Murine interstitial nephritis. VI. Characterization of the B cell response in anti-tubular basement membrane disease

In the present study we have examined the murine B cell response in anti-tubular basement membrane (alpha TBM) disease. Whereas only certain strains of mice are susceptible to the development of interstitial lesions after immunization with heterologous renal tubular antigen, all strains make anti-tubular basement membrane antibodies (alpha TBM-Ab), and all express the 3M-1 kidney antigen which is the target of disease. The magnitude of the alpha TBM-Ab response in serum and renal eluates, measured by radioimmunoassay against crude tubular antigen or affinity-purified 3M-1, also mapped independently of susceptibility. The fine specificity of epitope binding was further analyzed using a rat monoclonal alpha 3M-1 antibody to competitively inhibit the binding of renal eluate antibody to 3M-1. Maximum inhibition among nearly all tested strains ranged from 46 to 56% with no discernible difference between susceptible and nonsusceptible mice. Idiotypic representation of renal eluate alpha TBM-Ab was then evaluated by competitive inhibition using a polymorphic anti-idiotypic antisera. All mice examined possessed almost identical competitive inhibition patterns, indicating surprisingly similar idiotypic representation. Thus, in susceptible or nonsusceptible mice, neither the quantitative alpha TBM-Ab response, the epitopic fine specificity of that response, nor the idiotype of eluted alpha TBM-Ab serve as distinguishing markers for susceptibility to interstitial injury. Finally, passive transfer experiments with high-titered (greater than 1:10,000) alpha TBM-Ab from SJL mice were performed to test the hypothesis that alpha TBM-Ab alone may be sufficient for the induction of alpha TBM disease. Whereas this antiserum was capable of causing typical, severe alpha TBM disease in naive susceptible SJL mice, this treatment in allotype-identical, nonsusceptible B10.S(8R) mice was completely without effect. These data demonstrate, in conclusion, that, in the absence of appropriate susceptibility genes, alpha TBM-Ab are incapable of causing alpha TBM disease. The findings support previous observations that the ability of passively transferred alpha TBM-Ab to initiate interstitial injury is dependent on the host also expressing other susceptibility genes which promote the cooperative engagement of the cell-mediated immune response.     

Spontaneous interstitial nephritis in kdkd mice. II. Characterization of a tubular antigen-specific, H-2K-restricted Lyt-2+ effector T cell that mediates destructive tubulointerstitial injury

In this report, we have examined the effector T cell repertoire in the spontaneous interstitial nephritis of kdkd mice. Lymph node cells from nephritic kdkd mice are capable of transferring this disease into thymectomized, irradiated, and bone marrow-reconstituted CBA/Ca recipients. CBA/Ca mice do not spontaneously develop interstitial nephritis and are normally resistant to the adoptive transfer of nephritic cells, a resistance that in the short term can be attenuated with low-dose cyclophosphamide. We therefore used delayed-type hypersensitivity responses and direct transfer of immune cells under the renal capsule to characterize nephritogenic effector cells from kdkd donor mice. Lyt-2+, L3T4- T cells from the peripheral lymphoid organs of nephritic kdkd mice, after adoptive transfer into cyclophosphamide-pretreated CBA/Ca recipients, mediate an antigen-specific delayed-type hypersensitivity response to renal tubular basement membrane antigens. These cells are restricted by gene products in H-2Kk; they are also present in nephritic, but not in control kidneys. We have also observed this same phenotypic subpopulation of kdkd lymphocytes mediate a destructive interstitial renal lesion within 7 days of being placed under the kidney capsule of CBA/Ca mice. These findings suggest that T lymphocytes reactive to a parenchymal tubular antigen are of substantial importance in the development of spontaneous interstitial nephritis in kdkd mice.     

Murine interstitial nephritis. IX. Induction of the nephritogenic effector T cell repertoire with an antigen-specific T cell cytokine

The experiments presented in this report describe the biochemical and functional characteristics of a soluble Th cell factor (ThF) which can induce a nephritogenic effector T cell repertoire producing autoimmune interstitial nephritis. The ThF is Ag-specific, I-A-restricted, and comprises two chains noncovalently linked as a heterodimer. One chain at approximately 78,000 Mr is related to the TCR/Id and expresses a framework determinant (14-30) common to Ag-binding factors, and the other chain at approximately 82,000 Mr is I-A+. Together these chains can replace their parent cell by providing cognate help to precursor effector T lymphocytes in the presence of accessory cells, tubular Ag, and IL-2.     

Immunoregulation in experimental interstitial nephritis: immunization with renal tubular antigen in incomplete Freund's adjuvant induces major histocompatibility complex-restricted, OX8+ suppressor T cells which are antigen-specific and inhibit the expression of disease

We utilized a model of experimental interstitial nephritis induced by renal tubular antigen in complete Freund's adjuvant to examine a mechanism of immunologic tolerance produced by priming immunization with tubular antigen in incomplete Freund's adjuvant. Brown Norway rats primed with tubular antigen in incomplete adjuvant do not develop significant nephritis after challenge with antigen in complete adjuvant, and this tolerance can be transferred to naive recipients with donor T cells. These T cells also specifically suppress a delayed-type hypersensitivity response to soluble tubular antigen in recipients immunized to produce disease. This suppression is MHC-restricted and is mediated by OX8+ T cells which bind antigen and bear idiotypes cross-reactive with those on antibodies eluted from the tubular basement membrane. Despite the suppression of histologic disease, tolerized animals were able to produce significant titers of antibodies to tubular basement membrane. Our findings demonstrate an additional strategy for altering the natural history of immune-mediated renal disease, and further refine the characterization of the suppressive effect produced by incomplete Freund's adjuvant.     

Murine interstitial nephritis. IV. Long-term cultured L3T4+ T cell lines transfer delayed expression of disease as I-A-restricted inducers of the effector T cell repertoire

In the present study we observed that long-term cultures of tubular antigen-reactive L3T4+ T cells from immune SJL mice are able to adoptively transfer interstitial nephritis by 12 wk after i.v. injection. Lesions that develop under these conditions generally occur in the absence of anti-tubular basement membrane antibody formation. These cultured T cell lines are I-A restricted, require L3T4-associative interactions, and are tubular antigen specific, but do not share the phenotype, function, or H-2-restriction characteristics of Lyt-2+ nephritogenic effector T lymphocytes. Rather, our L3T4+ T cell lines, and phenotypically similar lymphocytes harvested from renal infiltrates, are inducers of this Lyt-2+ effector T cell repertoire. Such effector T cells, typically found in immune lymph nodes 4 to 7 days after immunization, can be induced in vitro within 5 days and will acutely transfer disease within another 5 days when placed under the kidney capsule. These findings collectively indicate that the time course for optimal effector cell differentiation and potential expression is relatively short. The immunologic inertia we previously observed between immunization or i.v. adoptive transfer and the development of cellular lesions, therefore, seems to reside in other systemic or interactional events beyond the timely formation of effector T cells.     

Heterologous antisera to Lyt-1+, 2- -derived antigen-binding factor detect a subfactor of an antigen-specific suppressor factor and cell surface proteins on Lyt-1+, 2- and Lyt-1-, 2+ T cells

Rabbits were immunized with TNP-specific Lyt-1+, 2- T cell-derived, antigen-binding proteins (PCI-F) released by T cells sensitized by skin painting with picrylchloride. The resulting antiserum (anti-PCI-F) bound to PCI-F and TNP-specific factors that suppressed delayed hypersensitivity (TSF) known to be comprised of PCI-F and Lyt-2+ -derived polypeptides released by cells sensitized by injection of trinitrobenzenesulfonic acid (TNBSF). Anti-PCI-F bound to T lymphocytes and 68,000 to 72,000 m.w. T cell surface proteins but not B cells on their surface proteins. Anti-PCI-F bound to both Lyt-1+ and Lyt-2+ T cells and surface proteins. A comparison of anti-PCI-F with anti-TSF indicates that anti-TSF contains specificity for Ly-2+ T cell-derived components of TSF and T cells not present in anti-PCI-F. The possibility of multiple isotypes of T cell receptors and antigen-binding molecules is discussed.     

Dual regulation of resistance against Toxoplasma gondii infection by Lyt-2+ and Lyt-1+, L3T4+ T cells in mice

Studies were performed to attempt to define the T cell subset responsible for resistance to Toxoplasma gondii. A temperature-sensitive mutant (ts-4) strain of T. gondii was used for immunization because it causes infection but does not persist in the host. Immunization with this strain induced marked resistance against lethal challenge infection with virulent strains of T. gondii in mice. The resistance could be transferred to normal recipient mice by i.v. injection of spleen cells from ts-4-immunized mice. Marked inhibition of cyst formation in the recipient mice was also noted. The protective activity of immune spleen cells was removed by pretreatment of the spleen cells with anti-Thy-1.2 and C, indicating that T cells are responsible for the observed protection. Pretreatment of immune spleen cells with anti-Lyt-2.2 and C completely ablated their protective effect; pretreatment with anti-Lyt-1.2 or anti-L3T4 and C had lesser effects on their ability to transfer resistance. The effect of anti-Lyt-1.2 was the same as that obtained with anti-L3T4. This suggested that one T cell subset that is partially responsible for protection has both Lyt-1.2 and L3T4 markers on the cell surface. These results indicate that there are substantial roles for both the Lyt-2+ and Lyt-1+, L3T4 T cell subsets in dual regulation of resistance against toxoplasma infection and that Lyt-2+ T cells are the principal mediator of the resistance.     

Lyt-2- T cell-independent functions of Lyt-2+ cells stimulated with antigen or concanavalin A

Approximately 30% of cytolytic Lyt-2+ clones from primed mice are able to proliferate autonomously, i.e., independent of IL 2 derived from Lyt-2- cells after antigenic stimulation. H-2K- or -D-restricted induction of Lyt-2+ cells to autonomous proliferation requires Ia+ stimulator cells. A strict correlation was observed between the ability of Lyt-2+ clones to proliferative autonomously and to induce DH. Eventually, the growth of all Lyt-2+ cytolytic clones becomes dependent on exogenous IL 2, and their ability to induce DH is lost. Small Lyt-2+ cells can also be induced in primary cultures by antigen or concanavalin A to proliferate in the absence of exogenous IL 2. The frequency of autonomously proliferating small Lyt-2+ cells is the same as that of small Lyt-2+ cells proliferating in the presence of exogenous IL 2. IL 2 derived from Lyt-2- cells can augment proliferation of Lyt-2+ cells, but is not obligatory.     

Murine interstitial nephritis. VII. Suppression of renal injury after treatment with soluble suppressor factor TsF1

We prepared soluble suppressor T cell factor (TsF1) from donor spleens harvested from mice primed with tubular antigen-derivatized lymphocytes to analyze both its functional interactions with a larger suppressor T cell network and its influence on the nephritogenic effector T cell response producing interstitial nephritis to a parenchymal antigen. Our findings indicate that TsF1 is antigen-specific, genetically restricted by I-J in its direct mediation of suppression, and capable of inhibiting the development of interstitial lesions. TsF1 also provides an inducing signal for the activation of effector Ts-2 suppressors following presentation by accessory cells. The induction of a Ts-2 effect, however, requires that the factor-presenting cell and the recipient of such cells share homology at I-J, and that the TsF1, the precursor Ts-2 cells, and the recipient of the Ts-2 effect share the same Igh-V allotype. Finally, the results of this current report clearly demonstrate a possible therapeutic role for soluble suppressor factors in the management of interstitial renal disease.     

Nonspecific inhibitor of DNA synthesis elaborated by T acceptor cells. I. Specific hapten- and I-J-driven liberation of an inhibitor of cell proliferation by Lyt-1-2+ cyclophosphamide-sensitive T acceptor cells armed with a product of Lyt-1+2+-specific suppressor cells

Lyt-1+2+ hapten-specific T suppressor cells (Ts) from mice injected and then painted with picryl or oxazolone derivatives produce hapten-specific T suppressor factors (TsF) in vitro. Stimulation by painting with contact sensitizer (which need not be specific) gives rise to Lyt-1-2+, I-J+, cyclophosphamide-sensitive T acceptor cells (Tacc). When the Tacc population is armed with TsF and then is exposed to specific antigen in the context of I-J-controlled determinants (antigen-presenting, haptenized spleen cells and Ts sharing the same I-J subregion), a nonspecific inhibitor of DNA synthesis (nsINH) appears in the supernatant. This inhibitor suppresses the primary DNA synthetic response to concanavalin A, lipopolysaccharide, and alloantigens in both syngeneic and allogeneic lymphocytes. The nsINH is only effective when added to lymphocyte cultures less than 8 hr after the stimulation with concanavalin A. The nsINH, however, affects neither primary nor secondary cytotoxicity in vitro. These data suggest the mouse immune system is capable of selective regulation of the response to specific antigen by the production of nonspecific soluble suppressor factor(s).     

Demonstration of antigen-specific suppressor cells in unresponsive nude mice: a model for the induction of antigen-specific suppressor cells

Nude spleen cells were rendered incapable of giving an in vitro response in the presence of TRF by pretreatment of nude mice with antigen, followed 1 week later by T-cell injection. It is shown that this unresponsiveness is caused by antigen-specific suppressor cells which affect not only the stimulation of nude spleen cells but also the activation of memory cells and the production of a T-cell-replacing factor. The appearance in nude mice of suppressing activity rather than helper activity, after administration of T cells, is dependent on the sequence of treatment. These results suggest a model for the induction of antigen-specific suppressor cells by activated B cells.     

The effect of anti-IFN-gamma antibody on the protective effect of Lyt-2+ immune T cells against toxoplasmosis in mice

The effect of an IFN-gamma mAb on the protective activity of immune T cells against Toxoplasma infection was examined in a murine model of toxoplasmosis. Mice that received anti-IFN-gamma antibody and immune spleen cells all died of toxoplasmosis after challenge with Toxoplasma tachyzoites. In contrast, mice that received normal IgG and immune spleen cells all survived the infection. The protective activity of Lyt-2+ immune T cells, previously shown to be the principal mediators of resistance against Toxoplasma in mice was completely ablated by the anti-IFN-gamma mAb. These results suggest that IFN-gamma is the major mediator of the resistance against Toxoplasma infection in mice which is conferred by immune T cells.     

A role for Lyt-2+ T cells in resistance to cutaneous leishmaniasis in immunized mice

The role of Lyt-2+ T cells in immunologic resistance to cutaneous leishmaniasis was analyzed by comparing infection patterns in resistant C57BL/6 mice and susceptible BALB/c mice induced to heal their infections after sub-lethal irradiation or i.v. immunization, with similar mice treated in vivo with anti-Lyt-2 antibodies. Administration of anti-Lyt-2 mAb resulted in a dramatic reduction in the number of lymphoid cells expressing the Lyt-2+ phenotype. Such treatment led to enhanced disease in both resistant C57BL/6 and irradiated BALB/c mice, as assessed by lesion size, but did not affect the capacity of these mice to ultimately resolve their infections. In contrast, anti-Lyt-2 treatment totally blocked the induction of resistance in i.v. immunized mice. These results suggest, that Lyt-2+ T cells may play a role in immunity to a Leishmania major infection and that their relative importance to resistance may depend on how resistance is induced.     

T suppressor factor activity is due to two separate molecules. The Lyt-1(-)2+I-J+ cells of mice primed with antigen make an antigen binding molecule which is only active when complemented by cofactor made by Lyt-1+2(-)I-J+ cells

Mice primed with picrylsulfonic acid (PSA) and then painted on the skin with picryl chloride produce antigen-specific T suppressor factor (TsF). In contrast unpainted primed mice fail to produce active TsF. This is not due to the absence of the antigen binding part of TsF but to the absence of a cofactor. This cofactor is (a) antigen nonspecific and occurs in potassium chloride extract of normal spleen cells. It also occurs in the 24 hr supernatant of normal cells modified by haptenisation with picryl or the unrelated NP antigen (4-hydroxy-3-nitrophenylacetyl), and in preparations of conventional TsF (PSA/PCl) from painted PSA-primed mice; (b) bears I-J determinants; and (c) is produced by Lyt-1+2(-)I-J+ cells. The antigen binding molecule occurs alone in the supernatant of PSA-primed mice. It lacks I-J determinants and has a molecular weight around 35,000 and 75,000. It is produced by Lyt-1(-)2+I-J+ cells and is only active when complemented by cofactor. However, the complementation is genetically restricted and the restriction maps to the I-J subregion of the MHC.     

Antigen-specific Lyt-2+ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.     

Regulation of granulomatous inflammation in murine schistosomiasis. III. Recruitment of antigen-specific I-J+ T suppressor cells of the granulomatous response by I-J+ soluble suppressor factor

Down-modulation of the schistosome egg-induced granulomatous response involves various interacting subsets of T suppressor (TS) lymphocytes. In the present study the inductive phase of the process of modulation was analyzed. A soluble, I-J+ granuloma TS cell recruiting factor (Gr-TSRF) derived from spleen cells of chronically infected mice is described. This factor eluted from immunoabsorbent columns coupled with anti-I-Jk alloantisera induced the recruitment and expansion of antigen-specific I-J+ TS cells from a TS precursor cell population in the spleens of acutely infected mice. The recruited TS cells suppressed the granulomatous response of normal recipients in a 2-day adoptive transfer model. The antigenic specificity of the recruited TS cells was demonstrated by their inability to suppress KLH-induced artificial granulomatous response. This mechanism of recruitment described in the current study and illustrated by adoptive transfer experiments is likely to be active in vivo in initiating the process of spontaneous modulation. The I-J+ Gr-TSRF and the I-J+ TS cell described in this paper, together with the previously described H-2 restricted I-C+ factor and the subsets of TS cells (THs, TSe, TSpr), indicate the existence of an intricate, regulatory pathway(s) that operates during the modulation of the granulomatous response.     

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). I. Analysis of bulk populations and establishment of Lyt-2+L3T4- and Lyt-2-L3T4+ bulk CTL lines

Murine allogeneic cytolytic T lymphocytes (CTLs), including long-term bulk CTL lines, were induced in I-region-incompatible combinations of strains in vitro in order to study the phenotypes of class II major histocompatibility complex (MHC) antigen-specific CTLs, as well as the possible functional involvement of accessory cell interaction molecules such as Lyt-2 and L3T4. This report shows that class II-specific allogeneic CTL populations consist of two types of T cells. Lyt-2+L3T4- (2+4-) and Lyt-2-L3T4+ (2-4+), in variable proportions depending on the strain combination, that in vitro bulk CTL lines with each of these phenotypes can be established, that the killing function of 2-4+ CTL is sensitive to the blocking effect of anti-L3T4 antibodies, suggesting functional involvement of this molecule in the CTL-target interaction, that anti-Lyt-2 antibodies fail to block killing by 2+4- cells, suggesting that such CTLs do not utilize this molecule in their killing function, and that while I-A-specific CTLs of both phenotypes are detectable, 2-4+ cells could not be detected among I-E-specific CTL populations.     

Cutting edge: increased expression of Bcl-2 in antigen-specific memory CD8+ T cells

Bcl-2 plays a critical role in regulating cell survival and apoptosis. We examined Bcl-2 expression in virus-specific CD8 T cells during the expansion, death, and memory phases of the T cell response following infection of mice with lymphocytic choriomeningitis virus (LCMV). Naive CD8 T cells expressed a basal level of Bcl-2 that was down-regulated in effector CD8 T cells just before the death phase. Bcl-2 levels remained low during the death phase but surviving memory CD8 T cells expressed higher levels of Bcl-2 than naive cells. These changes were shown to occur in LCMV TCR transgenic cells as well as virus-specific CD8 T cells in C57BL/6 and BALB/c mice identified by MHC class I tetramers. In all instances, memory CD8 T cells expressed higher levels of Bcl-2, suggesting that increased Bcl-2 expression plays a role in the long-term maintenance of memory CD8 T cells in vivo.