1. Radioactive iodide was covalently bound to living cells from normal mouse spleen and a variety of lymphoid tumours by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. About 3x10(5)-6x10(5) molecules of [(125)I]iodide/cell could be incorporated without affecting cell viability. 3. Electron-micrographic radioautography showed that the radioactive label was associated with the outer surfaces of the cells. 4. Radioiodinated proteins were solubilized in 9m-urea-0.2m-mercaptoethanol and analysed by gel-filtration and disc electrophoresis. 5. Comparison of distinct tumour lines by disc electrophoresis showed qualitative and quantitative differences in protein distribution patterns.

Knowledge of the nature of the receptor for antigen and its role in the specific activation of lymphocytes is essential to the elucidation of the mechanisms of immune recognition and differentiation. The fact that antibody formation (1-5) and cellmediated immunity (6-9) can be inhibited by antiglobulin reagents suggests that this receptor shares antigenic determinants with immunoglobulin. Complete analysis of the lymphocyte receptor for antigen requires isolation of the molecule and direct measurement of its binding properties for antigen. Furthermore, the manner in which the receptor is attached to the cell surface and the mechanism by which combination with antigen activates immune differentiation must be ascertained.As an initial step in this chain of biochemical proof, we have developed a method for the covalent attachment of radioactive iodine to accessible surface proteins of living lymphocytes (10, l l). A sufficient amount of radioisotope was incorporated to facilitate fractionation of these surface proteins by a variety of biochemical and immunological means. We previously reported the isolation of low molecular weight "yM immunoglobulin from the surfaces of murine and neonatal human thymic lymphocytes (12). Uhr and his colleagues (13, 14) have independently employed a similar approach to isolate "7S" "yM from splenic lymphocytes of BALB/c mice.In the present communication we report the radioiodination, isolation, and partial characterization of surface immunoglobin from neonatal human thymic lymphocytes and a variety of murine lymphocyte populations. These lymphocyte populations were chosen to provide information on the nature of surface immunoglobulin associated with bone marrow-derived lymphocytes (B ceUs) t and thymus-influenced lymphocytes (T cells). Materials and MethodsCell Sources and Methods of Preparatlon.--Male and female CBA/H/Wehi and (CBA ;K C57BL)F1 mice, weighing 20-25 g each, were used as a source of normal spleens and thymuses.

SUMMARYInjection of antigen into the ocular anterior chamber (AC) of a mouse eye (an immunologically privileged site) induces the activation of immunoregulatory NK1.1 + , CD4 ) CD8 ) , T-cell receptor (TCR) ab + thymocytes. These thymocytes transfer the suppression of delayed-type hypersensitivity (DTH) when injected into mice sensitized to the same antigen but do not effect the suppression of DTH. On the other hand, the immunized recipients of these transferred thymocytes produce splenic CD8 + T cells that effect the suppression of DTH. However, it is unclear whether the thymocytes transferred from the AC-injected donor differentiate into and/or activate CD8 + T-splenic suppressor cells. We therefore sought to determine the origin of splenic suppressor cells produced in the recipients of immunoregulatory thymocytes transferred from donors that receive an injection of antigen into the AC. CD45.1 + thymocytes from mice that received an AC injection of 2,4,6-trinitrobenzene sulphonic acid (TNP)-bovine serum albumin (BSA) were transferred to congenic CD45.2 + TNP-BSA-immune recipients. Spleen cells from the recipients were then sorted based on anti-CD45.1 or -CD45.2 antibody binding and assayed for suppressor cells. This was done by the injection of separated spleen cells into the footpad of TNP-BSA-immunized mice, concurrent with the induction of footpad swelling (contact sensitivity) of the footpad elicited by an epicutaneous application of picryl chloride. The systemic distribution of antigen after the injection of antigen into the AC was demonstrated by the injection of fluorescein or 125 I-labelled TNP-BSA into the AC. The results demonstrate that (i) splenic CD8 + T-suppressor cells produced in the immunized recipients of immunoregulatory thymocytes are derived from the CD45.2 recipient of the CD45.1 + thymocytes; (ii) the induction of recipient splenic suppressor T cells by the transferred immunoregulatory thymocytes requires that the recipient be immunized to the same antigen as that used to induce immunoregulatory thymocytes; (iii) antigen is introduced to the thymus after an injection of antigen into the AC; (iv) although the transfer of the suppression of DTH by regulatory thymocytes was not dependent on interleukin-4 (IL-4), CD4 + NK1.1 ) regulatory thymocytes from AC-injected donors enhanced the production of immunoglobulin G1 antibodies to TNP-BSA by an IL-4-dependent mechanism. These observations suggest that the adult thymus plays an active role in the induction and maintenance of anterior chamber-associated immune deviation as manifested by the generation of the suppression of cell-mediated immunity to exogenous antigen and the antigen-induced production of IgG1 antibodies.

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CD4(+)FoxP3(+) Tregs are essential mediators of the peripheral immune response to self-antigens. Accordingly, the homeostatic regulation of Treg activity and number would impact on the immune response to both self- and non-self antigens. Because the sympathetic nervous system (SNS) interacts chemically and physically with the central and peripheral immune system and exerts a direct influence on antigen-presenting cells and effector lymphocytes, we have investigated the effect of chemical ablation of the SNS on the number and function of peripheral Treg. Removal of murine peripheral sympathetic innervation by 6-hydroxydopamine induced an increase in splenic and lymph node CD4(+)FoxP3(+) Tregs by a TGF-beta-dependent mechanism. Further, this increase in Tregs coincides with an inhibition of the induction of experimental autoimmune encephalomyelitis. Our results demonstrate that the SNS is an important contributor to the maintenance of peripheral Treg and TGF-beta acts as a bridge between the immune system and the nervous system. Neurological events mediated by the SNS, such as a stress response, may affect the number of T cells that regulate an immune response. Additionally, targeting Tregs via the SNS may be a novel approach to the prevention or treatment of autoimmune diseases.

Cbl-b is an E3 ubiquitin ligase that negatively regulates T cell activation. Cbl-b−/− mice develop spontaneous autoimmunity, and Cbl-b dysregulation has been described in both murine and human autoimmune diseases. Although the mechanisms underlying the development of autoimmunity in Cbl-b−/− mice are not yet clear, we have reported that Cbl-b−/− CD4+CD25− effector T cells (Teffs) are resistant to CD4+CD25+ regulatory T cell (Treg)-mediated suppression in vitro and have suggested that this may be an important mechanism in the development of autoimmunity. To confirm the relevance of this resistance to autoimmune disease, we now show that Cbl-b−/− Teffs are resistant to suppression by Tregs in vivo and that this involves a resistance of truly naive Cbl-b−/− Teffs. Additionally, we show that Cbl-b−/− Tregs are fully functional in vivo, further suggesting that the regulatory abnormalities in Cbl-b−/− mice are related to defects in Teff, not Treg, function. To characterize the relevance of TGF-β sensitivity in Treg resistance, we examined in vivo Th17 generation and report that Cbl-b−/− mice are able to mount a normal Th17 response in vivo. As Cbl-b−/− Teffs have been shown to be insensitive to the suppressive effects of TGF-β in other in vivo models, the present results suggest that Cbl-b−/− Teffs demonstrate a context-dependent sensitivity to TGF-β in vivo. Overall, our results suggest that resistance to Tregs may be a bona fide mechanism underlying autoimmunity and that Cbl-b−/− mice offer unique approaches for studying the interrelationships between Treg function, TGF-β–mediated responses, and the development of autoimmunity.

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BackgroundProduction of the chemokine CCL2 by cells of the neurovascular unit (NVU) drives critical aspects of neuroinflammation. Suppression of CCL2 therefore holds promise in treating neuroinflammatory disease. Accordingly, we sought to determine if the compound bindarit, which inhibits CCL2 synthesis, could repress the three NVU sources of CCL2 most commonly reported in neuroinflammation – astrocytes, microglia and brain microvascular endothelial cells (BMEC) – as well as modify the clinical course of neuroinflammatory disease.MethodsThe effect of bindarit on CCL2 expression by cultured murine astrocytes, microglia and BMEC was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bindarit action on mouse brain and spinal cord in vivo was similarly investigated by qRT-PCR following LPS injection in mice. And to further gauge the potential remedial effects of bindarit on neuroinflammatory disease, its impact on the clinical course of experimental autoimmune encephalomyelitis (EAE) in mice was also explored.ResultsBindarit repressed CCL2 expression by all three cultured cells, and antagonized upregulated expression of CCL2 in both brain and spinal cord in vivo following LPS administration. Bindarit also significantly modified the course and severity of clinical EAE, diminished the incidence and onset of disease, and evidenced signs of disease reversal.ConclusionBindarit was effective in suppressing CCL2 expression by cultured NVU cells as well as brain and spinal cord tissue in vivo. It further modulated the course of clinical EAE in both preventative and therapeutic ways. Collectively, these results suggest that bindarit might prove an effective treatment for neuroinflammatory disease.