Activation of natural killer T cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes.

Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6-9). Thus, given that NKT cells respond to the alpha-galactosylceramide (alpha-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by alpha-GalCer might prevent the onset and/or recurrence of T1D. Here we show that alpha-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, alpha-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to alpha-GalCer. Protection from T1D by alpha-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet beta cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility that alpha-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.

Involvement of T-lymphocytes in the stimulatory effects of EMT6 tumors on medullary pluripotent stem cells of BALB/c mice.

Untreated EMT6 tumors and bone marrow of tumor-bearing BALB/c mice secrete factors capable of stimulating quiescent medullary splenic colony-forming units into cycle. Since the tumor population is heterogeneous, it is of interest to determine the nature of the cells which are involved in this phenomenon. Experiments with conditioned media from tumors of BALB/c and athymic mice show that EMT6 cells themselves may secrete a stimulating factor but that the presence of T-lymphocytes is necessary at some time in the development of the tumor. Likewise, the presence of the tumor is necessary for bone marrow to secrete these factors in our experimental model. This suggests reciprocal influences of the T-cells and the EMT6 tumor cells in the phenomena that we describe.

Influence of factors derived from EMT6 tumors and from bone marrow of tumor-bearing mice on tumor and bone marrow stem cell kinetics.

Untreated EMT6 tumors in BALB/c mice were used to assess the regulatory mechanisms of tumor growth in these animals. This tumor can be quantitated for clonogenic cells by in vitro techniques, and the hydroxyurea suicide method makes it possible to determine the kinetic status of the clonogenic cells. The untreated EMT6 tumor does not seem to have internal humoral regulatory mechanisms explaining tumor growth kinetics. However, the exponentially growing EMT6 experimental tumor releases a factor capable of stimulating quiescent splenic colony-forming units into cycle. This is also true of bone marrow taken from tumor-bearing mice. This study was made possible using an in vivo-in vitro technique which separates the effector cells from the responder cells by a Millipore filter floating on the culture medium. The relationship between tumor growth and normal hematopoietic tissue of the tumor-bearing animal is discussed.

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.

Influence of castration, alone or combined with thymectomy, on the development of diabetes in the nonobese diabetic mouse.

The nonobese diabetic (NOD) mouse is a recognized model for studying immunologically-mediated insulin-dependent diabetes mellitus. The disease appears with a greater preponderance in females than in males. Castration at weaning led to a significant increase in the prevalence of diabetes in NOD males, whereas a tendency to a decreased prevalence was observed in NOD females. Castration combined with thymectomy was found to further increase the prevalence of diabetes in NOD males, whereas in females castration reversed the effect of thymectomy. These results on changes in diabetes prevalence were corroborated by the degree of lymphocytic infiltration directed toward the pancreatic islets of Langerhans. Taken together these results indicate a direct relationship between the endocrine and immune systems, whereby orchidectomy has a deleterious effect on the immunopathogenesis of diabetes. In addition, we examined whether the distribution of lymphocyte subpopulations, mitogen reactivity, lymphokine production, and in vivo response to a thymus-dependent antigen, such as sheer red blood cell, were dependent or independent of the sex steroid environment.

Influence of iron-deficiency anemia on selected thymus functions in mice: thymulin biological activity, T-cell subsets, and thymocyte proliferation.

To define the effects of iron deficiency on thymulin biological activity, T-cell subsets, and thymocyte proliferation, C57BL/6 female mice at weaning were fed an iron-deficient diet (10 mg Fe/kg diet), an iron-sufficient diet (50 mg Fe/kg diet), or restricted amounts of the iron-sufficient diet (the pair-fed group) for 40 d. Iron deficiency did not reduce the concentration of either serum or intracytoplasmic thymulin. Although T-cell subsets in the thymus were not altered, both the cortical and medullar regions were depleted of thymocytes. In the spleen iron deficiency (but not underfeeding) significantly reduced the percentage of L3T4+ cells, of Lyt-2+ cells, and thus of the overall T-cell population. However, it did not affect the ratio of L3T4+ to Lyt-2+ T cells. Thymocyte proliferation was significantly reduced at the concanavalin A (Con A) dose (10 mg/L) that produced maximal stimulation in control and pair-fed mice but not at low (7.5 mg/L) or high (15 mg/L) Con A concentrations. We conclude that the impairment in immune functions associated with iron deficiency is not due to an impairment in thymic endocrine function but rather to decreased immunocompetent lymphocytes.

A one step cell sorting procedure for the enrichment of murine bone marrow CFU-S which is independent of their proliferative activity.

Murine bone marrow day 9 splenic colony-forming units (CFU-S) have been concentrated using a one step cell sorting technique. CFU-S were discriminated on the basis of their rather high forward light scatter intensity, their high affinity for the lectin WGA and the absence of a cell surface marker expressed by the majority of hematopoietic cells and recognized by the RA3-5B3 MoAb. This procedure permitted a 40-fold enrichment of quiescent or cycling CFU-S (obtained from normal and Ara-C-treated mice respectively) and did not alter their differentiation pathways towards the various myeloid lineages.

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.

Disorganization of thymic medulla precedes evolution towards diabetes in female NOD mice.

The thymic medulla is a complex microenvironment which plays a crucial role in central tolerance induction. Using a quantitative histological analysis of non-obese diabetic (NOD) mice, we show that the medulla undergoes several structural modifications during the course of the disease in NOD mice. Indeed, the majority of 70-day-old NOD mice show a scattering of medullary epithelial cells in the cortex which is associated with a reduction in the size of the medulla in heavily disorganized thymuses. The severity of this phenotype is shown to correlate with the subsequent appearance of diabetes in older female NOD mice. This trait is mainly controlled by non-major histocompatibility complex NOD genes since C57BL/6 H-2g(7) congenic mice have a normal medulla. It persists in conditions where effector lymphocytes that lead to diabetes are inhibited in periphery. These results suggest that primary alterations of the thymic stroma might play a role in the progression towards diabetes in NOD mice.

Role of interleukin-2 receptors in the suppressive effect of spleen cells from anti-CD3-treated mice.

In the present study, we demonstrate that unresponsive spleen T cells from mice injected with a low dose of anti-CD3 mAb (single 10 micrograms i.v. injection) significantly inhibit Con A-induced proliferation of normal spleen cells. The induction of this phenomenon requires in vivo activation since spleen cells from mice injected with the F(ab')2 fragment of anti-CD3 mAb fail to promote it. Suppression of normal T cell proliferation is concomitant with increased expression of IL-2 receptor on spleen cells from anti-CD3-treated mice. It disappears within 3 days when IL-2R has returned to background levels. A normal proliferative response to Con A can be restored when high concentrations of IL-2 are added together with the "suppressor" cells. Taken together, these data support the notion that activated spleen cells from anti-CD3-injected mice exert their inhibitory effect by competing for the IL-2 generated during culture.

Persistence of stem cell activity within the murine thymus after transfer of a bone marrow fraction enriched in CFU-S.

Mechanisms involved in prothymocyte migration, differentiation and self-commitment were investigated. We used a murine bone marrow fraction isolated on a discontinuous Ficoll gradient and enriched 10-20 times in CFU-S activity, and studied its fate after intrathymic transfer over a period of 200 days. In order to assess their hemopoietic activity, chimeric thymuses were intravenously transferred to secondary lethally irradiated hosts and both day 8 and day 12 spleen colonies were evaluated. The results show that transfer of a stem cell enriched fraction leads to long-term repopulation of the thymuses and that the input of progenitors is regulated by the size of the intrathymic precursor pool. Furthermore, stem cells can locate within the irradiated thymus and remain in a primitive stage for several months.

Synergistic effect of colony-stimulating factors and IL-2 on prothymocyte proliferation linked to the maturation of macrophage/dendritic cells within L3T4-Lyt-2-Ia-Mac- cells.

Bone marrow-derived precursors colonize the thymus, where they constitute the minor L3T4-Lyt2- subset which can give rise to all thymocyte subpopulations. We show in the present paper that L3T4-Lyt2- population depleted of Ia+, Mac-1+ cells contain pluripotent hemopoietic stem cells (CFU-S) and granulocyte-macrophage colony-forming cells (GM-CFC). Addition of GM-CSF to the culture medium leads to the production of adherent and nonadherent cells of the macrophage-monocyte lineage. L3T4-Lyt2- cells poorly respond to IL-2 in vitro, but the addition of either rIL-3 or rGM-CSF allows the IL-2 response of L3T4-Lyt2- cells. This response is at least partly mediated by maturation of double-negative cells for L3T4 and Lyt-2 Ag into cells able to produce IL-1.

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.

Restoration by serum thymic factor of colony-forming unit (CFU-S) entry into DNA synthesis in thymectomized mice after T-dependent antigen treatment.

Adult thymectomy prevents stimulation of hematopoietic stem cells as measured by colony-forming units (CFU-S) if T-dependent antigens are injected, but not when T-independent antigens are used. This can be observed as soon as 10 to 15 days after thymectomy. The serum thymic factor can restore CFU-S response to T-dependent antigens in thymectomized mice.