The suppressor activity of the spleen cells from bone marrow chimeras prepared with total-lymphoid irradiation was analyzed in vitro. The chimeric spleen cells lacked responsiveness to host-type, but not to thirdparty, antigens in the mixed-leukocyte reaction (MLR) as judged by (3H)thymidine incorporation and the generation of cytolytic cells. When the donor-type chimeric spleen cells were used as cocultured cells in the MLR, modest nonspecific suppression of (3H)thymidine incorporation and potent antigen-specific suppression of the generation of the cytolytic cells was observed. The donor-type suppressor cells may play an important role in preventing graft-versus-host disease in vivo.
Abstract Systemic lupus erythematosus is an autoantibody mediated disease that causes immune complex glomerulonephritis as well as injury to multiple organs. Invariant natural killer T cells (iNKT) from non-autoimmune strains of mice regulate and suppress graft versus host disease, transplantation immunity and autoimmunity. We have shown that iNKT cells from lupus prone NZB/W mice are abnormal and promote rather than suppress spontaneous in vitro immunoglobulin and autoantibody secretion of autoreactive B cells. To determine the role of iNKT cells in the induction and augmentation of immune cell activation, we examined cytokine production of purified iNKT cells from NZB/W and control C57BL/6 mice in vitro. We found that upon stimulation of NKT stimulatory ligand, α-galactosylceramide, young NZB/W splenocytes secreted much higher IFNγ, IL-4 and IL-17 compared with C57BL/6 mice. With activation of CD3 and CD28, sorted NZB/W splenic iNKT cells, but not conventional T cells, also produced dramatically higher IFNγ and IL-4 than controls. Interestingly, we found the expression of Ly108, a SLAM family member (SLAMF6), affected cytokine production, and promoted help for immunoglobulin secretion by autoreactive B cells. Sorted NZB/W Ly108+ iNKT cells produced less IL-17 but more efficiently helped B cells secrete IgG but not IgM as compared to Ly108- iNKT cells. In conclusion, expression of the SLAM family member, Ly108, promoted the interaction of NZB/W iNKT cells with autoreactive B cells.
Abstract We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and β2m−/− mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23− CD43−) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in β2m−/− mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both β2m−/− and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the β2m-dependent form of CD1. They further suggest that a β2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.
Twenty-six patients participated in a randomized, double-blind study of the efficacy of total lymphoid irradiation in the treatment of intractable rheumatoid arthritis. All 26 patients, for whom therapy with gold compounds and penicillamine had failed, would ordinarily have been considered candidates for cytotoxic or antimetabolite drug therapy. Thirteen patients randomly assigned to receive full-dose total lymphoid irradiation (2000 rad) and 11 patients assigned to receive control low-dose total lymphoid irradiation (200 rad) completed radiotherapy. Alleviation of joint disease activity was significantly greater in the high-dose group as judged by morning stiffness, joint tenderness, and functional assessment (global composite score) at 3 and 6 months after radiotherapy. The high-dose group had a marked reduction in both T-lymphocyte function and numbers, but this finding was not observed in the low-dose group. Complications seen in the high-dose but not low-dose group included transient neutropenia, thrombocytopenia, pericarditis, and pleurisy.
Abstract Lupus‐prone NZB/W F1 mice develop glomerulonephritis after T helper cell‐dependent isotype switching of autoantibody secretion from IgM to IgG at about 6 months of age. We compared innate immune natural killer (NK) T cells and conventional T cells for their capacity to help spontaneous in vitro immunoglobulin and autoantibody secretion of innate immune (B‐1 and marginal zone) and conventional (follicular) B cell subsets from NZB/W F1 mice. We found that purified NKT cells not only increased spontaneous secretion of IgM and IgM anti‐double‐stranded (ds)DNA antibodies by B‐1 and marginal zone B cells, but also facilitated secretion of IgG anti‐dsDNA antibodies predominantly by B‐1 B cells. Few IgM or IgG anti‐dsDNA antibodies were secreted by follicular B cells, and conventional T cells failed to provide potent helper activity to any B cell subset. All combinations of T and B cell subsets from normal C57BL/6 mice failed to generate vigorous IgM and IgG secretion. NZB/W NKT cell helper activity was blocked by anti‐CD1 and anti‐CD40L mAb. In conclusion, direct interactions between innate immune T and B cells form a pathway for the development of IgM and IgG lupus autoantibody secretion in NZB/W mice.
Ten patients with lupus nephritis and marked proteinuria (3.9 g or more/d) that did not respond adequately to treatment with prednisone alone or prednisone in combination with azathioprine were treated with total lymphoid irradiation in an uncontrolled feasibility study. Within 6 weeks after the start of total lymphoid irradiation, the serum albumin level rose in all patients in association with a reduction in the serum level of anti-DNA antibodies, an increase in the serum complement level, or both. Improvement in these variables persisted in eight patients followed for more than 1 year, with the stabilization or reduction of the serum creatinine level. Urinary leakage of albumin was substantially reduced in all patients. Side effects associated with radiotherapy included transient constitutional complaints in ten patients, transient blood element depressions in three, localized viral and bacterial infections in four, and ovarian failure in one. The results suggest that total lymphoid irradiation may provide an alternative to cytotoxic drugs in the treatment of lupus nephritis.
Abstract An in vitro system was established in which single‐cell suspensions of lymphocytes and synovial cells from the joints of patients with rheumatoid arthritis were cultured and produced an outgrowth of an organized inflammatory tissue with an extracellular matrix and capsule. The tissue outgrowth, which had histologic features of pannus, required the addition of mycobacterial antigen and interleukin‐2 to the tissue culture medium and was dependent upon the presence of T lymphocytes and their interaction with synovial fibroblasts.
Introduction: Immune tolerance and persistent mixed chimerism can be achieved reproducibly after combined organ and hematopoietic cell transplantation in mice conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG). Over the last decade, we studied the safety and reproducibility of this approach in clinical kidney transplantation. Methods: Twenty-five patients, 14 men and 11 women, age range 22 to 61 years, have undergone HLA-mismatched (n=7) and matched (n=18) living donor kidney transplantation followed by conditioning with 10 doses of TLI and 5 doses of ATG. This was followed by infusion of donor CD34+ hematopoietic progenitor cells and T cells. Criteria for withdrawal of ant-rejection drug therapy at ≥ 6 months were development of transient chimerism (in the first 6 HLA-mismatched patients) or stable chimerism for at least 6 months (in the 18 HLA-matched and the 7th HLA-mismatched patient), and absence of rejection and graft versus host disease. Results: All patients had excellent graft function at last observation, one month to 11 years following transplantation. Of the first 6 HLA-mismatched patients, enrolled in 2000-2003, two developed transient chimerism and were withdrawn from anti-rejection drugs. Both subsequently developed acute rejection, one at 3.5 months and one at 5.5 months after drug withdrawal, necessitating resumption of anti-rejection drug therapy, and graft function returned to baseline. Of the 18 HLA-matched patients, enrolled in 2005-2012, three patients had acute rejection that was easily reversed and one had immediately recurrent focal segmental glomerulosclerosis that has gone into long-term remission; all 4 remain on immunosuppression. Eleven of the 18 have been withdrawn from anti-rejection drugs, 3 have been observed for five months and 8 for two to four years without evidence of subsequent rejection. Patients 16 and 17 have stable chimerism and are in the midst of drug withdrawal, and patient 18 was recently transplanted. The 7th HLA-mismatched patient, enrolled in 2010, developed only transient chimerism and remains on immunosuppression. Conclusion: TLI and ATG safely promotes the development of chimerism and tolerance in HLA-matched kidney and hematopoietic cell transplantation, without serious risk to patient or allograft in the short or intermediate term, and no HLA matched or mismatched grafts have been lost to acute or chronic rejection in the past 11 years. We continue to enroll patients in the HLA-matched protocol. Based on clinical studies of hematopoietic cell transplantation in haplotype-matched patients with leukemia and lymphoma, we have modified the protocol to enhance the development of stable chimerism and tolerance in HLA-mismatched transplantation. We are actively enrolling patients into the HLA-mismatched protocol.
