. Host protective immunity involves participation of both humoral and cellular responses. However, the mechanisms involved in immune evasion are not clear and remain under investigation. One of these mechanisms could be associated with the ability of parasite proteases to modulate or interfere with the inflammation process, which is initiated by expression of pro-inflammatory cytokines such as chemokines. To further clarify the potential role of cysteine proteases in modulating chemokine-mediated functions, we have analysed the ability of Entamoeba histolytica cysteine protease 2 (EhCP2) to have an effect on the chemotaxis of leucocytes by chemokine cleavage. We find that EhCP2 is capable of cleaving chemokines CCL2, CCL13 and CXCL8, and the resulting proteolysis products modulate the chemotaxis of leucocytes when compared to that induced by intact chemokine. Thus, the extracellular activity of the cysteine proteases affects chemokine-mediated responses and could be considered as part of the mechanisms used by Entamoeba histolytica to circumvent the host immune responses.

Papel de las quimiocinas en el desarrollo de los linfocitos T / Role of Chemokines in T cell development

Las quimocinas juegan un papel muy importante en el desarrollo tímico y la migración específica a órganos linfoides. Las quimiocinas son citocinas quimioatrayentes de bajo peso molecular y ejercen su función al interaccionar con receptores de siete dominios transmembranales asociados a proteínas G heterotriméricas. Como consecuencia de la unión de la quimiocina con su receptor se activa la vía Jak/Stat, que parece ser crucial para la generación de respuestas funcionales tales como la quimiotaxis y la movilización de calcio intracelular. La expresión diferencial de quimiocinas y sus receptores durante los distintos estadíos de diferenciación de los timocitos permite la migración dirigida de timocitos inmaduros desde la región subcapsular hasta la médula, pasando a través de la corteza. Además, existen evidencias de que las quimocinas también están implicadas en el reclutamiento de progenitores hasta el timo así como en la salida de los timocitos maduros hacia órganos linfoides secundarios. La generación de ratones deficientes para quimiocinas y/o sus receptores ha sugerido que ciertas quimiocinas pueden tener un papel redundante, mientras que otras dan lugar a un fenotipo muy definido, como el caso del receptor CXCR4 y su ligando CXCL12 cuya deficiencia resulta letal a nivel embrionario (AU)

Analysis of the individual role of the TCRzeta chain in transgenic mice after conditional activation with chemical inducers of dimerization.

Signaling through the TCR/CD3 complex plays a critical role in T-cell development and activation. Gene-targeted mice lacking particular components of this complex show arrested T-cell development in the thymus. As all TCR/CD3 components are required for efficient surface expression of the complex, it is difficult to assess the specific signaling role of each receptor component. To overcome this problem, we designed a strategy to examine the specific role(s) of individual receptor chains. A chimeric protein, containing binding domains for chemical inducers of dimerization fused to the cytoplasmic tail of TCRzeta, was generated. Activation of the chimeric receptor after stimulation with chemical dimerizers in Jurkat cells showed tyrosine phosphorylation of the TCRzeta chain chimera, recruitment of phosphorylated Zap70, and generation of NFAT in a reporter assay. Analysis of thymocytes from transgenic mice expressing this chimeric receptor showed that intracytoplasmic crosslinking of the chimera induced tyrosine phosphorylation of the protein, as well as a slow and very weak calcium mobilization response. However, this signaling did not lead to increased expression of activation markers, T-cell proliferation, or apoptosis. In addition, stimulation of thymocytes in suspension or in fetal thymic organ cultures with chemical inducers of dimerization did not lead to alterations in positive or negative selection. We conclude that signaling through the TCRzeta chain alone is not sufficient to generate downstream events leading to full T-cell activation or thymocyte selection; instead, additional CD3 components must be required to induce a functional response in primary thymocytes and peripheral T cells.

Breaking immunologic ignorance to an antigenic peptide of simian virus 40 large T antigen.

Expression of antigenic proteins in the periphery may result in immunologic tolerance, immunologic ignorance, or autoimmunity. It is not clear why some Ags induce tolerance, whereas others activate Ag-reactive T cells. The physical nature of the Ag, the developmental timing of Ag expression, the priming of reactive lymphocytes, and the level of Ag expression are possible factors determining the immunologic response to extra-thymic Ags. Expression of the whole SV40 large T Ag (SV40-T) induces transformation of T antigen-expressing cells in vivo, and this phenomenon has been postulated to be the triggering event that leads to autoimmunity in some transgenic mouse models. Here we present a model in which a nononcogenic, yet antigenic, fragment of the SV40-T (SV40-Tfrag) is expressed specifically in pancreatic islet beta-cells. In contrast to whole SV40-T transgenic mice, SV40-Tfrag mice that are also transgenic for a TCR specific for the SV40-T are ignorant of Ag in vivo. They do not respond in vivo to the tissue-specific SV40-Tfrag Ag even after priming, but are fully responsive in vitro. This immunologic ignorance cannot be broken after activated SV40-T reactive T cells are transferred into sublethally irradiated mice expressing the islet-specific SV40-Tfrag. However, similar adoptive transfer experiments in mice co-expressing B7-1 and SV40-Tfrag on islet cells specifically lead to Ag recognition and diabetes. This demonstrates that in some circumstances the presence of (primed) reactive T cells is not sufficient to break tolerance; rather, costimulation is additionally required to elicit an autoimmune response. This also suggests that SV40-T-induced cellular transformation is important for the autoimmune response directed against SV40-T in other tissue-specific transgenic models.

B7-1 expression by a non-antigen presenting cell-derived tumor.

The existence of a naturally occurring immunosurveillance against neoplastic cells is controversial. A difficulty with this concept is that tumor-specific antigen-reactive T cells would not be expected to become activated after encountering tumor cells, since T cells that bind to antigen in the absence of the costimulation provided by antigen-presenting cells may be inactivated. We studied a transgenic model of tumorigenesis where T cells reactive to a particular tumor-specific antigen are lost prior to the development of non-antigen-presenting cell-derived tumors; therefore, the tumors that develop are not subjected to immunosurveillance. We found that a tumor cell line derived from one such tumor expresses the T-cell costimulatory molecule B7-1, the expression of which is normally restricted to antigen-presenting cells. In addition, we found that several immortalized cell lines, which are nontumorigenic and thus have suffered only early genetic events in the tumorigenesis process, express B7. This suggests that a host cell can be induced to express surface B7-1 molecules after suffering an oncogenic insult, which might possibly be a primary mechanism of immunosurveillance against tumors.

The liver eliminates T cells undergoing antigen-triggered apoptosis in vivo.

Deletion of mature peripheral T cells may result from TCR ligation by bacterial enterotoxins, endogenous provirus-encoded superantigens, and peptide antigens. But the ultimate fate of deleted T cells is not clear. Using a line of T cell receptor transgenic mice injected with antigenic peptide, we have documented that peripheral deletion is accompanied by the induction of abortive T cell activation followed by the disappearance of transgene-positive T cells. As these T cells disappear from the lymph nodes and spleen, they accumulate in the liver, where they undergo apoptosis. This is likely to be a general clearance pathway for T cells that are programmed to undergo apoptosis in vivo, and it may further explain the expansion of the intrahepatic T cell pool in mice with genetic defects in the T cell apoptosis mechanism, such as the lpr mutant.

T cells are responsive to the simian virus 40 large tumor antigen transgenically expressed in pancreatic islets.

The qualities of a peripheral Ag that determine whether T cells will be tolerant of or responsive to it are poorly understood. To approach this problem, we studied the T cell response in a line of transgenic mice selectively expressing an oncoprotein in the islets of Langerhans. The SV40 large tumor Ag (SV40-T) is directed to islet beta-cells in Rip1-Tag3 (RT3) mice by a hybrid insulin promoter-SV40-T construct. Ag is first detected on these cells between 10 and 12 wk after birth. RT3 mice were bred with mice expressing a transgenic rearranged TCR recognizing SV40-T in the context of the class I MHC molecule, H-2Kk. T cell response in the resultant RT3/TCR-double transgenic mice was then analyzed. T cells are fully responsive to SV40-T in RT3/TCR-transgenic mice, and T cells infiltrate the islets of both RT3 and RT3/TCR-transgenic mice. This work demonstrates that T cells may remain responsive to self-Ag expressed outside the thymus, and that this responsiveness may result in autoimmunity. The developmentally delayed expression or the oncogenic nature of SV40-T in the RT3-transgenic mice may be important in determining this T cell response.

Reevaluation of autoantibodies to islet cell membrane in IDDM. Failure to detect islet cell surface antibodies using human islet cells as substrate.

Since their demonstration in 1975, ICSAs have been proposed as serological markers and pathogenic elements in IDDM. ICSAs are detected in the sera of most newly diagnosed IDDM patients by indirect IFL that uses viable preparations of rat islet or insulinoma cells as substrate, but they also can be detected by using human insulinoma or fetal islet cells. We have tried to demonstrate ICSAs in the sera of 31 newly diagnosed diabetic patients, including 6 positive samples on human fetal islet cells, which used their natural target for the first time: normal human islet cells. In spite of using different types of preparations of these cells (i.e., freshly dispersed cell suspensions, monolayer cultures, or dispersed islets after culture), ICSAs could not be detected by IFL under the UV microscope, nor by flow cytometry. In contrast, 9 of 29 of the sera gave a positive staining on the RIN rat insulinoma cells. In an attempt to establish whether the putative ICSA autoantigen is present in the surface of human islet cells in the diabetic pancreas, the insulitis microenvironment was emulated by exposing the islets to three types of stress: 1) cytokines (IFN-gamma and TNF-alpha); 2) heat shock; and 3) hyperglycemia. However, diabetic sera failed again to recognize membrane antigens on the islet cells after either of these treatments. Neither were islet cells from a newly diagnosed diabetic patient stained by its autologous serum (ICA titer > 80 JDF U). These results suggest that ICSA autoantigen is not expressed in the membrane of human islet cells and therefore raises doubts about their proposed pathogenic role.