Autoreactive T cells in MRL/Mpr-lpr/lpr mice. Characterization of the lymphokines produced and analysis of antigen-presenting cells required

Lymph node cells from 4-wk-old MRL/Mp-lpr/lpr mice, but not from MRL/Mp-+/+ mice, when cultured in vitro for 5 to 7 days, will spontaneously proliferate and produce IL-2. We examined the expression of several cell surface Ag on lymph node cells from MRL/Mp-lpr/lpr mice before and after in vitro culture. There is an increase in the expression of Thy-1, L3T4, IL-2R, T cell activating protein, T cell receptor, and T3 complex on the surface of cultured cells. Cultured cells produced IL-3, IFN-gamma, and small but detectable amounts of IL-1 in addition to IL-2. Gamma irradiation of APC from young MRL/Mp-lpr/lpr mice or treatment of APC with a mAb (J11D) and C, completely abrogated their stimulatory capacity. These experiments suggest that B cells are the predominant APC responsible in the activation of autoreactive T cells in MRL/Mp-lpr/lpr mice. Lymph node cells from C57BL/6-lpr/lpr or C3H-lpr/lpr mice were unable to spontaneously proliferate or produce IL-2. Lymph node cells from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr) F1 mice or (C3H-lpr/lpr x MRL/Mp-lpr/lpr) F1 mice did proliferate and produced IL-2 after in vitro culture. Using T cells from these F1 animals and APC from each parental haplotype, we found that APC from MRL/Mp-lpr/lpr mice induced more proliferation and greater amounts of IL-2, when compared to APC from F1 animals. APC from C57BL6-lpr/lpr mice or C3H-lpr/lpr were unable to induce spontaneous proliferation and IL-2 production. Therefore, B cells from MRL/Mp-lpr/lpr mice appear to possess unique features that enable them to activate autoreactive T cells more effectively than B cells from other mice bearing the lpr/lpr gene.     

IL-21 receptor is required for the systemic accumulation of activated B and T lymphocytes in MRL/MpJ-Fas(lpr/lpr)/J mice

MRL/MpJ-Fas(lpr/lpr)/J (MRL(lpr)) mice develop lupus-like disease manifestations in an IL-21-dependent manner. IL-21 is a pleiotropic cytokine that can influence the activation, differentiation, and expansion of B and T cell effector subsets. Notably, autoreactive CD4(+) T and B cells spontaneously accumulate in MRL(lpr) mice and mediate disease pathogenesis. We sought to identify the particular lymphocyte effector subsets regulated by IL-21 in the context of systemic autoimmunity and, thus, generated MRL(lpr) mice deficient in IL-21R (MRL(lpr).IL-21R(-/-)). Lymphadenopathy and splenomegaly, which are characteristic traits of the MRL(lpr) model were significantly reduced in the absence of IL-21R, suggesting that immune activation was likewise decreased. Indeed, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R-deficient MRL(lpr) mice. Correspondingly, we observed a significant reduction in autoantibody titers. Activated CD4(+) CD44(+) CD62L(lo) T cells also failed to accumulate, and CD4(+) Th cell differentiation was impaired, as evidenced by a significant reduction in CD4(+) T cells that produced the pronephritogenic cytokine IFN-γ. T extrafollicular helper cells are a recently described subset of activated CD4(+) T cells that function as the primary inducers of autoantibody production in MRL(lpr) mice. Importantly, we demonstrated that T extrafollicular helper cells are dependent on IL-21R for their generation. Together, our data highlighted the novel observation that IL-21 is a critical regulator of multiple pathogenic B and T cell effector subsets in MRL(lpr) mice.     

Gammadelta T cells promote a Th1 response during coxsackievirus B3 infection in vivo: role of Fas and Fas ligand

Fas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL(+/+) mice infected with CVB3 develop severe myocarditis, a dominant CD4(+) Th1 (gamma interferon [IFN-gamma(+)]) response to the virus, and a predominance of gammadelta T cells in the myocardial infiltrates. MRL lpr/lpr and MRL gld/gld mice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4(+) Th2 (interleukin-4 [IL-4(+)]) phenotype to CVB3. Spleen cells from virus-infected wild-type, lpr, and gld animals proliferate equally to virus in vitro. Adoptive transfer of gammadelta T cells from hearts of CVB3-infected MRL(+/+) mice (FasL(+)) into infected MRL gld/gld recipients (FasL(-)/Fas(+)) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRL lpr/lpr recipients (FasL(+)/Fas(-)) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial gammadelta T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4(+) T cells. These results demonstrate that Fas/FasL interactions conferred by gammadelta T cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.     

Mesenchymal stem cell transplantation inhibits abnormal activation of Akt/GSK3β signaling pathway in T cells from systemic lupus erythematosus mice

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by activation and proliferation of autoreactive T cells and B cells. We examined changes in cell cycle progression of T cells from MRL/lpr mice with or without allogenic bone marrow mesenchymal stem cells (BMMSCs) treatment and analyzed the expression of cell cycle associated proteins. In addition, the Akt/GSK3β protein kinase cascade was studied. We demonstrated that high-dose MSCs transplantation effectively ameliorated disease activity in MRL/lpr mice. BMMSCs treatment inhibited G1/S transition of the abnormal lupus T lymphocytes. Moreover, it increased the expression of p21(WAF1/CIP1) and p27(Kip1) and decreased the expression of CDK2. Furthermore, high-dose MSCs inhibited abnormal activation of the Akt/GSK3β signaling pathway of T cells from MRL/lpr mice. Our results suggest that high-dose BMMSCs transplantation successfully treated MRL/lpr lupus mice by inhibiting abnormal activation of Akt/GSK3β signaling pathway of T cells.     

Biased expression of variable region gene families of the immunoglobulin heavy chain in autoimmune-prone mice

We have examined usage of variable region gene families of the immunoglobulin heavy chain (VH gene family) in spleens of MRL/MpJ-1pr/lpr (MRL/lpr), (NZB x NZW)F1, and BXSB mice by Northern analysis using various VH probes, including the VHPAR gene which we cloned and identified as a gene encoding the heavy-chain variable region of antipoly(ADP-ribose) antibody. The amount of VHS107 family mRNA was almost constant for the same amount of splenic crude RNA in autoimmune-prone and normal mice, while concentrations of other family mRNAs were elevated in autoimmune-prone mice. For example, per splenic RNA the VHPAR family was expressed in MRL/lpr mice 10 times more than in their normal counterpart, MRL/MpJ-+/+ (MRL/+) mice. These results indicate the bias of VH gene usage in autoimmune-prone mice. Expression of the VHS107 family was depressed from an early life stage of MRL/lpr and male BXSB mice. Furthermore, the expression of IL-4 and IL-5 were quantitatively compared, as B cell differentiation factor was thought to be produced by abnormally proliferative T cells in lymph nodes of MRL/lpr mice. We could not, however, observe overproduction of IL-4 and IL-5 mRNA in the lymph nodes.     

B cells are required for lupus nephritis in the polygenic, Fas-intact MRL model of systemic autoimmunity.

B cells are required for both the expression of lupus nephritis and spontaneous T cell activation/memory cell accumulation in MRL-Faslpr mice (MRL/lpr). Autoimmunity in the MRL/lpr strain is the result of Fas-deficiency and multiple background genes; however, the precise roles of background genes vs Fas-deficiency have not been fully defined. Fas-deficiency (i.e., the lpr defect) is required in B cells for optimal autoantibody expression, raising the possibility that the central role for B cells in MRL/lpr mice may not extend to MRL/+ mice and, thus, to lupus models that do not depend on Fas-deficiency ("polygenic lupus"). To address this issue, B cell-deficient, Fas-intact MRL/+ mice (JHd-MRL/) were created; and disease was evaluated in aged animals (>9 mo). The JHd-MRL/+ animals did not develop nephritis or vasculitis at a time when the B cell-intact littermates had severe disease. In addition, while activated/memory CD4+ and CD8+ T cells accumulated in B cell-intact mice, such accumulation was substantially inhibited in the absence of B cells. This effect appeared to be restricted to the MRL strain because it was not seen in B cell-deficient BALB/c mice (JHd-BALB) of similar ages. The results indicate that B cells are essential in promoting systemic autoimmunity in a Fas-independent model. Therefore, B cells have an important role in pathogenesis, generalizable to lupus models that depend on multiple genes even when Fas expression is intact. The results provide further rationale for B cell suppression as therapy for systemic lupus erythematosus.     

Monoclonal autoantibodies with interleukin 3-like activity derived from a MRL/lpr mouse

Hybridomas that secrete monoclonal antibodies with interleukin 3 (IL-3)-like activity were established from spleen cells of a nonimmunized autoimmune MRL/lpr mouse. Five of the monoclonal antibodies thus obtained bound selectively to IL-3-dependent cells and supported their growth. These monoclonal antibodies inhibited the binding of IL-3 to FDC-P2 cells and vice versa. Thus, these antibodies were probably directed to IL-3 receptor sites, or at least to some cell surface proteins related to the growth of the IL-3-dependent cells. These MRL/lpr-derived monoclonal antibodies reacted strongly with cells from bone marrow, spleen, and lymph node of MRL/lpr mice, but minimally with such cells of MRL/+ or BALB/c mice. The findings were consistent with our earlier suggestion that the IL-3-like activity in MRL/lpr sera is not caused by IL-3 itself but is associated with IgG that is probably an autoantibody directed to the IL-3 receptor.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.     

Histone deacetylase 9 deficiency protects against effector T cell-mediated systemic autoimmunity

Co-repressor histone deacetylase 9 (HDAC9) plays a key role in the development and differentiation of many types of cells, including regulatory T cells. However, the biological function of HDAC9 in T effector cells is unknown. Systemic autoimmune diseases like lupus, diabetes, and rheumatoid arthritis have dysfunctional effector T cells. To determine the role of HDAC9 in systemic autoimmunity, we created MRL/lpr mice with HDAC9 deficiency that have aberrant effector T cell function. HDAC9 deficiency led to decreased lympho-proliferation, inflammation, autoantibody production, and increased survival in MRL/lpr mice. HDAC9-deficient mice manifested Th2 polarization, decreased T effector follicular cells positive for inducible co-stimulator, and activated T cells in vivo compared with HDAC9-intact MRL/lpr mice. HDAC9 deficiency also resulted in increased GATA3 and roquin and decreased BCL6 gene expression. HDAC9 deficiency was associated with increased site-specific lysine histone acetylation at H3 (H3K9, H3K14, and H3K18) globally that was localized to IL-4, roquin, and peroxisome proliferator-activated receptor-γ promoters with increased gene expression, respectively. In kidney and spleen, HDAC9 deficiency decreased inflammation and cytokine and chemokine production due to peroxisome proliferator-activated receptor γ overexpression. These findings suggest that HDAC9 acts as an epigenetic switch in effector T cell-mediated systemic autoimmunity.     

Autoimmune MRL/lpr mice sera contain IgG with interleukin 3-like activity

The spleen cells, thymocytes, and bone marrow cells of autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice do not constitutively produce interleukin 3 (IL-3), but these mice had IL-3-like activity in their sera. MRL/lpr sera supported the growth of the IL-3-dependent cell lines FDC-P2 and DA-1 but not the growth of IL-2-dependent T-572 cells. This IL-3-like activity increased with age. Biochemical analysis of the MRL/lpr sera by anion-exchange chromatography, gel filtration on a Superose 12 column, the binding to protein-A and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the serum factor with the IL-3-like activity was not IL-3 itself but was associated with IgG. Flow cytometric analysis also showed that the serum level of the Ig capable of binding FDC-P2 cells was high in MRL/lpr but not in MRL/+ mice. We suggest that IL-3 is not responsible for lymphoid hyperplasia, contrary to a previous report; rather auto-antibodies directed toward the IL-3 receptor may act pathogenically in MRL/lpr mice.     

T cell reactivity to MHC class II-bound self peptides in systemic lupus erythematosus-prone MRL/lpr mice

The epitopes recognized by pathogenic T cells in systemic autoimmune disease remain poorly defined. Certain MHC class II-bound self peptides from autoimmune MRL/lpr mice are not found in eluates from class II molecules of MHC-identical C3H mice. Eleven of 16 such peptides elicited lymph node cell and spleen cell T cell proliferation in both MRL/lpr (stimulation index = 2.03-5.01) and C3H mice (stimulation index = 2.03-3.75). IL-2 and IFN-gamma production were detected, but not IL-4. In contrast to what was seen after immunization, four self peptides induced spleen cell proliferation of T cells from naive MRL/lpr, but not from C3H and C57BL/6.H2(k), mice. These peptides were derived from RNA splicing factor SRp20, histone H2A, beta(2)-microglobulin, and MHC class II I-A(k)beta. The first three peptides were isolated from I-E(k) molecules and the last peptide was bound to I-A(k). T cell responses, evident as early as 1 mo of age, depended on MHC class II binding motifs and were inhibited by anti-MHC class II Abs. Thus, although immunization can evoke peripheral self-reactive T cells in normal mice, the presence in MRL/lpr mice of spontaneous T cells reactive to certain MHC-bound self peptides suggests that these T cells actively participate in systemic autoimmunity. Peptides eluted from self MHC class II molecules may yield important clues to T cell epitopes in systemic autoimmunity.     

Regulation of T cell-dependent autoantibody production by a gammadelta T cell line derived from lupus-prone mice

Lupus-prone (MRLxC57BL/6) F(1) mice lacking gammadelta T cells show more severe lupus than their T cell-intact counterparts, suggesting that gammadelta T cells down-modulate murine lupus. To determine the mechanisms for this effect, we assessed the capacity of gammadelta T cell lines derived from spleens of alphabeta T cell-deficient MRL/Mp-Fas(lpr) (MRL/Fas(lpr)) mice to down-regulate anti-dsDNA production generated by CD4(+)alphabeta T helper cell lines and activated B cells from wild-type MRL/Fas(lpr) mice. One line, GD12 (gd TCR(+), CD4(-)CD8(-)), had the capacity to reduce anti-dsDNA production in a contact-dependent manner. GD12 also killed activated MRL/Fas(lpr) (H-2(k)) B cells, with less cytolysis of resting B cells than that generated by in comparison to cytokine-matched gammadelta T cell lines. In addition, GD12 also killed activated B cells derived from C57BL/6-Fas(lpr) (H-2(b)) or beta(2)-microglobulin (beta(2) M)-deficient MRL/Fas(lpr) mice, suggesting cytolysis was neither MHC- nor CD1-restricted. Killing by GD12 was inhibited by anti-TNFalpha and anti-TNF-R1, and partially blocked by anti-gd TCR Fab fragments, but not by anti-FasL, anti-TNF-R2 (p75) or concanamycin A. IL-10 produced by GD12 also partially inhibited alphabeta Th1-dependent but not alphabeta Th2-dependent autoantibody production. These findings prove that we have identtified a gammadelta T cell line that suppresses autoantibody synthesis by alphabeta T-B cell collaboration in vitro.     

Ontogeny and function of B220+ L3T4+ T-cell subset of MRL/Mp-lpr/lpr mice

T-cell-enriched populations obtained from lymph nodes (LNs) of 4-month-old MRL/Mp-lpr/lpr (MRL-lpr), C3H/HeJ-lpr/lpr (C3H-lpr), and C3H/HeJ-gld/gld (C3H-gld) mice were studied for the expression of B220, L3T4, and Lyt 2 antigens. A new B220+ L3T4+ phenotype was demonstrated in T-cell populations of these mice by two-color flow cytometry with phycoerythrin-conjugated monoclonal antibodies (MoAb) to L3T4 and FITC-anti-B220 MoAb. The generation of the T subset was apparently under the influence of the lpr or gld gene, since it could not be demonstrated in T-cell-enriched populations of MRL/Mp- +/+ and normal C3H mice. The expression level of L3T4 antigen on the T subset was lower than that on B220- L3T4+ cells, while the level of B220 antigen was similar to that of B220+ L3T4- or B220+ Lyt 2- cells. The B220+ L3T4+ phenotype appeared in LNs and spleens, but not in thymuses, of MRL-lpr mice as early as 2 months of age. Its proportion to whole LN T cells at this age was equivalent to that observed in 4-month-old mice. Functional studies on FACS-sorted cell populations revealed that the T subset similar to B220+ L3T4- cells possessed deficiencies in the IL-2-IL-2 receptor system. The appearance of the T subset with an intermediate phenotype and its functional defects suggests that lpr and gld genes in these autoimmune mice exert their influences on the ontogeny and function of L3T4+ T cells and contribute to the induction of early lupus.     

Evidence for the existence of two parallel differentiation pathways in the thymus of MRL lpr/lpr mice.

MRL mice homozygous for the lpr/lpr gene develop a massive lymphadenopathy caused by the accumulation of CD4-CD8-, Thy-1-positive T cells that express B220. This phenotypically unusual T cell population coexists with normal, B220- T cells in lpr/lpr animals. To investigate the origin and differentiation pathway of B220+ T cells, the expression of a panel of developmentally regulated cell surface markers including TCR, CD4, CD8, Thy-1, and B220 was examined. Thymocytes and peripheral T lymphocytes from lpr/lpr mice were analyzed by four-color flow cytometry. The results showed that both B220+ and B220- thymocytes contained all of CD4-CD8-, CD4+CD8+, and CD4 or CD8 single positive T cell subpopulation in the lpr thymus. Expression of the V beta 11 TCR, measured by flow cytometry and reverse polymerase chain reaction, was demonstrated in lpr thymus. However, the number of T cells expressing V beta 11 was greatly reduced in both the B220+ and B220- T cell populations in lymph node, spleen, and liver. Taken together, the data provide evidence for maturation and selection of a distinct population of B220+ T cells in the thymus of MRL lpr/lpr mice.     

Autoimmune CD4+ T cells interfere with immune tolerance to a thymic-independent antigen

The ability of autoimmune T cell subsets to interfere with tolerization of B cells can be studied by using thymic-independent Ag. We have defined an abnormality within the CD4+ T cell compartment in young NZB and MRL-lpr/lpr mice by studying tolerance of spleen and B cells to the thymic independent Ag, fluorescein-Brucella abortus. Tolerization of spleen cells is defective in MRL-lpr/lpr mice, but not MRL-+/+ or C3H.lpr mice, suggesting that the defect requires both the autosomal MRL background and the lpr gene to be present. T enriched cells from NZB mice and from MRL-lpr/lpr mice (but not MRL-+/+ or C3H.lpr mice) reverse tolerance in spleen cells from [NZB X DBA/2]F1 and C3H/HeJ mice, respectively. This interference is removed by treatment with anti-CD4 antibody and C. Supernatants from cultured T cells of NZB and MRL-lpr/lpr mice also prevent tolerance in spleen cells of [NZB X DBA/2]F1 and MRL-+/+ mice, respectively, unless CD4+ cells are removed prior to T cell culture. Removal of T cells from NZB and MRL-lpr/lpr spleen cells allows normal tolerization of B cells, which is abrogated by the addition of syngeneic T cells or cultured T cell supernatants. This effect also depends on the presence of CD4+ T cells. These studies show that in MRL-lpr/lpr mice, through interaction of the lpr and MRL background genes in a T cell subset, and in NZB mice, CD4+ T cells interfere with B cell tolerance to a thymic-independent Ag.     

Selective elimination of non-lpr lymphoid cells in mice undergoing lpr-mediated graft-vs-host disease

The transfer of lpr BM stem cells into lethally irradiated non-lpr recipients (including the congenic MRL/+ differing only at the lpr locus) causes GVHD characterized by a wasting syndrome. In this study we investigated the interaction between the autoimmune (lpr) and normal (A-Thy) B, T, and RBC cell lineages in two types of radiation chimeras: MRL/lpr plus A-Thy----(MRL/lpr X A-Thy)F1 and MRL/+ plus A-Thy----(MRL/lpr X A-Thy)F1. Analysis of B cell repopulation by competitive RIA of serum Igh-1 allotype showed that both the MRL and the A-Thy donor cells initially engrafted. However, by 2 to 4 mo post-transplantation the normal A-Thy allotype was barely detectable (reduced greater than 2 orders of magnitude), whereas the autoimmune MRL/lpr allotype persisted at normal levels. Similarly, investigation of the donor origin of peripheral blood T cells by two-color flow cytometry showed that by 8 mo post-transplantation normal A-Thy T cells had been eliminated and only MRL/lpr T cells were present in the circulation. In contrast, erythrocytes from both the MRL/lpr and A-Thy donor strains successfully engrafted the F1 recipients and persisted until the termination of the study. Control chimeras transplanted with a mixture of MRL/+ plus A-Thy BM were stably engrafted with both donor strains in both the erythroid and lymphoid populations. Additional experiments in which either B6/lpr or MRL/lpr (and B6/+ or MRL/+ control) BM cells were transferred into (MRL/lpr X B6/+)F1 and (MRL/lpr X B6/lpr)F1 recipients demonstrated that the development of GVHD was not simply due to increased alloreactivity by the lpr donor cells. In these chimeras only the recipients heterozygous (but not homozygous) for the lpr gene developed lpr-GVHD, although both types of recipients had identical genotypes except at the lpr locus.     

Unresponsiveness of MRL/MP-lpr/lpr mice to antigen given subcutaneously in adjuvant: partial restoration of response after local injection of B cells

MRL/lpr mice were used as a model for seeking information on the role of B cells as antigen-presenting cells in vivo. In confirmation of the finding of other workers, MRL/lpr mice with pronounced lymphadenopathy failed to undergo T cell priming in lymph nodes (LN) after s.c. injection of keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA): thus, in contrast to normal mice or young MRL/lpr mice, the LN cells from older MRL/lpr mice failed to give secondary T proliferative responses to the injected antigen in vitro. Because previous work from this laboratory has shown that B cells play a major role in priming T cells in LN of normal mice, we tested the possibility that the lack of T cell priming to KLH seen in older MRL/lpr LN reflected the relative paucity of B cells. To test this idea, MRL/LPR mice were injected s.c. with B cells taken from normal or young MRL/lpr mice 1 day before priming with KLH/CFA. In the case of old (20 to 24 wk) MRL/lpr mice with massive lymphadenopathy, prior injection of B cells had virtually no effect in promoting LN priming. In marked contrast, injection of B cells into mice exhibiting less-pronounced LN enlargement (mice tested at 14 to 16 wk) was highly effective in restoring LN priming; before B cell injection, the LN of these mice contained only 2 to 5% B cells. These findings add to the evidence that T cell priming in LN is heavily dependent on the presence of B cells.