Extreme Beta-Cell Deficiency in Pancreata of Dogs with Canine Diabetes

The pathophysiology of canine diabetes remains poorly understood, in part due to enigmatic clinical features and the lack of detailed histopathology studies. Canine diabetes, similar to human type 1 diabetes, is frequently associated with diabetic ketoacidosis at onset or after insulin omission. However, notable differences exist. Whereas human type 1 diabetes often occurs in children, canine diabetes is typically described in middle age to elderly dogs. Many competing theories have been proposed regarding the underlying cause of canine diabetes, from pancreatic atrophy to chronic pancreatitis to autoimmune mediated β-cell destruction. It remains unclear to what extent β-cell loss contributes to canine diabetes, as precise quantifications of islet morphometry have not been performed. We used high-throughput microscopy and automated image processing to characterize islet histology in a large collection of pancreata of diabetic dogs. Diabetic pancreata displayed a profound reduction in β-cells and islet endocrine cells. Unlike humans, canine non-diabetic islets are largely comprised of β-cells. Very few β-cells remained in islets of diabetic dogs, even in pancreata from new onset cases. Similarly, total islet endocrine cell number was sharply reduced in diabetic dogs. No compensatory proliferation or lymphocyte infiltration was detected. The majority of pancreata had no evidence of pancreatitis. Thus, canine diabetes is associated with extreme β-cell deficiency in both new and longstanding disease. The β-cell predominant composition of canine islets and the near-total absence of β-cells in new onset elderly diabetic dogs strongly implies that similar to human type 1 diabetes, β-cell loss underlies the pathophysiology of canine diabetes.     

Induction of diabetes with signs of autoimmunity in primates by the injection of multiple-low-dose streptozotocin

Aim

To develop a preclinical large animal model of autoimmune diabetes to facilitate the translational research of autoimmune diabetes in human.

Materials and methods

Nine young rhesus monkeys received multiple-low-dose (MLD) intravenous injections of streptozotocin for five consecutive days, followed by two additional boosting injections of STZ given 1 week apart. The induction of autoimmune diabetes was evaluated by regular metabolic testing, serological assessment of islet-reactive autoantibodies and histological examination of pancreatic tissues.

Results

Seven of nine treated animals became diabetic with moderate hyperglycemia initially and more severe hyperglycemia thereafter. All diabetic animals exhibited severely impaired glucose tolerance, limited islet function, and required insulin therapy to maintain relatively normal glucose metabolism and healthy status. Serological tests showed that all diabetic monkeys developed autoantibodies specifically against insulin and islet antigens. Furthermore, histological examination of the pancreata from diabetic animals revealed evidence of specific destruction of islet β cells and islets infiltrated with T lymphocytes. Overt and persistent diabetes can be induced in young rhesus monkeys by the injection of MLD-STZ, and autoimmune responses to pancreatic islet cells seem to be involved in the development of glucose intolerance and diabetes.

Conclusion

These data indicate for the first time that autoimmune diabetes can be induced in primates; this may serve as a valuable preclinical model for studying the pathogenesis of and potential therapies for autoimmune diabetes in humans.     

T cell response to preproinsulin I and II in the nonobese diabetic mouse

Immunization against insulin, insulin B chain, or B chain peptide B(9-23) (preproinsulin peptide II(33-47)) prevents diabetes in the nonobese diabetic (NOD) mouse. Whether or not peptide II(33-47) is the only proinsulin determinant recognized by CD4 T cells remains unclear. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four proinsulin epitopes within the islet cell infiltrate of prediabetic female NOD mice. These epitopes were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice with individual peptides in CFA. Immunogenic epitopes were found on both isoforms of insulin, especially proinsulin II, which is the isoform expressed in the thymus. The autoimmune response to proinsulin represented only part of the immune response to islet cells within the islet cell infiltrate in 15-wk-old NOD mice. This is the first systematic study of preproinsulin T cell epitopes in the NOD mouse model.     

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell–tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c⁺ expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.     

Immunohistochemical Demonstration of Leptin in Pancreatic Islets of Non-Obese Diabetic and CD-1 mice: Co-localization in Glucagon Cells and its Attenuation at the Onset of Diabetes

Leptin is a 16 kD polypeptide hormone produced predominantly by white adipose tissue and exerts profound effects on food intake and energy balance. More recent studies have shown extra sites of leptin production in human and rodent tissues and have ascribed additional roles for the hormone, e.g., in immune and reproductive functions. A role for the hormone has also been implicated in insulin-dependent diabetes mellitus in the non-obese diabetic (NOD) mouse. However, whether leptin originates from islet cells of the mouse is not known. Here dual-label immunohistochemistry was employed to examine leptin expression in islet cells, and its distribution and cellular sources in pancreatic sections of female NOD/Ak and CD-1 mice of various ages. For comparison, leptin immunolabelling was examined in adult pancreatic sections from male NOD/Ak CD-1, Balb/c and FVB/N mice and female severe combined immunodeficient CB. 17 mice. Pancreatic tissues from adult female guinea pig, sheep and cattle and neonatal pigs were also studied. Our results show that in the day 1 NOD and CD-1 mice, leptin immunolabelling was observed in selective glucagon cells within the developing islets while at days 15 and 22, it became more intense and co-incident. This pattern of staining was maintained at days 40, 90, 150 and 250. In the female NOD mouse, leptin was absent in intra-islet immune cells. Its expression was variable in islets from male NOD and CD-1 mice. In spontaneously diabetic female NOD mice and following acceleration of diabetes with cyclophosphamide, despite the persistence of strong immunolabelling for glucagon in the re-distributed alpha cells, leptin expression was either absent, diminished or present in only a proportion of alpha cells. The reduction in leptin labelling was often associated with diabetic islets which had insulitis in association with only a small number of residual beta cells. Leptin expression was absent in guinea pig, ovine, bovine and neonatal porcine islet cells, despite the expression of intensely labelled glucagon cells. The present results demonstrate leptin co-localization in glucagon cells of the mouse islet. Its expression diminishes in the presence of inadequate insulin. Leptin produced within the mouse islet may have bi-directional influences on leptin and insulin regulation and may play local functions in islet development and metabolism.     

The natural killer T-cell ligand alpha-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. V alpha 14-J alpha 15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of V alpha 14 NKT cells by alpha-galactosylceramide (alpha-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that alpha-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of alpha-GalCer to suppress interferon-gamma but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because alpha-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that alpha-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.     

Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing β cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2rγ^null mice. The selective destruction of pancreatic islet β cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total β-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the β cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet β cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4⁺ T cell infiltration and clonal expansion, and the mouse islet β-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet β cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.     

Importance of persistent cellular and humoral immune changes before diabetes develops: prospective study of identical twins.

OBJECTIVES--To determine the pattern of cellular and humoral immune changes associated with insulin dependent diabetes before diabetes develops. DESIGN--Prospective study over 10 years of 25 non-diabetic identical twins of patients with insulin dependent diabetes. The non-diabetic twins were followed up either till they developed diabetes or to the end of the study. SETTING--Teaching hospital. SUBJECTS--25 non-diabetic identical cotwins of patients with diabetes; 46 controls of the same sex and similar age tested over the same period. Of the 25 twins (total follow up 144 patient years), 10 developed diabetes (prediabetic twins); the remainder were followed up for a mean of 7.7 years. MAIN OUTCOME MEASURES--Results of glucose tolerance tests or fasting blood glucose concentrations at each sample point. Measurements of activated T lymphocytes, expressing the HLA-DR antigen, islet cell antibodies, and insulin autoantibodies in samples. RESULTS--All 10 prediabetic twins had both cellular and humoral changes initially and in most samples before diabetes was diagnosed (activated T lymphocytes in 39/40, islet cell antibodies in 45/47, and insulin autoantibodies to islet cells and insulin were detected infrequently (in 8/54, 6/69, and 0/69 samples, respectively). The combination of cellular and humoral (islet cell antibodies or insulin autoantibodies) immune changes were detected in all 10 of the prediabetic twins but in only one of the 15 non-diabetic twins (P < 0.001). The positive predictive value in this cohort of increased percentages of activated T cells and the presence of antibodies to islet cells or insulin on two consecutive occasions was 100%. CONCLUSION--Most of the twins had cellular or humoral immune changes at some stage. A combination of cellular and humoral immune changes and their tendency to persist is highly predictive of insulin dependent diabetes and distinguishes twins who develop diabetes from those who do not.     

Branched-chain amino acid-enriched diet: effects on insulin secretion and cellular immune aggression

Several reports have demonstrated that high-protein diets may have beneficial effects on experimental models of diabetes and have raised the possibility that branched-chain amino acids could play a role in these protective effects. We investigated the effect of a normoproteic, branched-chain amino acid-enriched diet (experimental diet) on insulin secretion from C57BL/6N mice transferred with splenocytes from diabetic syngeneic donors. Mice previously fed with the experimental or control diet received three intraperitoneal injections, every other day, of 5 x 107 viable mononuclear splenocytes obtained from control or diabetic donors. Results showed that mice fed with the experimental diet and transferred with "diabetic" splenocytes presented: i) normoglycemia, and (ii) significantly higher levels in both phases of glucose-induced insulin secretion and normal values of arginine-glucose-induced insulin secretion. To evaluate the in vitro cellular immune aggression, dispersed mouse islet cells were co-cultured with splenocytes from syngeneic diabetic mice. First, dispersed islet cells from mice on the experimental or control diet were co-cultured with splenocytes from control or diabetic mice on a commercial diet. In the presence of "diabetic splenocytes, dispersed islet cells from mice on the experimental diet presented a significantly lower in vitro cellular immune aggression. On the other hand, "diabetic" splenocytes from mice fed with the experimental diet produced a significantly reduced cellular immune aggression on dispersed islet cells. Our results showed that feeding branched-chain amino acids increased the capacity of beta cells to withstand a functional assault and diminished the extent of in vitro cellular immune aggression.     

Islet Autoimmunity Identifies a Unique Pattern of Impaired Pancreatic Beta-Cell Function,Markedly Reduced Pancreatic Beta Cell Mass and Insulin Resistance in Clinically Diagnosed Type 2 Diabetes

There is a paucity of literature describing metabolic and histological data in adult-onset autoimmune diabetes. This subgroup of diabetes mellitus affects at least 5% of clinically diagnosed type 2 diabetic patients (T2DM) and it is termed Latent Autoimmune Diabetes in Adults (LADA). We evaluated indexes of insulin secretion, metabolic assessment, and pancreatic pathology in clinically diagnosed T2DM patients with and without the presence of humoral islet autoimmunity (Ab). A total of 18 patients with at least 5-year duration of clinically diagnosed T2DM were evaluated in this study. In those subjects we assessed acute insulin responses to arginine, a glucose clamp study, whole-body fat mass and fat-free mass. We have also analyzed the pancreatic pathology of 15 T2DM and 43 control cadaveric donors, using pancreatic tissue obtained from all the T2DM organ donors available from the nPOD network through December 31, 2013. The presence of islet Ab correlated with severely impaired β-cell function as demonstrated by remarkably low acute insulin response to arginine (AIR) when compared to that of the Ab negative group. Glucose clamp studies indicated that both Ab positive and Ab negative patients exhibited peripheral insulin resistance in a similar fashion. Pathology data from T2DM donors with Ab or the autoimmune diabetes associated DR3/DR4 allelic class II combination showed reduction in beta cell mass as well as presence of autoimmune-associated pattern A pathology in subjects with either islet autoantibodies or the DR3/DR4 genotype. In conclusion, we provide compelling evidence indicating that islet Ab positive long-term T2DM patients exhibit profound impairment of insulin secretion as well as reduced beta cell mass seemingly determined by an immune-mediated injury of pancreatic β-cells. Deciphering the mechanisms underlying beta cell destruction in this subset of diabetic patients may lead to the development of novel immunologic therapies aimed at halting the disease progression in its early stage.     

Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.     

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined ¹. This is particularly true in autoimmune diseases and cancer². Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes ^1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays ⁶. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.     

Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.

Type 1 diabetes is an autoimmune disease in which the insulin-producing pancreatic beta cells are destroyed at an early age by an immune process that involves both CD4 and CD8 T lymphocytes. The identification of autoantigens in diabetes is very important for the design of antigen-specific immunotherapy. By screening a pancreatic islet cDNA library, we have identified the autoantigen recognized by highly pathogenic CD8 T cells in the non-obese diabetic mouse, one of the best animal models for human diabetes. This is the first identification, to our knowledge, of a CD8 T-cell epitope in an autoimmune disease. The peptide recognized by the cells is in the same region of the insulin B chain as the epitope recognized by previously isolated pathogenic CD4 T cells. This has very important implications for the potential use of insulin in preventative therapy.     

Autoimmunity to both proinsulin and IGRP is required for diabetes in nonobese diabetic 8.3 TCR transgenic mice

T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8(+) T cells specific for IGRP(206-214) (NOD8.3 mice) develop accelerated diabetes that requires CD4(+) T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP(206-214)-specific CD8(+) T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP(206-214)-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4(+) T cell help to activate IGRP(206-214)-specific T cells.     

Secreted antigens of the amastigote and promastigote forms of Leishmania infantum inducing a humoral response in humans and dogs.

To study the antigens secreted by promastigote and amastigote forms of Leishmania infantum which are able to induce a humoral response in human patients and dogs, we have carried out immunoprecipitation assays with different supernatants of in vitro cultured parasites, metabolically labelled with [35S]methionine, using serum samples from human patients and dogs. In addition, some metabolic labelling experiments were performed daily during the in vitro culture parasite's life cycle to follow the time course excretion-secretion of parasitic antigens. The results demonstrated that the two different hosts developed an antibody response against secreted antigens of both stages of Leishmania infantum. Nevertheless, the humoral response directed against the excreted-secreted antigens of the promastigote forms was qualitatively and quantitatively different when we compare the human and the dog immune responses. On the other hand, when the excreted-secreted antigens of the amastigote forms are immunoprecipitated with either human or canine immune serum, the humoral response is similar. In addition, the time course study showed that excretion-secretion of antigens was qualitatively and quantitatively modulated during the parasitic in vitro life cycle.     

Presence of residual beta cells and co-existing islet autoimmunity in the NOD mouse during longstanding diabetes: a combined histochemical and immunohistochemical study

During type 1 diabetes, most beta cells die by immune processes. However, the precise fate and characteristics of beta cells and islet autoimmunity after onset are unclear. Here, the extent of beta cell survival was determined in the non-obese diabetic (NOD) mouse during increasing duration of disease and correlated with insulitis. Pancreata from female NOD mice at diagnosis and at 1, 2, 3 and 4 weeks thereafter were analysed immunohistochemically for insulin, glucagon and somatostatin cells and glucose transporter-2 (glut2) and correlated with the degree of insulitis and islet immune cell phenotypes. Insulitis, although variable, persisted after diabetes and declined with increasing duration of disease. During this period, beta cells also declined sharply whereas glucagon and somatostatin cells increased, with occasional islet cells co-expressing insulin and glucagon. Glut2 was absent in insulin-containing cells from 1 week onwards. CD4 and CD8 T cells and macrophages persisted until 4 weeks, in islets with residual beta cells or extensive insulitis. We conclude that after diabetes onset, some beta cells survive for extended periods, with continuing autoimmunity and expansion of glucagon and somatostatin cells. The absence of glut2 in several insulin-positive cells suggests that some beta cells may be unresponsive to glucose.     

Differential expression and localization of neuropeptide Y peptide in pancreatic islet of diabetic and high fat fed rats

Neuropeptide Y (NPY) inhibits insulin secretion. Increased numbers of pancreatic islet cells expressing NPY have been observed in type 1 diabetic rats. To understand the functional significance of NPY expression in islet cells, we investigated the effects of high fat feeding and diabetic conditions on the expression and location of NPY expressing cells in normal and diabetic rats. Twenty rats were maintained on either normal chow (ND) or a high fat dietary regimen (HFD) for 4 weeks. In half of each group, type 1 or type 2 diabetes (groups T1DM and T2DM, respectively) was induced by injection of streptozotocin. At 8 weeks rats were euthanized and the pancreases were processed for immunofluorescence labeling (NPY/insulin, NPY/glucagon, NPY/somatostatin, and NPY/pancreatic polypeptide). Compared with the ND group, HFD rats had significantly fewer alpha cells, but beta cells were similar, while T1DM and T2DM rats showed significant increases in the proportions of alpha, delta, and PP cells. Robust increases in NPY-positive islet cells were found in the HFD, T1DM, and T2DM rats compared with ND controls. In ND rats, 99.7% of the NPY-positive cells were PP cells. However, high fat feeding and diabetes resulted in significant increases in NPY-positive delta cells, with concomitant decreases in NPY-positive PP cells. In summary, high-fat feeding and diabetes resulted in changes in the hormonal composition of pancreatic islet and increased number of NPY-expressing islet cells. Under diabetic conditions NPY expression switched from predominantly a characteristic of PP cells to predominantly that of delta cells. This may be a factor in reduced pancreatic hormone secretion during diabetes.     

Characterization of porcine T lymphocytes and their immune response against viral antigens.

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4+CD8- T helper cells and CD4-CD8+ cytolytic T lymphocytes, extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-CD4-CD8- T cell receptor (TcR)-gamma delta+ T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4+CD8- and CD4+CD8+. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4+CD8+ T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4-CD8+ T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6- cells which showed spontaneous cytolytic activity and CD6+ MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.