Lymphocyte Islet Research Articles

Lymph node sharing between pancreas, gut, and liver leads to immune crosstalk and regulation of pancreatic autoimmunity

Lymph nodes (LNs) are critical sites for shaping tissue-specific adaptive immunity. However, the impact of LN sharing between multiple organs on such tailoring is less understood. Here, we describe the drainage hierarchy of the pancreas, liver, and the upper small intestine (duodenum) into three murine LNs. Migratory dendritic cells (migDCs), key in instructing adaptive immune outcome, exhibited stronger pro-inflammatory signatures when originating from the pancreas or liver than from the duodenum. Qualitatively different migDC mixing in each shared LN influenced pancreatic β-cell-reactive Tcells to acquire gut-homing and tolerogenic phenotypes proportional to duodenal co-drainage. However, duodenal viral infections rendered non-intestinal migDCs and β-cell-reactive Tcells more pro-inflammatory in all shared LNs, resulting in elevated pancreatic islet lymphocyte infiltration. Our study uncovers immune crosstalk through LN co-drainage as a powerful force regulating pancreatic autoimmunity.

Administration of metrnl delays the onset of diabetes in non-obese diabetic mice.

Type 1 diabetes is a chronic metabolic disease characterized by hyperglycemia due to progressive destruction of pancreatic beta cells via autoimmune attack. Meteorin-like protein (metrnl) is a secreted protein homologous to the neurotrophin metrn and it is induced after exercise in the skeletal muscle. In our paper published previously, we showed that the serum level of metrnl was significantly correlated with the lipid profile, glucose profile and insulin resistance. In this experiment, we asked whether intravenous administration of metrnl could delay the onset of diabetes in non-obese diabetic (NOD) mice. 4-week-old NOD mice were injected intravenously with metrnl. Blood glucose levels were measured weekly. Insulitis scoring, intraperitoneal glucose tolerance test, adoptive T cell transfer, flow cytometry analysis and real-time PCR were performed to investigate the underlying mechanism. The results showed that intravenous administration of metrnl delayed the onset of diabetes in NOD mice. Histology of pancreas showed a decreased infiltration of leukocytes, which was in association with augmentation of regulatory T cells, suppression of autoreactive T cells and altered cytokine secretion. To sum up, the present study showed that intravenous administration of metrnl ameliorated islet lymphocyte infiltration and modulated immune cell responses, raising the possibility that it might be beneficial in improving islet function clinically.

Lymphocytes in autoimmune-resistant mice may protect the islets through IL-10 production

Abstract We have identified T cells and B cells in the islets of autoimmune resistant mice, yet the role of these lymphocytes is unknown. In the islets of autoimmune resistant C57BL/6 mice, local antigen receptor signaling was assessed using Nur77-GFP mice. Nur77-GFP expression was increased in islet CD8+ T cells and B cells suggesting that these lymphocyte subsets receive local antigenic stimulus. Islet T cell and B cell populations expressed the regulatory cytokine IL-10, assessed using the B6.TIGER reporter mouse. T cell populations lacked expression of the pro-inflammatory cytokine IFN-γ. These findings indicate that islet T cell populations are likely playing a regulatory role. In addition, cultured whole islets produced IL-10 protein, but not IFN-γ protein. To test the stability of the islet lymphocyte populations under inflammatory conditions, mice were exposed to metabolic stress through a diet-induced obesity (DIO) model. Obesity and type 2 diabetes (T2D) are associated with circulating inflammatory cytokines. In T2D patients, islet lymphocytes have been identified, though the function of these populations is largely unknown. C57BL/6 mice were fed a high fat diet (DIO) or a matched control diet (Control) starting at 5 weeks of age, and then analyzed at 16–25 weeks of age. The number and phenotype of lymphocytes in the islets were similar in DIO and control mice. Notably, under the conditions of obesity-associated systemic inflammation, islet lymphocytes in the DIO mice maintained IL-10 production. From these data, we propose that islet lymphocytes play a regulatory role to protect the islets from systemic inflammation through IL-10 production.

Characterization of resident lymphocytes in human pancreatic islets.

The current view of type 1 diabetes (T1D) is that it is an immune-mediated disease where lymphocytes infiltrate the pancreatic islets, promote killing of beta cells and cause overt diabetes. Although tissue resident immune cells have been demonstrated in several organs, the composition of lymphocytes in human healthy pancreatic islets have been scarcely studied. Here we aimed to investigate the phenotype of immune cells associated with human islets of non-diabetic organ donors. A flow cytometry analysis of isolated islets from perfused pancreases (n = 38) was employed to identify alpha, beta, T, natural killer (NK) and B cells. Moreover, the expression of insulin and glucagon transcripts was evaluated by RNA sequencing. Up to 80% of the lymphocytes were CD3+ T cells with a remarkable bias towards CD8+ cells. Central memory and effector memory phenotypes dominated within the CD8+ and CD4+ T cells and most CD8+ T cells were positive for CD69 and up to 50-70% for CD103, both markers of resident memory cells. The frequency of B and NK cells was low in most islet preparations (12 and 3% of CD45+ cells, respectively), and the frequency of alpha and beta cells varied between donors and correlated clearly with insulin and glucagon mRNA expression. In conclusion, we demonstrated the predominance of canonical tissue resident memory CD8+ T cells associated with human islets. We believe that these results are important to understand more clearly the immunobiology of human islets and the disease-related phenotypes observed in diabetes.

Hyaluronan: A Mediator of Islet Dysfunction and Destruction in Diabetes?

Hyaluronan (HA) is an extracellular matrix (ECM) component that is present in mouse and human islet ECM. HA is localized in peri-islet and intra-islet regions adjacent to microvessels. HA normally exists in a high molecular weight form, which is anti-inflammatory. However, under inflammatory conditions, HA is degraded into fragments that are proinflammatory. HA accumulates in islets of human subjects with recent onset type 1 diabetes (T1D), and is associated with myeloid and lymphocytic islet infiltration, suggesting a possible role for HA in insulitis. A similar accumulation of HA, in amount and location, occurs in non-obese diabetic (NOD) and DORmO mouse models of T1D. Furthermore, HA accumulates in follicular germinal centers and in T-cell areas in lymph nodes and spleen in both human and mouse models of T1D, as compared with control tissues. Whether HA accumulates in islets in type 2 diabetes (T2D) or models thereof has not been previously described. Here we show evidence that HA accumulates in a mouse model of islet amyloid deposition, a well-known component of islet pathology in T2D. In summary, islet HA accumulation is a feature of both T1D and a model of T2D, and may represent a novel inflammatory mediator of islet pathology.

Maternal obesity exacerbates insulitis and type 1 diabetes in non-obese diabetic mice

Accompanying the dramatic increase in maternal obesity, the incidence of type 1 diabetes (T1D) in children is also rapidly increasing. The objective of this study was to explore the effects of maternal obesity on the incidence of T1D in offspring using non-obese diabetic (NOD) mice, a common model for TID. Four-week-old female NOD mice were fed either a control diet (10% energy from fat, CON) or a high-fat diet (60% energy from fat) for 8 weeks before mating. Mice were maintained in their respective diets during pregnancy and lactation. All offspring mice were fed the CON to 16 weeks. Female offspring (16-week-old) born to obese dams showed more severe islet lymphocyte infiltration (major manifestation of insulitis) (P<0.01), concomitant with elevated nuclear factor kappa-light-chain-enhancer of activated B cells p65 signaling (P<0.01) and tumor necrosis factor alpha protein level (P<0.05) in the pancreas. In addition, maternal obesity resulted in impaired (P<0.05) glucose tolerance and lower (P<0.05) serum insulin levels in offspring. In conclusion, maternal obesity resulted in exacerbated insulitis and inflammation in the pancreas of NOD offspring mice, providing a possible explanation for the increased incidence of T1D in children.

A stochastic mathematical model to study the autoimmune progression towards type 1 diabetes

The integrity of the interactions and the 3D architecture among beta cell populations in pancreatic islets is critical for proper biosynthesis, storage and release of insulin. The aim of this study was to evaluate the effect on electrophysiological signalling of beta cells that is produced by progressive lymphocytic islet cell infiltration (insulitis), by modelling the disruption of pancreatic islet anatomy as a consequence of insulitis and altered glucose concentrations. On the basis of histopathological images of murine islets from non-obese diabetic mice, we simulated the electrophysiological dynamics of a 3D cluster of mouse beta cells via a stochastic model. Progressive damage was modelled at different glucose concentrations, representing the different glycaemic states in the autoimmune progression towards type 1 diabetes. At 31% of dead beta cells (normoglycaemia) and 69% (hyperglycaemia), the system appeared to be biologically robust to maintain regular Ca(2+) ion oscillations guaranteeing an effective insulin release. Simulations at 84%, 94% and 98% grades (severe hyperglycemia) showed that intracellular calcium oscillations were absent. In such conditions, insulin pulsatility is not expected to occur. Our results suggest that the islet tissue is biophysically robust enough to compensate for high rates of beta cell loss. These predictions can be experimentally tested in vitro by quantifying space and time electrophysiological dynamics of animal islets kept at different glucose gradients. The model indicates the necessity of maintaining glycaemia within the physiological range as soon as possible after diabetes onset to avoid a dramatic interruption of Ca(2+) pulsatility and the consequent drop of insulin release.

Preparatory studies of composite mesenchymal stem cell islets for application in intraportal islet transplantation

Background. Low engraftment and adverse immune reactions hamper the success rate of clinical islet transplantation. In this study, we investigated the capacity of human mesenchymal stem cells (MSCs) to adhere to human islets of Langerhans and their effects in immune modulation and during blood interactions in vitro.Methods. Composite MSC–islets were formed by suspension co-culture, and the phenotype was evaluated by confocal microscopy. Islet function was assessed by dynamic insulin release in response to glucose in vitro. Mixed lymphocyte–islet reactions (MLIR) and the tubing blood loop model were utilized as in vitro tools to analyse the effect of MSCs on the innate and adaptive immune reactions triggered by the islets.Results. MSCs rapidly adhered to islets and spread out to cover the islet surface. Insulin expression and secretion were sustained with the MSC coating. MSC-coated islets showed unaffected reactions with blood in vitro in comparison to control islets. Furthermore, MSCs suppressed lymphocyte proliferation induced by islet cells in MLIR.Conclusion. We conclude that it is possible to create composite MSC–islets to enable delivery of the MSCs by utilizing the adhesive capacity of the MSCs. This could have beneficial immunosuppressive effects in optimizing pancreatic islet transplantation.

Islet lymphocyte subsets in male and female NOD mice are qualitatively similar but quantitatively distinct

Islet-infiltrating lymphocytes of individual male and female non-obese diabetic (NOD) mice were examined with the purpose of determining the differences that lead to a predominance of diabetes in female versus males NOD mice. When normalized for the amount of islet lymphocytes recovered, the infiltrating lymphocytes of female NOD mice were indistinguishable from those of male NOD mice. The only observed difference was that islet inflammation progressed at an increased rate in female compared to male NOD mice. There was no difference in the composition of islet infiltrates in male and female NOD mice. Unexpectedly, the ratio of CD4+:CD8+ T cells was tightly controlled in the islets throughout diabetogenesis. The frequency of IL-4+ CD4+ T cells started high but quickly fell to 3% of the population that was maintained with increasing inflammation. A significant portion of the CD8+ T cells were islet-specific glucose-6-phosphatase catalytic subunit-related protein specific in both male and female NOD mice and this population was antigen experienced and increased at high levels of islet inflammation. Surprisingly, a large pool of antigen inexperienced naïve T cells was detected in the islets. We conclude the underlying immunological processes in both male and female NOD mice are similar while the rates differ and the presence of naïve T cell in the islets may contribute to epitope spreading.

Evaluation of the effect of microencapsulation and immuno-modulation on islet immunogenicity in vitro.

The complexity of the transplantation model in microencapsulated islet transplantation suggests the use of an in vitro model for the analysis of the effect of encapsulation and graft pretreatment on islet immunogenicity. In this study, the mixed lymphocyte islet culture is applied to islets encapsulated in barium alginate beads, showing a significant reduction of the cellular response compared with non-encapsulated islets. Moreover, the effect of low-temperature culture as the immuno modulatory principle is shown on encapsulated and non-encapsulated islets. The data suggest that encapsulation reduces but not totally impedes the immunological interaction between the encapsulated tissue and the surrounding immune cells and that the stimulatory potency of the encapsulated islet may be modified by immuno-altering pretreatment.

Islet lymphocyte subsets in male and female NOD mice are qualitatively similar but quantitatively distinct

Islet lymphocyte subsets in male and female NOD mice are qualitatively similar but quantitatively distinct

Primer: immunity and autoimmunity.

For nonimmunologists, a daunting and rapidly evolving immunologic vocabulary, our incomplete understanding of both normal and abnormal immune function, and multiple interrelated complex immune cellular pathways can be a barrier to using basic immunology to understand and improve care for patients with type 1 diabetes. Our task in this review is to introduce current immune concepts specifically relevant to type 1 diabetes. Because our understanding is not complete, as evidenced perhaps by our lack of standard immunotherapy to prevent type 1 diabetes, we can only provide a partial framework. Perhaps the simplest framework (which may be wrong) is to consider the development of type 1 diabetes as the balance between regulatory and effector T lymphocytes. We know that in the absence of a major portion of regulatory T lymphocytes in a rare syndrome caused by mutation of the FOXP3 gene, most infants (even neonates) develop type 1 diabetes. Central to the development of type 1 diabetes are T lymphocytes with specific T-cell receptors that recognize islet molecules. When a T-cell is activated through its receptor, it can orchestrate protection from infection or autoimmunity, depending on the target. Other T-cells can suppress or enhance autoimmunity (either in general or only for T lymphocytes in islets). The activation of a T-cell involves multiple different cell types and genes, as we will discuss. The modern era of immunology began with the clonal selection theory independently expressed by David W. Talmage and Sir Frank Macfarlane Burnet (1,2). The clonal selection theory postulates that a foreign antigen entering the body binds to one unique antibody selected from an unlimited repertoire of antibodies formed early in the organism's life. This explains how the immune system is able to recognize and respond to a virtually inestimable number of foreign antigens. The immune system is a …

Rotavirus Infection Accelerates Type 1 Diabetes in Mice with Established Insulitis

Infection modulates type 1 diabetes, a common autoimmune disease characterized by the destruction of insulin-producing islet beta cells in the pancreas. Childhood rotavirus infections have been associated with exacerbations in islet autoimmunity. Nonobese diabetic (NOD) mice develop lymphocytic islet infiltration (insulitis) and then clinical diabetes, whereas NOD8.3 TCR mice, transgenic for a T-cell receptor (TCR) specific for an important islet autoantigen, show more rapid diabetes onset. Oral infection of infant NOD mice with the monkey rotavirus strain RRV delays diabetes development. Here, the effect of RRV infection on diabetes development once insulitis is established was determined. NOD and NOD8.3 TCR mice were inoculated with RRV aged > or = 12 and 5 weeks, respectively. Diabetes onset was significantly accelerated in both models (P < 0.024), although RRV infection was asymptomatic and confined to the intestine. The degree of diabetes acceleration was related to the serum antibody titer to RRV. RRV-infected NOD mice showed a possible trend toward increased insulitis development. Infected males showed increased CD8(+) T-cell proportions in islets. Levels of beta-cell major histocompatibility complex class I expression and islet tumor necrosis factor alpha mRNA were elevated in at least one model. NOD mouse exposure to mouse rotavirus in a natural experiment also accelerated diabetes. Thus, rotavirus infection after beta-cell autoimmunity is established affects insulitis and exacerbates diabetes. A possible mechanism involves increased exposure of beta cells to immune recognition and activation of autoreactive T cells by proinflammatory cytokines. The timing of infection relative to mouse age and degree of insulitis determines whether diabetes onset is delayed, unaltered, or accelerated.

Ingested (Oral) SIRS Peptide 1-21 Suppresses Type 1 Diabetes in NOD Mice

Type 1 diabetes (T1D) is a chronic disorder that results from autoimmune destruction of the insulin-producing pancreatic beta cell. The nonobese diabetic (NOD) mouse is a model of the human autoimmune disease T1D. Soluble immune response suppressor (SIRS) is a nonspecific protein suppressor of immune response produced by immunomodulatory T cells stimulated by type I interferon (IFN). SIRS inhibits antibody responses in vivo, lipopolysaccharide (LPS)-induced fever, and delayed-type hypersensitivity (DTH) responses. Previous investigators have isolated the N-terminal sequence of SIRS protein consisting of 21 amino acids. Mice ingesting 1 microg SIRS peptide 1-21 showed significant delayed onset of T1D and a decreased frequency of T1D compared with mock-fed and 10-microg-fed mice and a significant decrease in islet inflammation. There were significant decreases in islet lymphocyte chemokine production of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein-1 gamma (MIP-1 gamma), regulated upon activation, normal T cell-expressed, and presumably secreted (RANTES), and stromal cell-derived factor-1 (SDF-1) in the SIRS-fed mice, factors important in migration of inflammatory cell into the islets. Ingested (oral) SIRS peptide inhibits clinical T1D by decreasing target organ cellular migration of islet destructive populations by suppression of islet lymphocyte chemokine secretion.

Virally induced inflammation triggers fratricide of Fas-ligand-expressing beta-cells.

Tissue-specific expression of Fas-ligand (Fas-L) can provide immune privilege by inducing apoptosis of "invading" lymphocytes expressing Fas. However, accelerated diabetes has been reported in transgenic mice expressing Fas-L in islets (RIP-Fas-L) as a result of Fas-dependent fratricide of beta-cells after transfer of diabetogenic clones. Here we studied whether Fas-L could protect islets from autoaggressive CD8 lymphocytes in a transgenic model of virally induced diabetes (RIP-LCMV-NP transgenic mice), in which the autoaggressive response is directed to a viral nucleoprotein (NP) expressed as a transgene in beta-cells. Indeed, disease incidence after viral (lymphocytic choriomeningitis virus [LCMV]) infection was reduced by approximately 30%, which was associated with a decrease of autoaggressive CD8 NP-specific lymphocytes in islets and pancreatic draining lymph nodes. However, surprisingly, a high degree (50%) of diabetes was seen in mice that expressed only Fas-L but not the viral transgene (NP) in beta-cells after infection with LCMV. This was due to induction of Fas on beta-cells after LCMV infection of the pancreas, resulting in Fas/Fas-L-mediated fratricide. Thus, although Fas-L can lend some immune privilege to islet cells, local virus-induced inflammation will induce Fas on beta-cells, leading to their mutual destruction if Fas-L is present. Expression of Fas-L therefore might not be protective in situations in which viral inflammation can be expected, resulting in Fas induction on the targeted cell itself.

Removal of CD45+ cells from human fetal pancreas alters immunogenicity in vitro

Human fetal pancreas (HFP) is a potential source of islets for the treatment of diabetes mellitus with the potential for growth and differentiation after transplantation. However, because of the small mass of a given HFP, multiple donors would be required for transplantation, thereby increasing the immunological challenge to the recipient. In this study, we investigate the contribution of hematopoietic cells to the immunogenicity of HFP. Single cell suspensions of HFP were depleted of CD45+ cells using antibody-conjugated magnetic beads. In vitro mixed lymphocyte islet cultures were established using with CD45-depleted or nondepleted HFP. Depletion of CD45+ cells resulted in the low levels of IFNγ production at early time points (day 4), which increased to near normal levels by day 7. The development of donor-specific CTL was not consistently inhibited by CD45 cell depletion. The data suggests that CD45+ cells within HFP are capable of stimulating immune responses by the direct pathway of antigen presentation, but that the indirect pathway is also involved in the development of CTL. The inhibition of early IFNγ release, however, may be beneficial for the survival of transplanted islets. Therefore, the combination of CD45 depletion strategies with standard immunosuppressive drug therapies could result in better long-term survival of transplanted islets.

A Dual Role for TNF-α in Type 1 Diabetes: Islet-Specific Expression Abrogates the Ongoing Autoimmune Process When Induced Late but Not Early During Pathogenesis

We report here that islet-specific expression of TNF-alpha can play a dual role in autoimmune diabetes, depending on its precise timing in relation to the ongoing autoimmune process. In a transgenic model (rat insulin promoter-lymphocytic choriomeningitis virus) of virally induced diabetes, TNF-alpha enhanced disease incidence when induced through an islet-specific tetracycline-dependent promoter system early during pathogenesis. Blockade of TNF-alpha during this phase prevented diabetes completely, suggesting its pathogenetic importance early in disease development. In contrast, TNF-alpha expression abrogated the autoimmune process when induced late, which was associated with a reduction of autoreactive CD8 lymphocytes in islets and their lytic activities. Thus, the fine-tuned kinetics of an autoreactive process undergo distinct stages that respond in a differential way to the presence of TNF-alpha. This observation has importance for understanding the complex role of inflammatory cytokines in autoimmunity.

Low dose streptozotocin-induced diabetes in rat insulin promoter-mCD80-transgenic mice is T cell autoantigen-specific and CD28 dependent.

Although transgenic mice expressing murine B7-1 (mCD80) on their pancreatic beta cells under the rat insulin-1 promoter (RIP-mCD80(+) mice) rarely develop spontaneous beta cell destruction and diabetes, we have previously reported the transgene-dependent induction of profound insulitis and lethal diabetes following multiple low dose injections of the beta cell toxin streptozotocin (MLDS) in RIP-mCD80(+) mice. Here, we have further characterized this MLDS-induced diabetes model using the RIP-mCD80(+) mice and now demonstrate that disease is critically dependent on T cell signaling via CD28. Thus, although naive RIP-mCD80(+) and nontransgenic littermates have comparable gross beta cell mass, and immediately following MLDS induction the mice display similar degrees of insulitis and decrements in the beta cell mass, only transgenic mice continued to destroy their beta cells and develop insulin-dependent diabetes mellitus. Strikingly, MLDS-induced diabetes was completely prevented in CD28-deficient mice (RIP-mCD80(+)CD28(-/-)) due to abrogation of leukocytes infiltrating their pancreatic islets. We further characterized MLDS-induced diabetes in the RIP-mCD80(+) mice by demonstrating that the MLDS-induced lymphocytic islet infiltrate contained a substantial frequency of autoantigen-specific, IFN-gamma-secreting, CD8(+) T cells. We conclude that MLDS-induced beta cell destruction and subsequent insulin-dependent diabetes mellitus in RIP-mCD80(+) mice is T cell-mediated as it involves both Ag-specific recognition of self-target molecules in the inflamed pancreatic islet (signal 1) and is CD28 costimulation dependent (signal 2).

Recognition of islet-directed peripheral blood lymphocytes in vitro before rejection of allogeneic grafted islets.

A co-culture of splenic lymphocytes with allogeneic pancreatic islets [i.e., mixed lymphocyte islet co-culture (MLIC)] for 96 hr leads to reduction of beta-cells and to an allospecific induction of major histocompatibility complex (MHC) class II antigens on beta-cells. The intent of our investigation was to determine whether peripheral blood lymphocytes (PBL) obtained from allogeneic islet-grafted BB/OK rats (=sensitization in vivo) cause similar alterations to donor-specific islet cells. PBL prepared before transplantation, before (at day 7) and after islet rejection were co-cultured for 24 hr with donor-specific islets. PBL obtained at any time before and after transplantation caused reduction of beta-cells and enhancement of intercellular adhesion molecule-1(+)/beta-cells. Induction of MHC class II+ beta-cells was most pronounced with PBL obtained before rejection. Down-regulation of major histocompatibility complex class I+ beta-cells was caused by PBL that had been obtained from grafted animals only; it was most pronounced before islet rejection and has never been observed with lymphocytes from nongrafted normoglycemic rats. The 24-hr MLIC is capable of recognizing functionally active, donor-specific lymphocytes and is able to distinguish between the effects of sensitized and nonsensitized lymphocytes.

Effect of interleukin-10 on human anti-porcine xenogeneic cellular response in vitro.

Pigs are being used as an alternative source of tissues for humans and we are interested in the xenotransplantation of fetal pig islet-like cell clusters (ICC) into type 1 diabetic patients. Interleukin-(IL) 10 is a Th2 cytokine with immunosuppressive properties that down-regulate the cell-mediated response. In this study, we evaluated the effects of recombinant human IL-10 on human anti-pig xenogeneic cellular response in mixed lymphocyte culture (MLC) and in mixed islet lymphocyte culture (MILC). Human peripheral blood mononuclear cells as responder cells were cultured in one-way MLC with pig and human peripheral blood mononuclear cells as stimulant cells in xeno and allo-MLC, respectively, and also with fetal pig ICCs in MILC. IL-10 was added at the time of culture. The addition of IL-10 significantly inhibited the xeno-MLC (human anti-pig) in a dose-dependent manner, the percentage inhibition being 36, 60, and 73% at 1, 10, and 50 ng/ml, respectively. Inhibition in xeno-MLC was significantly lower than that of the allo-MLC (human anti-human) at all concentrations used, the percentage inhibition of the latter being 58, 84, and 92% at 1, 10, and 50 ng/ml, respectively. Further, the addition of IL-10 also significantly inhibited the proliferation of human peripheral blood mononuclear cells when they were cocultured with fetal pig ICCs, the inhibition being 59, 72, and 80% at 1, 10, and 50 ng/ml, respectively. IL-10 was not toxic to ICCs as determined by 3H-thymidine incorporation over 5 days culture. Preincubation of IL-10 with the pig stimulant cells or the human responder cells did not confer additional benefit in the inhibition of xeno-MLC. IL-10 needs to be present at the start or at an early stage (within 4 hr) in the xeno-MLC because if the addition of IL-10 was delayed by 4 hr, the effect was lost. Next, the production of cytokines was examined in MLC and MILC. In xeno-MLC, levels (pg/ml) of tumor necrosis factor-alpha (TNF-alpha) (163+/-17), interferon-gamma (IFN-gamma) (278+/-60), IL-5 (24+/-10), IL-6 (2959+/-923), and IL-10 (17+/-2) were produced in greater amounts than autologous controls (P<0.05). The levels of TNF-alpha, IFN-gamma, IL-6, and IL-10 but not IL-5 were significantly (P<0.05) lower in xeno-MLC than those produced in allo-MLC. All of these cytokines were also produced in MILC when human peripheral blood mononuclear cells (PBMC) were cocultured with ICCs, levels (pg/ml) being TNF-alpha (308+/-47), IFN-gamma (93+/-17), IL-5 (6.2+/-3), IL-6 (5649+/-421), and IL-10 (122+/-18). No detectable levels of IL-2 and IL-4 were produced in the MLC and in MILC. Addition of IL-10 significantly inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 76, 96, 100, and 93%, respectively, in xeno-MLC. Addition of IL-10 also significantly (P<0.05) inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 88, 91, 100, and 96%, respectively, in MILC. Exogenous addition of IL-2 was partially able to reverse the effect of IL-10 although addition of TNF-alpha had no effect on xeno and allo-MLC. Synergism was seen between IL-10 and cyclosporine in the inhibition of xeno and allo-MLC. Taken together, the results demonstrated that IL-10 has an immunomodulatory role to play in the inhibition of cellular immune responses associated with the xenotransplantation of fetal pig ICCs.