Administration of sulfatide to ameliorate type I diabetes in non-obese diabetic mice.

The endogenous glycosphingolipid sulfatide is a ligand for CD1d-restricted type II natural killer T (NKT) lymphocytes. Through the action of these cells,sulfatide treatment has been shown to modulate the immune response in mouse models for autoimmune diseases, infections and tumour immunity. Sulfatide exists naturally in different organs including the pancreas, where sulfatide colocalizes with insulin within the Langerhans islet b-cells, targets for the immune destruction in type 1 diabetes (T1D). Human T1D patients, but not patients with type 2 diabetes nor healthy individuals, have autoantibodies against sulfatide in serum, suggesting that sulfatide induces an immune response in the natural course of T1D in humans. Here, we investigate sulfatide as an autoantigen and a modulator of autoimmune disease in the murine model forT1D, the non-obese diabetic (NOD) mice. We demonstrate that aged NOD mice displayed serum autoantibody reactivity to sulfatide; however, this reactivity did not correlate with onset of T1D. Repeated administration of sulfatide did not result in an increase in serum reactivity to sulfatide. Moreover, a multidose sulfatide treatment of female NOD mice initiated at an early (5 weeks of age),intermediate (8 weeks of age) or late (12 weeks of age) phase of T1D progression did not influence the incidence of disease. Thus, we demonstrate that a fraction of NOD mice develop autoantibody reactivity to sulfatide; however, we fail to demonstrate that sulfatide treatment reduces the incidence of T1D in this mouse strain.

Immunomodulatory type II natural killer T lymphocytes in health and disease.

Natural killer T (NKT) lymphocytes are αß T cells activated by lipid-based ligands presented on the non-polymorphic CD1d-molecule. Type I NKT cells that carry an invariant Vα14 (in the mouse) or Vα24 (in humans) T cell receptor α-chain rearrangement have received significant attention for their involvement in a diversity of immune reactions. Their sister population, CD1d-restricted type II NKT cells, has been more difficult to study because of the lack of molecular markers that specify these cells. In the last few years, however, significant progress has been made, demonstrating that type II NKT cells have unique functions in immune responses to tumours and infections, in autoimmunity, obesity and graft-versus-host disease. Type II NKT cells appear more frequent than type I NKT cells in humans and accumulate in certain diseases such as ulcerative colitis, hepatitis and multiple myeloma. Recently, novel type II NKT cell ligands have been identified, and it is becoming clear that the type II NKT cell population may be oligoclonal. Here, we review the recent progress in the study of type II NKT cells, supporting the view that type II NKT cells may be attractive targets for immunotherapy.

CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice.

Rather unexpectedly, major histocompatibility complex class II-deficient mice have a significant population of peripheral CD4+ T lymphocytes. We have investigated these cells at the population and clonal levels. CD4+ T lymphocytes from class II-deficient animals are thymically derived, appear early in ontogeny, exhibit the phenotype of resting memory cells, are potentially functional by several criteria, and have a diverse T cell receptor repertoire. They do not include substantially elevated numbers of NK1.1+ cells. Hybridomas derived after polyclonal stimulation of the CD4+ lymphocytes from class II-deficient animals include a subset with an unusual reactivity pattern, responding to splenocytes from many mouse strains including the strain of origin. Most members of this subset recognize the major histocompatibility complex class Ib molecule CD1; their heterogeneous reactivities and T cell receptor usage further suggest the involvement of peptides and/or highly variable posttranslational modifications.

Unique invariant natural killer T cells promote intestinal polyps by suppressing TH1 immunity and promoting regulatory T cells.

CD1d-restricted invariant natural killer T (iNKT) cells are known as potent early regulatory cells of immune responses. Besides the established roles in the regulation of inflammation and autoimmune disease, studies have shown that iNKT cells have important roles in tumor surveillance and the control of tumor metastasis. Here we found that the absence of iNKT cells markedly decreased the total number of intestinal polyps in APCMin/+ mice, a model for colorectal cancer. Polyp iNKT cells were enriched for interleukin-10 (IL-10)- and IL-17-producing cells, showed a distinct phenotype being CD4+, NK1.1- CD44int, and PD-1lo, and they were negative for the NKT cell transcription factor promyelocytic leukemia zinc-finger. The absence of iNKT cells was associated with a reduced frequency of regulatory T (Tregs) cells and lower expression levels of FoxP3 protein and transcript uniquely in the polyps, and a switch to an inflammatory macrophage phenotype. Moreover, in iNKT cell-deficient APCMin/+ mice, expression of T-helper (TH) 1-associated genes, such as IFN-γ and Nos2, was increased in polyps, concomitantly with elevated frequencies of conventional CD4+ and CD8+ T cells in this tissue. The results suggest that a population of regulatory iNKT cells locally promote intestinal polyp formation by enhancing Treg cells and immunosuppression of antitumor TH1 immunity.

Improved preparation of the integral membrane proteins of human red cells, with special reference to the glucose transporter.

Human red cell membranes were isolated and partially stripped of peripheral proteins by gel filtration of hemolysates on a Sepharose CL-4B column at pH 8 connected in tandem to a Sepharose CL-6B column at pH 10.5. The eluted material was washed by centrifugations, once at pH 10.5 and twice at pH 12. In this way, water-soluble proteins and peripheral membrane proteins were thoroughly removed, and 0.2 g of integral membrane proteins could be prepared within 10 h from 0.2 litre of red cells. The exposure to high pH did not lower the D-glucose transport activity, and electrophoretically pure glucose transport protein could be isolated from this preparation. Gel filtration in sodium dodecyl sulfate separated the integral membrane components into four fractions, one of them containing 4.5-material; gel electrophoresis showed about 14 zones and two-dimensional electrophoresis resolved up to 100 mostly minor components, among which the glucose transporter focused around pH 7. However, purified glucose transporter focused around pH 8. Glucose and nucleoside transport proteins were co-purified in active form on DEAE-cellulose and a fraction isolated by adsorption to Mono Q was used for immunization of mice and production of monoclonal antibodies. One hybridoma produced antibodies that reacted with material in the 4.5-region, possibly the glucose transport protein, and not with band 3-material. Upon two-dimensional electrophoresis of integral membrane components that had been solubilized with octyl glucoside the immunoreactive and the silver-stained 4.5-material focused in a broad range from pH 6 to pH 9. A possible explanation for this heterogeneity might be interaction between the glucose and nucleoside transport proteins and negatively charged lipids.

The natural killer T lymphocyte: a player in the complex regulation of autoimmune diabetes in non-obese diabetic mice.

Manipulation of the immune response to specifically prevent autoaggression requires an understanding of the complex interactions that occur during the pathogenesis of autoimmunity. Much attention has been paid to conventional T lymphocytes recognizing peptide antigens presented by classical major histocompatibility complex (MHC) class I and II molecules, as key players in the destructive autoreactive process. A pivotal role for different types of regulatory T lymphocytes in modulating the development of disease is also well established. Lately, CD1d-restricted natural killer T (NKT) lymphocytes have been the subject of intense investigation because of their ability to regulate a diversity of immune responses. The non-classical antigen presenting molecule CD1d presents lipids and glycolipids to this highly specialized subset of T lymphocytes found in both humans and mice. From experimental models of autoimmunity, evidence is accumulating that NKT cells can protect from disease. One of the best studied is the murine type 1 diabetes model, the non-obese diabetic (NOD) mouse. While the NKT cell population was first recognized to be deficient in NOD mice, augmenting NKT cell activity has been shown to suppress the development of autoimmune disease in this strain. The mechanism by which CD1d-restricted T cells exert this function is still described incompletely, but investigations in NOD mice are starting to unravel specific effects of NKT cell regulation. This review focuses on the role of CD1d-restricted NKT cells in the control of autoimmune diabetes.

Trypsin does not reconstitute responsiveness to lipopolysaccharide in the strain C3H/HeJ, but is a B-cell mitogen-like lipopolysaccharide, stimulating a different subpopulation.

The effect of trypsin on mouse spleen cells and enriched B cells, added alone or together with lipopolysaccharide (LPS), was investigated. With trypsin, proliferation in serum free spleen cell cultures was 2-6 times greater than the background using cells from LPS responder strains, and 2-4 times the background with cells from the C3H/HeJ strain. Trypsin also induced the formation of a low number of IgM plaque forming cells (PFC). When added together with LPS, trypsin increased the proliferation caused by LPS alone by 10-50% with cells from LPS responder strains and by 50-100% with cells from the LPS non-responder strain C3H/HeJ. Trypsin enhanced proliferation in cultures maximally stimulated by LPS. The increased proliferation obtained when trypsin was added to LPS-stimulation of cells from the C3H/HeJ strain, was therefore not interpreted as a reconstitution of the LPS response. We conclude that trypsin has a moderate mitogenic effect on mouse B cells, stimulating the cells to proliferate and secrete IgM. The mechanism of action is unknown, but is different and independent from the action of LPS.

Interleukin 2, 4 and 5 are sequentially produced in mitogen-stimulated murine spleen cell cultures.

Lymphokine production was analyzed in murine spleen lymphocytes stimulated with different T cell mitogens. Using in situ hybridization, frequencies of cells and the kinetics of production of interleukin (IL) 2, 4 and 5 were analyzed. The different mitogens varied in their ability to induce the three interleukins. IL2 was most successfully induced with a high dose of the calcium ionophore A23187 combined with phorbol 12-myristate 13-acetate (PMA). Significant frequencies of cells containing IL4 or IL5 mRNA were found among cells stimulated with an anti-CD3 antibody together with PMA, or pokeweed mitogen. The combination of anti-CD3 and PMA induced relatively high frequencies of all three cytokines. The production was sequential with the highest levels of IL2 mRNA present during the first 24 h, IL4 mRNA reaching a peak on day 2 and finally IL5 peaking on day 3. When cells that had been stimulated with mitogens in vitro were restimulated, the lymphokines were produced more rapidly. The order of production was maintained with IL2 mRNA reaching a maximum already at 3 h of culture, IL4 mRNA at 8 h and IL5 mRNA at 24 h.

Differential regulation of Ly49 expression on CD4+ and CD4-CD8- (double negative) NK1.1+ T cells.

The pan-NK cell marker NK1.1, present in some mouse strains, is also found on a subset of TCRalphabeta+ lymphocytes termed NKT cells. These cells are primarily CD4+ or CD4-CD8- (double negative, DN), and both NKT subpopulations contain cells reactive with the MHC class I-like molecule CD1d. Murine NK cells express clonally distributed inhibitory receptors of the Ly49 family that bind to different alleles of MHC class I molecules and transmit negative signals regulating NK cell function. Ly49 receptors are also found on TCRalphabeta+ NK1.1+ T cells. To investigate the potential role of inhibitory Ly49 markers in the regulation of NKT cells, we have done a thorough analysis of their expression on different NKT populations. The CD4+ and DN NK1.1+ T cell subsets have traditionally been dealt with as one NK1.1+ T cell population, but we found dramatic differences between these two NKT cell subsets. We demonstrate here expression of Ly49 receptors on DN, but not on CD4+, NK1.1+ T cells in spleen and liver. Absence of the specific MHC class I ligand in the host was associated with elevated levels of expression and, to a greater extent than has been found for NK cells, increased the frequencies of Ly49-positive cells within the DN subset, while CD4+ NK1.1+ cells remained negative. In the thymus and bone marrow both NK1.1+ T cell subsets contained high frequencies of Ly49-positive cells. Results from in vitro stimulation of DN NKT cells further suggest that activation and expansion of NKT cell subsets are regulated by the Ly49 receptors.

Diverse CD1d-restricted T cells: diverse phenotypes, and diverse functions.

Invariant CD1d-restricted T cells express NK cell markers and use a limited TCR repertoire. Here, we describe a second CD1d-restricted T cell subset that uses a diverse TCR repertoire. These T cells can also express NK cell markers and function similarly to invariant T cells. The antigens recognized by the diverse subset are likely to be different from those recognized by invariant TCRs. The variable NK1.1 antigen expression on these T cell populations limits its usefulness in identifying CD1d-restricted T cells. Lastly, the discovery of antigens recognized by diverse CD1d-restricted T cells will provide insight into their role in normal and pathological immune responses.

Commitment to lymphokine profile during primary in vitro stimulation.

We have shown previously that different conditions in primary in vitro SEB stimulation of resting CD4+ T cells, could induce selectively production of either IFN gamma or IL4 and IL10. The present investigation shows that the priming conditions also decide which lymphokines will be produced during restimulations. The cells that had been induced to produce mainly IFN gamma during the primary SEB stimulation, or during stimulation with IL4 and IL10, synthesized the same lymphokines at restimulation, regardless of stimuli and were unaffected by exogenous IL4. Thus, at an early stage of primary SEB stimulation, cells became committed to produce a certain pattern of lymphokines which remained throughout the period of in vitro culture testing.

Manipulation of the superantigen-induced lymphokine response. Selective induction of interleukin-10 or interferon-gamma synthesis in small resting CD4+ T cells.

The production of several lymphokines by freshly isolated CD4+ T cells has been analyzed at the single-cell level, after stimulation with staphylococcal enterotoxin B (SEB). High frequencies of cells producing interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were induced, but very low frequencies of CD4+ T cells produced IL-4, IL-5 or IL-10 in response to SEB. Exogenously added IL-4 markedly altered the lymphokine profile induced during primary SEB stimulation. IFN-gamma production was reduced, while a high fraction of cells contained IL-10 and IL-4 after activation in the presence of IL-4. We further demonstrate that IL-4 and IL-10 or IFN-gamma production was selectively induced in resting, high-density CD4+ T cells during primary stimulation, by SEB + IL-4 or SEB. Under conditions where both IL-10 and IFN-gamma were produced, most cells contained only one of the two lymphokines.

Interleukin 4 and interferon gamma production in restimulated CD4+ and CD8+ cells indicates memory type responsiveness.

Interleukin 4 (IL-4) and interferon gamma (IFN-gamma) production was analysed in murine spleen cells during primary and secondary mitogen stimulation in vitro. The kinetics, frequency and phenotype of single lymphokine-producing cells were studied by combining intracytoplasmatic immunofluorescence and surface staining. Both IL-4 and IFN-gamma was produced by CD4+ as well as CD8+ cells, however 75-80% of IL-4 producers were CD4+ and 90% of IFN-gamma+ cells were CD8+. In primary stimulations, concanavalin A (Con A) activation or anti-CD3 antibody together with phorbol 12-myristate 13-acetate (PMA) induced different patterns of lymphokine production. Approximately the same frequency of IFN-gamma+ cells was induced by both stimulation procedures but the kinetics was different with a peak at 30 h using Con A and at 52 h using anti-CD3 and PMA. IL-4 production peaked at 52 h, but the frequency of IL-4+ cells was 8-10 times higher after stimulation by anti-CD3 and PMA than after Con A stimulation. During restimulation of the mitogen activated cells, lymphokines were rapidly produced; both IL-4 and IFN-gamma production peaked at 8-11 h. Only a small increase in the frequency of IL-4+ cells was seen, at most two to three times. No evidence for a major shift of lymphokines produced between primary and secondary stimulations could be found. Instead, the pattern of lymphokine production induced by the primary stimulus was dominant also in secondary cultures irrespective of stimulation condition.

Helper interleukins are produced by both CD4 and CD8 splenic T cells after mitogen stimulation.

We have earlier described (Cardell, S. and Sander, B., Eur. J. Immunol. 1990. 20:389) mitogen-induced production of interleukin (IL)2, IL4 and IL5 mRNA by murine spleen cells, analyzed by in situ hybridization. In the present study we have investigated the potential of CD8 T cells to produce these interleukins, normally associated with the helper function of CD4 T cells. When concanavalin A (Con A)-activated spleen cells were restimulated with Con A and phorbol 12-myristate 13-acetate (PMA), higher levels of IL2, IL4 and IL5 mRNA were induced, as detected both by increased frequencies of positive cells, and by more mRNA per cell. Four-to-six-day Con A blasts were enriched for CD4+ or CD8+ T cells, and restimulated with Con A and PMA. Both CD4 and CD8 cells were found to produce all three kinds of mRNA when restimulated. The frequencies of IL2 mRNA-containing CD8 cells were half of those found for CD4 cells (3.5% as compared to 7%). On the average 1% of the CD8 cells were induced to produce IL4 and IL5 mRNA, while 9% and 3% of the activated CD4 cells contained IL4 and IL5 mRNA, respectively. CD4 and CD8 cells displayed different sensitivities to the reagents when tested alone. Con A induced the synthesis of IL4 and IL5 in CD4 cells, but not CD8 cells, independently of PMA. PMA alone induced extensive thymidine incorporation in CD8 cells, but not in CD4 cells, in the absence of detectable lymphokine mRNA. The results suggest that some CD8 cells have the capacity to give help in immune responses, by secretion of IL2, IL4 and IL5.

Primary stimulation of CD4+ cells in the presence of IL-4 or IFN-gamma alters the frequencies of cytokine-producing cells at restimulation.

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8+ cells and of exogenously added cytokines (interleukin (IL)-2, IL-4 and interferon (IFN)-gamma) on the cytokine production in splenic CD4+ T cells. IL-2, IL-4, IL-5 and IFN-gamma production in CD4+ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8+ cells. On day 5 the cells were restimulated with mitogen alone and analysed to evaluate the short-term development of cytokine-producing cells in such cultures. Preactivation in the presence of either exogenous IL-4 or IFN-gamma led to an increased production of IL-4 and IFN-gamma respectively at restimulation, and the effects of both IL-4 and IFN-gamma were augmented by IL-2. After preactivation in the presence of IL-2 and IL-4, every third CD4+ cell could be induced to produce IL-4. Exogenous IL-4 or IFN-gamma further decreased each other's production. Depletion of CD8+ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8+ cells will influence lymphokine production in CD4+ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4+ and CD8+ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines.

Qualitative shift of lymphokine production in response to stimulation, as a consequence of preactivation in vivo or in vitro.

Lymphokine production, analysed at the single cell level, was compared in resting and primed T-cell populations. Cells were preactivated in vitro by repeated mitogen stimulations, or isolated as large, low density cells naturally activated in vivo, from normal spleens of unimmunized animals. A similar qualitative shift in the pattern of lymphokines synthesized after restimulation was found as a result of in vivo and in vitro preactivation of cells. Repeated stimulations in vitro resulted in a qualitative shift in the lymphokines produced in response to activation, from a dominance of IL-2 during the first and second culture, to a dominance of IL-4 and IL-5 in the later stimulations. In vivo activation lead to a similar separation of lymphokine production as primarily IL-2 was made by small resting cells, while large cells preferentially produced IL-4 and IL-5. IFN-gamma was produced by both small and large cells. Preactivation in vitro lead to a more rapid appearance of lymphokines during restimulation. In contrast, the in vivo naturally activated cells responded with a slow onset of lymphokine production when stimulated in vitro.

Differential regulation of lymphokine production in mitogen-stimulated murine spleen cells.

Activation of murine spleen cells in vitro with soluble anti-CD3 monoclonal antibody and phorbol 12-myristate 13-acetate (PMA) induced an initial production of interleukin 2 (IL2), interferon-gamma (IFN-gamma), IL4 and IL5, followed by a refractory state during which the T cells did not produce lymphokines when stimulated with some common mitogens. The refractory state was long-lasting, depended on the presence of anti-CD3 and PMA but could be reverted by incubation in fresh medium. Pokeweed mitogen (PWM) differed from other mitogens tested, since stimulation by PWM and PMA induced lymphokine production and proliferation also in the refractory cells. Furthermore, PWM stimulation selectively induced IL4 and IFN-gamma production but not IL2 and IL5, as detected by intracellular cytokine-specific immunofluorescence and in situ hybridization for mRNA. The results indicate differential regulation of lymphokine production in primary lymphocytes.

Characterization of subpopulations of T-cell receptor intermediate (TCRint) T cells.

CD1-autoreactive T cells of two types have been demonstrated among T cells expressing the T-cell receptor (TCR) alphabeta at intermediate levels (TCRint cells). One type constitutes a major fraction of the natural killer (NK)1.1+ TCRint population in C57BL/6 (B6) mice and carries a restricted TCR composed of an alpha-chain with an invariant Valpha14-J281 rearrangement, and a beta-chain using Vbeta8. 2, 7 or 2. The second type utilises a variety of TCR and was derived from CD4+ cells in mice lacking MHC class II. To increase our understanding of the two different CD1-reactive subsets, we have investigated and compared the populations of origin: NK1.1+ and NK1. 1- TCRint subsets from MHC class II-deficient mice and CD4+NK1.1+ T cells from B6 mice. The three TCRint populations shared a phenotype indicating previous activation, and contained low frequencies of cells expressing NK receptors of the Ly49 family. In contrast to control CD4+ cells, the three TCRint subsets produced high amounts of interleukin (IL)-4 and interferon (IFN)-gamma after activation. Importantly, no IL-10 could be detected in either TCRint population, implying a distinct function for these cells, different from those of conventional CD8+ and CD4+ cells, including the typical T-helper 2 (Th2) cell. Analysis of TCR expression indicated that the proportion of cells using the semi-invariant Valpha14/Vbeta8.2-type TCR was lower in NK1.1+ cells from MHC class II-negative mice than in CD4+NK1.1+ B6 cells. Further, usage of the Valpha14-J281 rearrangement was also demonstrated among NK1.1- TCRint cells.