Immunology of Diabetes Society T-Cell Workshop: HLA class II tetramer-directed epitope validation initiative.

BACKGROUND: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study. METHODS: A tetramer-based assay was used in five participating centres to measure T-cell reactivities to DR0401-restricted epitopes. Three participating centres concurrently performed ELISPOT or immunoblot assays. Each centre used blind-coded, centrally distributed peptide and tetramer reagents. RESULTS: All participating centres detected responses to auto-antigens and the positive control antigen, and in some cases cloned the corresponding T cells. However, response rates varied among centres. In total, 74% of patients were positive for at least one islet epitope. The most commonly recognized epitope was GAD270-285. Only a minority of the patients tested by tetramer and ELISPOT were concordant for both assays. CONCLUSIONS: This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course.

Lymph node T-cells do not optimally transfer diabetes in NOD mice.

The nonobese diabetic mouse in a model of spontaneous development of autoimmune type I diabetes. The disease can be induced in young, irradiated recipients by injecting splenic T-cells from diabetic donors. The adoptive transfer of diabetes requires the presence of both CD4+ and CD8+ splenic T-cell subsets. To test whether diabetogenic cells distribute in other lymphoid organs of diabetic mice, we first analyzed lymph node cells. Lymph node cells were much less efficient in transferring diabetes than splenocytes. This inefficacious transfer was not attributable to the absence of hematopoietic precursors or a lack of macrophages. Lymph node cells did not protect from the transfer of diabetes by splenocytes, indicating the absence of suppressor cells. Although CD8+ lymph node T-cells seemed functionally comparable to CD8+ splenocytes, CD4+ lymph node T-cells failed to cooperate with CD8+ splenocytes to transfer diabetes. Our study suggests that diabetogenic cells are not evenly distributed in the different lymphoid organs. This may reflect a differential migration pattern of pathogenic T-cells in this animal model.

Two different mechanisms for the inhibition of rosette formation in mice.

The vast majority of spleen T cells (T.sRFC) which spontaneously bind to sheep red blood cells (SRBC) in an antigen-specific fashion express the Thy-1+, CD3+, CD8+ phenotype. Inhibition of rosetting by antibodies to surface molecules occurs via distinct mechanisms according to the antibody. CD8 and CD3 molecules are located in proximity to SRBC receptors and steric hindrance is the most likely explanation for the inhibition of rosetting by antibodies to these molecules. On the other hand, anti-Thy-1 antibody bound to T.sRFC induces a dynamic process involving intracellular cAMP, and which results in the inaccessibility of SRBC receptors. Thymulin could restore normal sensitivity of T.sRFC from adult thymectomized (A.Tx) mice to all inhibitory antibodies whatever the mechanism by which they hinder rosette formation. These results reinforce the idea that thymulin may act on membrane characteristics.

Expression of growth hormone receptors in murine lymphoid cells analyzed by flow cytofluorometry.

We have analyzed the expression of GH receptors (GHR) in murine lymphoid organs using flow cytofluorometry with biotinylated bovine GH and specific fluorescein isothiocyanate-labeled monoclonal antibodies defining distinct lymphoid cell populations. GHR were widely expressed in al murine hematopoietic tissues and in fetuses, newborns, and 3- and 7-week-old animals. In the bone marrow, all hematopoietic lineages expressed variable levels of GH receptors, whereas in the thymus, this expression was mainly seen in CD4-, CD8-, CD4+CD8+, and CD8+ subpopulations. At the periphery, 50% of splenocytes and peripheral blood lymphocytes and 20% of lymph node cells were GHR positive, with a wider receptor expression on B cells and macrophages (approximately 50%) than on T cells (approximately 20%), where the labeling was seen on both CD4+ and CD8+ cell subsets. Interestingly, the proportion of GHR-bearing CD4+ and CD8+ splenocytes significantly increased after T cell activation with Concanavalin A or anti-CD3. Finally, we demonstrated that all peripheral T cells expressing GHR also expressed PRL receptors. Our study provides a molecular basis to study the factors controlling GHR expression and to better understand the influence of GH in the regulation of immune function.

Neuroendocrine control of thymic hormonal production. I. Prolactin stimulates in vivo and in vitro the production of thymulin by human and murine thymic epithelial cells.

The thymic epithelium is responsible for the secretion of thymic peptides, which intervene in some steps of intra- and extrathymic T cell differentiation. Recent data suggest that thymic hormone secretion is modulated by the neuroendocrine network, comprising thyroid, adrenals, and gonads. However, the role of the pituitary gland in this regulation is still poorly understood. In the present paper we studied the in vivo and in vitro influences of PRL on the secretion of thymulin, one of the chemically defined thymic hormones, by thymic epithelial cells (TEC). When injected daily (20-100 micrograms/20 g) in young or old C57BL/6 mice, PRL induced a specific increase in thymulin synthesis and secretion, respectively, measured by the number of thymulin-producing cells in the thymus and the peripheral levels of the hormone. This stimulation was dose dependent and reversible after the end of treatment. Similar findings have been made in animals with pituitary dwarfism, known to have low levels of circulating thymulin. This stimulatory effect was also observed in primary cultures of human and mouse TEC when PRL (10(-7) to 10(-8) M) was applied to culture supernatants, thus suggesting that PRL could act directly on TEC. In addition, we induced in vivo experimental hypoprolactinemia, treating mice with bromocriptine, a dopamine receptor agonist that inhibits pituitary PRL secretion. Bromocriptine treatment (100-200 micrograms/20 g) yielded a significant decrease in thymulin secretion that could be reversed by coincident treatment with PRL. In the light of previous observations that bovine GH can also increase thymulin production in aged dogs, we performed a series of experiments in vitro to evaluate whether GH has a direct effect on TEC. We observed that only human GH preparations that are known to have a PRL-like effect were efficient in stimulating thymulin biosynthesis and release into the culture supernatants. The effects of PRL on TEC were not restricted to thymic hormone production. We observed that TEC proliferation, as well as the numbers of a TEC subset defined by the expression of cytokeratins 3 and 10, could also be increased by PRL treatment. All these findings show that the pituitary gland directly affects TEC in terms of cytoskeletal and secretory protein expression as well as cell cycle.

Prolactin receptor expression in human hematopoietic tissues analyzed by flow cytofluorometry.

PRL receptor (PRL-R) expression has been analyzed in human hematopoietic tissues using flow cytofluorometric analysis with a series of biotinylated monoclonal antibodies (mAbs) directed against the extracellular domain of the rat liver PRL-R. In the thymus, more than 75% of cells were labeled by the anti-PRL-R mAb. Regarding PRL-R expression in the four T-cell subsets defined by CD4/CD8 expression, the majority of cells expressed low receptor levels, whereas a minority of double negative (CD4-CD8-) and single positive CD4+ cells were strongly labeled by the anti-PRL-R mAb. In the peripheral blood, an average of 80% of lymphoid cells, comprising all B-cells, all monocytes, and 75% of T-cells, were consistently PRL-R positive. Regarding T-cell subsets, similar percentages of PRL-R+ cells were observed in CD4+ and CD8+ peripheral lymphocytes (70-75%), and the density of labeling per cell was significantly lower than that occurring in B-cells or monocytes. Interestingly, the intensity of labeling significantly increased in peripheral T-cells after T-cell activation. The ubiquitous distribution of PRL-R in bone marrow stem cells, B-cells, monocytes, and T-cells was confirmed by the positive staining obtained in a set of human lymphoid cell lines. These data along with those showing that the PRL gene is specifically expressed in human T-cells suggest that lymphocyte PRL may act in a paracrine or autocrine fashion in both central and peripheral lymphoid organs.

Growth hormone stimulates the proliferation of activated mouse T lymphocytes.

A modulatory role for GH on immune function has been suggested, but hormonal effects have been difficult to demonstrate with isolated cells. We have recently shown that GH receptors are present in murine hematopoietic tissues, with a lower receptor number in T lymphocytes than in B cells or macrophages. The binding of bovine GH (bGH) to murine splenocytes is increased after T cell activation with either concanavalin A or anti-CD3 antibody. In the present study, we show that bGH is able to stimulate the proliferation of activated murine T cells. Splenocytes were stimulated with either Con A or anti-CD3 antibody; addition of the mitogen resulted in increased [3H]thymidine uptake. When added together with the mitogen to the culture medium, bGH was able to further stimulate thymidine uptake. A bell-shaped dose-response curve was observed. bGH was able to increase cell proliferation by 2.5-fold over the effect of anti-CD3 alone. The amplitude of the bGH response was greater in unfractionated splenocytes than in purified T lymphocytes or thymocytes. Splenocytes were also stimulated by lipopolysaccharide, a B cell-specific mitogen; no change in the level of bGH binding was observed during activation of B cells, and no effect of bGH on the proliferative response of splenocytes to lipopolysaccharide was detected. The GH proliferative effect on T lymphocytes is probably direct and not through locally produced insulin-like growth factor I, because insulin-like growth factor I did not affect the cell proliferation when added at concentrations ranging from 10(-9)-10(-7) M. Ovine PRL was also able to stimulate [3H]thymidine uptake in splenocytes and thymocytes, and a synergistic effect was observed when bGH and ovine PRL were added together at 10(-8) M. Our findings support the biological significance of the GH receptors identified in murine T lymphocytes and confirm the role of GH in the regulation of immune functions.

Growth hormone (GH) and prolactin receptors in human peripheral blood mononuclear cells: relation with age and GH-binding protein.

GH receptors (GHRs) and PRL receptors (PRLRs) were studied in human peripheral blood mononuclear cells (PBMC) using flow cytometry, biotinylated anti-GH receptor monoclonal antibody 10B8, and biotinylated human PRL. Variations of GHR and PRLR expression and the relationship of plasma GHBP and GH receptor in PBMC subsets were examined as a function of age and sex. By double immunofluorescence staining, we show that about 30% of total cells express GH receptors, with a low expression in T cells, whereas almost all B cells and monocytes are GH receptor positive. Four age groups were defined among the 64 normal volunteers, aged 12 to 85 yr, who were included in the study. The percentage of PBMC expressing GH receptors is significantly lower in group 2 (20-40 yr) than in group 1 (12-20 yr) and group 4 (>60 yr). In T cells, monocytes and B cells, no significant changes are detected in either the percentage of GH receptor positive cells or in the GH receptor level per cell. The level of PRLRs expressed in PBMC is significantly higher in age group 2 than in age group 4. A negative correlation is observed between plasma GHBP and the percentage of PBMC expressing GH receptors. These results suggest that regulation of GH receptors in lymphocytes and in other target cells could be different.

Growth hormone and its receptor are expressed in human thymic cells.

GH has been shown to modulate various functions of the thymus. We now demonstrate the production of human GH (hGH) by human thymic cells, and the expression of GH receptors in thymic epithelial cells (TEC) and in thymocytes at different stages of differentiation. The presence of hGH messenger RNA was shown by RT-PCR in both human thymocytes and in primary cultures of TEC. Moreover, immunoreactive hGH material was detected in culture media of thymocytes and TEC with the use of a sensitive immunoradiometric assay. GH receptor gene expression was shown in TEC in primary cultures and in fetal and postnatal TEC lines as well as in thymocytes. By immunocytochemistry, the presence of GH receptors in the various TEC preparations was confirmed. In cytofluorometric studies with the use of a biotinylated anti-GH receptor monoclonal antibody, we could show that GH receptors are predominantly expressed by immature thymocytes: over 90% of CD3- CD4- CD8- CD19- CD34+ CD2- cells (a phenotype characterizing the most immature T cell progenitors in the thymus) were GH receptor positive. Our results provide a molecular basis for an autocrine/paracrine mode of action of GH in the human thymus.

Growth hormone and insulin-like growth factor-I stimulate hormonal function and proliferation of thymic epithelial cells.

We have investigated the role of GH and insulin-like growth factor-I (IGF-I) in controlling the secretion of thymulin, a hormone produced by thymic epithelial cells (TEC). Thymulin plasma concentrations (mean +/- SD) were increased in 21 patients with acromegaly compared to those in 30 controls, as assessed by bioassay (4.24 +/- 0.97 vs. 2.67 +/- 0.87; P less than 0.001) and RIA (561 +/- 241 vs. 315 +/- 113 pg/L; P less than 0.01). Good correlations were observed between plasma levels of thymulin and IGF-I (P less than 0.001). In vitro experiments demonstrated that both recombinant human GH and IGF-I significantly increased thymulin production in culture supernatants of normal human TEC and a rat TEC line. In parallel, IGF-I also significantly stimulated the proliferation of human TEC, as measured by bromodeoxyuridine incorporation. Additionally, the stimulatory effect of GH on thymulin production was abrogated by both an anti-IGF-I antibody and an anti-IGF-I receptor antibody. These results support a role for GH and IGF-I in the control of thymic hormonal function in man and suggest that the effect of GH may be mediated by local secretion of IGF-I within the thymus.

Reduced ability of hypothalamic and pituitary extracts from old mice to stimulate thymulin secretion in vitro.

There is substantial evidence that growth hormone (GH) is particularly important in the control of the age-related decline of thymus function. It was therefore of interest: (a) to assess the overall capacity of tissue extracts from mediobasal hypothalamus (MBH), anterior pituitary (AP) and testis, obtained from young (3 months, Yc), middle-aged (13 months, MAc) and old (18 months, Oc) intact C57BL/6 mice to stimulate in vitro the release of thymulin, a Zn-bound immunoregulatory thymic peptide, from pure cultures of mouse thymic epithelial cells (TEC); (b) to perform the same evaluation utilizing MBH, AP and testicular extracts from mice of the same age-range but treated for 45 days with a sc dose of ovine GH (2 micrograms/g body wt) known to stimulate thymulin secretion in vivo. Pituitary hormones were measured by heterologous rat RIAs, whereas thymulin release was estimated by a rosette assay. Untreated animals showed a significant age-dependent increase in the AP content of follicle stimulating hormone but not in other AP hormones. In both control and treated animals, pituitary GH content decreased significantly with age. MBH extracts from C57BL/6 males evidenced thymulin-releasing activity on mouse TEC lines. This activity was maximal in the MBH from young animals and declined with the age of the MBH donors. The thymulin-releasing activity of MBHs from GH-treated mice was higher than that of the control animals and showed a less pronounced decline with age. AP extracts from the same animals showed a higher thymulin-releasing activity than did MBH preparations. This activity showed a progressive age-associated reduction in the APs from untreated mice, whereas in the GH-treated group, an age-related decline was only seen in the old donors. Control testicular extracts had little effect on thymulin release whereas GH treatment induced a definite thymulin-release inhibiting activity in the testicular homogenates of our animals which increased progressively with the age of the testis donors. We conclude that the MBH, AP and testis of the young mouse contain factors able to affect directly the endocrine activity of the thymic epithelium. The amount of these substances declines with age and seems to be modulated by GH.

Isolation of leukocytes infiltrating the islets of Langerhans of diabetes-prone mice for flow cytometric analysis.

A simple method was developed for flow cytometric immunofluorescence analysis of cells infiltrating the islets of Langerhans in diabetes-prone rodents. Pancreases were submitted to collagenase P digestion and, in order to minimize collagenase action on leukocytes, islets were isolated before digestion was completed, that is, when exocrine tissue still remained around the islets. Islets, maintained in medium supplemented with sodium azide to prevent modulation of surface markers, were then pressed through a metal sieve and the cell suspension filtered through two different nylon screens. Cells were washed before immunofluorescence staining. Comparative phenotyping of spleen cells submitted to a similar treatment showed that, provided the collagenase P batch was screened, this procedure did not alter leukocyte surface markers and allowed multicolor analysis of the islet infiltrating cells.

Growth hormone receptors and immunocompetent cells.

Growth hormone plays a significant role in regulation of the humoral and cellular immune responses in physiological as well as pathological situations. This role is exerted via the existence of specific receptors on cells of the immune system. Using flow cytofluorometry and biotinylated bovine GH, we have recently analyzed the expression of GH receptors (GHRs) in murine lymphoid organs. GHRs are widely expressed in all murine hematopoietic tissues and in fetuses, newborns, and 3- and 7-week-old animals. In the bone marrow, all hematopoietic lineages express variables levels of GH receptors, whereas in the thymus, this expression is mainly seen in CD4-, CD8-, CD4+CD8+, and CD8+ subpopulations. At the periphery, 50% of splenocytes and peripheral blood lymphocytes and 20% of lymph node cells are GHR positive, with a wider receptor expression on B cells and macrophages (approximately 50%) than on T cells (approximately 20%), where the labeling is seen on both CD4+ and CD8+ cell subsets. Interestingly, the proportion of GHRs bearing CD4+ and CD8+ splenocytes is increased after T-cell activation with Con A or anti-CD3. Finally, all peripheral T cells expressing GHRs also express prolactin receptors. These data provide a molecular basis to study the factors controlling GHR expression and regulation of immune function.

Neuroendocrine control of thymic hormonal production. II. Stimulatory effects of endogenous opioids on thymulin production by cultured human and murine thymic epithelial cells.

Data have now accumulated to strongly demonstrate that several neuropeptides, including endogenous opioids, can have immunomodulatory functions. Most of the studies have so far focused on the direct action of these substances on lymphocytes. We decided to investigate whether thymic epithelial cells (TEC) - the major component of the thymic microenvironment - could also be modulated by endogenous opioids. Primary cultures of human and murine TEC were subjected to several opioids (alpha-beta- or gamma-endorphins, as well as met- or leuenkephalins) applied in concentrations ranging from 10(-6) to 10(-9) M. On the following days we measured the levels of thymulin (a chemically-defined thymic hormone known to stimulate some steps of T-cell differentiation) in the culture supernatants, as well as the numbers of thymulin containing cells, evaluated by immunofluorescence with an anti-thymulin monoclonal antibody. After treatment of TEC cultures with beta-endorphin or leu-enkephalin a significant increase in the levels of thymulin in the culture media was observed, paralleled by a rise in the percentage of thymulin containing cells. In addition, this stimulatory effect was dose-dependent. Preincubation of the opioids with the specific antibodies abrogated the opioid-induced stimulatory effect on TEC. Moreover, naloxone, an opioid receptor antagonist, blocked the effect of beta-endorphin on thymulin production, suggesting that the effect of this neuropeptide on epithelial cells was mediated by an opioid receptor. Importantly, no effect on thymulin production was observed with the other opioids used, whatever the dose. These results suggest that, at least in vitro, beta-endorphin and leu-enkephalin stimulate the hormonal function of the thymic epithelium. These findings lead to the general concept that the modulatory role of endogenous opioids on the immune system is not restricted to lymphocytes but can also take place at the level of cells belonging to T-cell differentiating microenvironments.

Specific binding sites for growth hormone in cultured mouse thymic epithelial cells.

Growth Hormones bound specifically to murine Thymic epithelial cells, which represent the major component of thymic micro-environment and can be modulated by pituitary hormones. The Kds found with human growth hormone and bovine growth hormone were 0.14 and 0.27 nM with a Bmax 0.56 and 0.35 fmol/10(6) cells respectively. Competition experiment analysis showed ED50 of 0.24 nM for hGH, 0.46 nM for rGH, 0.71 nM for bGH, 11.8 nM for hPRL and 11.2 nM for oPRL. No specific binding of [125I]-oPRL was observed under the same conditions. Both hPRL and bGH showed a negative regulatory effect on the number of the hGH binding sites when incubated with the culture for three days. The presence of GH receptors on Thymic epithelial cells provides biochemical evidence for the effect of GH on thymic function.

Prolactin receptors and the immune system.

The existence of a physiological immunoneuroendocrine network clearly contributing to homeostasis has now been demonstrated. In this context, nervous, endocrine, and immune systems communicate with each other, using common mediators and respective receptors. An interesting aspect of this network involves the interactions between prolactin (PRL) and the immune system. Prolactin plays a significant role in regulation of the humoral and cellular immune responses in physiological as well as pathological situations, such as autoimmune diseases. This role is exerted via the existence of specific receptors on cells on the immune system. Recently, using monoclonal antibodies (mAbs) raised against the extracellular domain of the rat liver PRL-receptor (PRL-R), we demonstrated by immunochemistry and molecular biology the presence of functional PRL receptors on thymic epithelial cells, one of the major components of the thymic microenvironment, which significantly influences early events in T-cell differentiation. Furthermore, using analytical fluocytometry, we showed that human and murine lymphoid cells also expressed PRL receptors. In both of the primary lymphoid organs, namely the thymus and bone marrow, more than 80% of cells expressed this receptor. In the periphery, all B cells and macrophages and 70% of T cells, with similar percentages of CD4+ and CD8+ cells, were PRL-R+. The density of receptors was lower on T cells than on B cells and macrophages, but this density was significantly enhanced following stimulation by T cell mitogens. These data, together with the demonstration of PRL production by thymocytes led to the hypothesis that, in addition to the classical endocrine pathway, autocrine and paracrine PRL/PRL-R interactions may exist in both central and peripheral lymphoid organs, and involve lymphocytes and microenvironmental cells.

Characterization of peripheral regulatory CD4+ T cells that prevent diabetes onset in nonobese diabetic mice.

The period that precedes onset of insulin-dependent diabetes mellitus corresponds to an active dynamic state in which pathogenic autoreactive T cells are kept from destroying beta cells by regulatory T cells. In prediabetic nonobese diabetic (NOD) mice, CD4+ splenocytes were shown to prevent diabetes transfer in immunodeficient NOD recipients. We now demonstrate that regulatory splenocytes belong to the CD4+ CD62Lhigh T cell subset that comprises a vast majority of naive cells producing low levels of IL-2 and IFN-gamma and no IL-4 and IL-10 upon in vitro stimulation. Consistently, the inhibition of diabetes transfer was not mediated by IL-4 and IL-10. Regulatory cells homed to the pancreas and modified the migration of diabetogenic to the islets, which resulted in a decreased insulitis severity. The efficiency of CD62L+ T cells was dose dependent, independent of sex and disease prevalence. Protection mechanisms did not involve the CD62L molecule, an observation that may relate to the fact that CD4+ CD62Lhigh lymph node cells were less potent than their splenic counterparts. Regulatory T cells were detectable after weaning and persist until disease onset, sustaining the notion that diabetes is a late and abrupt event. Thus, the CD62L molecule appears as a unique marker that can discriminate diabetogenic (previously shown to be CD62L-) from regulatory T cells. The phenotypic and functional characteristics of protective CD4+ CD62L+ cells suggest they are different from Th2-, Tr1-, and NK T-type cells, reported to be implicated in the control of diabetes in NOD mice, and may represent a new immunoregulatory population.

L-selectin(-/lo) and diabetogenic T cells are similarly distributed in prediabetic and diabetic nonobese diabetic mice.

We have previously shown that in the nonobese diabetic (NOD) mouse, an experimental model for autoimmune insulin-dependent diabetes, spleen diabetogenic T cells are contained within the T-cell subpopulation that express no or low levels of L-selectin. This phenotype characterizes activated/memory T cells. In the present study, we have compared the distribution of autoreactive T cells to that of L-selectin -/lo T cells in prediabetic and diabetic mice. Activated/memory T cells were found in decreasing concentrations in the bone marrow (BM), spleen, peritoneum, lymph nodes, and blood. This distribution correlated perfectly with that of T cells capable of transferring diabetes into syngeneic nondiabetic recipients. In diabetic mice, the highest levels of diabetogenic cells were observed in the spleen and BM. The peritoneum and lymph nodes contained intermediate frequencies of autoreactive cells. In the peripheral blood, the number of autoreactive T cells was variable, usually low; in some cases, they were undetectable. These cells were rare in the thymus. Diabetes-transferring cells were present in the spleen and BM of prediabetic mice. In these animals, diabetogenic cells were present in the blood circulation with frequencies higher than in diabetic mice, suggesting that after disease onset, when almost all target beta cells have disappeared, the recirculation of autoreactive cells is greatly decreased and finally stops. These observations: (a) suggest that T cells from BM of prediabetic and diabetic patients may be a better source of diabetogenic cells than the blood for analyzing diabetes-associated responses or for generating diabetogenic T-cell clones, and (b) point up the risk of using healthy autoimmune-prone donors for BM transplantation.

Strain-dependent migration of CD4 and CD8 lymphocyte subsets to lymph nodes in NOD (nonobese diabetic) and control mice.

Subpopulations of lymphoid cells were compared with respect to their ability to migrate into peripheral lymphoid organs of nonobese diabetic (NOD) mice and various strains of control mice. In short-term, in vivo homing studies, no major differences in the pattern of homing of B and T cells were observed among all mouse strains studied. On the other hand, CD4 cells localized consistently more efficiently than CD8 cells in both PP and LN of adult NOD and BALB/c mice, whereas both populations migrated roughly equivalently in LN of adult DBA/2, CBA, and C57BL/6 mice. No age-dependent differences in the homing of CD4 and CD8 cells were observed in BALB/c mice. On the contrary, in 2-week-old NOD mice, CD4 and CD8 cells migrated equally well. The preferential entry of CD4 cells in adult NOD and BALB/c did not result from increased blood transit time of CD8 cells. On the other hand, the preferential migration of CD8 cells was observed in the liver, whereas the two T-cell subsets migrated equally well in the lungs. The differences in the homing characteristics of CD4 and CD8 cells among NOD, BALB/c, and C57BL/6 mice were not related to modifications in the level of expression of adhesion molecules such as MEL-14, LFA-1, and Pgp-1.