Characteristics of a spontaneous monoclonal thymocytotoxic antibody from New Zealand Black mice: recognition of a specific NTA determinant

Naturally occurring thymocytotoxic autoantibodies (NTA) have been described both in humans and in mice with SLE, and have been reported to be preferentially reactive with T suppressor as compared to T helper cells. However, although NTA has been shown by some groups of investigators to induce autoantibodies in normal strains of mice, other researchers have suggested that NTA has only a minor, if any, role in murine lupus. We have been studying the characteristics of a monoclonal antibody (TC-17) derived from the fusion of 4-mo-old NZB spleen cells with P3-X63-AG8.653 plasmacytoma cells. This monoclonal IgM reagent is cytotoxic for approximately 40% of total thymocytes, 50% of cortical thymocytes, less than 1% of cortisol-resistant thymocytes, 10% of splenocytes and lymph node cells, and less than 3% of bone marrow and fetal liver cells. The thymocytotoxicity can be absorbed by thymocytes but not by brain cells. Although NZB, NZW, NFS, and BALB/c thymocytes all manifest reactivity with TC-17, there was considerable difference between strains with respect to antigen density; NZB thymocytes have the highest density. By FACS analysis, TC-17 occurs independently of Lyt-1, Lyt-2, and T helper cell-specific antigens, and is more prevalent on larger proliferating thymocytes. TC-17 augments the response to SRBC but does not influence responses to TI-1 (TNP-BA) or TI-2 (DNP-Ficoll) antigen and production of LPS-induced B cell colonies. We believe that TC-17 recognizes a new T cell antigen, probably one involved in T cell differentiation. Because this monoclonal NTA reacts with only 40% of thymocytes, and is not absorbed with brain, it would not have been detected in mouse sera by using previously published methods. NTA are a heterogeneous group of autoantibodies; some specificities such as TC-17 went unrecognized in the past, and may be important either for disease pathogenesis or for secondary immunologic abnormalities.     

Autoantibodies to the thymus and skin epithelium in NZB/N mice and (NZBxNZW)F1 hybrids

Antibodies reacting with thymus and skin epithelial cells were revealed by indirect immunofluorescence in sera of NZB/N mice and (NZB X NZW)F1 hybrids (B/W) 1-2 and 4-5 months of age. Similar antibodies were not found in sera of BALB/c mice. The inhibition experiments with DNA have shown that antibodies reacting with the thymus and skin epithelium differ from those reacting with the cellular nucleus. Positive reactions with the epithelium were obtained in all thymus and skin tissue samples of humans, guinea-pigs and NZB/N, B/W and BALB/c mice, including autologous tissues of NZB/N and B/W mice. Thus, antibodies reacting with thymus and skin epithelial tissues belong to autoantibodies. These autoantibodies are revealed during the first month of life before the onset of autoimmune processes. The role of these autoantibodies in the damage of thymus epithelium and the development of immunoregulatory disturbances, typical of autoimmune processes, needs further study.     

A peripheral and central T cell antigen recognized by a monoclonal thymocytotoxic autoantibody from New Zealand black mice

Naturally occurring thymocytotoxic autoantibodies (NTA) have been suggested to be the cause of thymic atrophy and T cell disorders in human and murine lupus. Definitive studies on NTA's role in the induction of SLE, however, have been lacking due to the lack of a pure source of NTA. Although it is clear that NTA are a heterogeneous group of antibodies, the nature of their antigens has remained obscure. We report the characteristics of a monoclonal NTA, designated SAG-3, which appears more reflective of the activities previously reported of serum NTA than other NTA-secreting clones. SAG-3 is an IgM autoantibody cytotoxic for 80 to 90% of thymocytes, 20 to 25% of splenic lymphocytes, 25 to 30% of lymph node cells, and less than 3% cortisol-resistant thymocytes, bone marrow, and fetal liver cells. SAG-3 is murine-specific without reactivity towards rat, hamster, or guinea pig, and appears very early in thymic development, on day 17 fetal thymocytes. SAG-3 is equally cytotoxic against several strains of mice, including both Thy-1.1 and Thy-1.2 allotypes, and the cytotoxicity is absorbed by brain but not liver cells. Reactive thymocytes occurred throughout the cortical regions of the thymus, indicating preferential affinity towards immature thymocytes. Although the serologic activities of SAG-3 suggest that Thy-1 alloantigen is its target, SAG-3 antigen is found to be distinct from Thy-1 and also from Lyt-1, Lyt-2, or L3T4 antigens. The binding of SAG-3 to thymocytes could be competitively inhibited by NTA-positive NZB sera.     

Induction of autoimmune phenomena in normal mice treated with natural thymocytotoxic autoantibody

Natural thymocytotoxic autoantibody (NTA) developed spontaneously in New Zealand Black (NZB) mice consists of two autoantibodies in terms of target cell specificity. One of the autoantibodies, NTA-2, is strongly cytotoxic only against desialized lymphocytes, whereas the other one, NTA-1, is cytotoxic against both intact thymocytes and asialolymphocytes. To study the pathogenic role of NTA in murine autoimmunity, DBA/2 mice were injected every other day with affinity-purified NTA (NTA-1, NTA-2). Control mice received normal mice sera (NMS) or saline. After 20 days of treatment, spleen cells from DBA/2 mice treated with NTA-1 or NTA-2 showed a significant increase in the number of anti-ssDNA plaque-forming cells and IgM-producing cells. Sera from NTA-treated mice showed greater DNA binding than sera from control mice did. The levels of proteinuria were moderately increased in NTA-2-treated mice. Con A responsiveness of thymocytes was markedly reduced in NTA-2-treated mice. On the other hand, Con A-activated spleen cells from both control and NTA-treated mice equally suppressed anti-SRBC antibody production in vitro, suggesting that NTA treatment didn't affect the direct precursors of suppressor T cells. Finally, prior absorption of NTA-1 by thymocytes prevented its ability to induce anti-DNA antibodies; however, prior absorption of NTA-2 by thymocytes didn't affect its activity.     

Developmental abnormalities of terminal deoxynucleotidyl transferase positive bone marrow cells and thymocytes in New Zealand mice: effects of prostaglandin E1

The enzyme TdT was used as a marker with which to study the ontogeny of primitive lymphopoietic cells in NZ strain mice. A marked accumulation of abnormally large, rapidly proliferating TdT+ cells was seen in the subcapsular region of the thymus cortex in the NZB and NZB/W mice. This abnormal accumulation of TdT+ thymocytes was most pronounced in the NZB/W hybrid and persisted for at least the first 16 wk of life. In addition, significantly elevated percentages of TdT+ bone marrow cells (presumptive prothymocytes) were present in NZB, NZW, and NZB/W mice between 1 and 4 wk of age, with the highest mean peak levels occurring in the NZB strain. Treatment of both normal and adrenalectomized BALB/c and NZB/W mice with pharmacologic doses (7 to 10 mg/kg) of PGE1 caused a marked, dose-dependent decrease in thymus weight and thymus cell number within 12 to 18 hr. Histologic and cell separation studies showed that this was due to the selective depletion of PNA+ TdT+ cortical thymocytes. Similarly, PGE1 caused a reversible, dose-dependent decrease in the percentage of TdT+ bone marrow cells. In contrast, PGF2 alpha, which is not therapeutically active against autoimmunity in NZB/W mice, had no detectable effect on TdT+ bone marrow cells or thymocytes in BALB/c or NZB/W mice. These results directly document the existence of abnormalities in the development of lymphopoietic precursor cells in the bone marrow and thymus cortex of NZ strain mice prior to the onset of autoimmune phenomena. The results also raise the possibility that the therapeutic efficacy of exogenous PGE1 in autoimmune NZ strain mice may be related, at least in part, to its ability to rectify the abnormal development of these early lymphoid cells.     

Delineation of spontaneous erythrocyte autoantibody responses of NZB and other strains of mice.

Four anti-erythrocyte autoantibody responses (anti-X, anti-HB, anti-HOL, and anti-I) that occur spontaneously in mice have been characterized with regard to antigenic specificities, predominant immunoglobulin class, and pathogenetic importance. Each autoantibody response exhibits specificity for an independent erythrocyte membrane autoantigen (X, HB, HOL, or I) or a soluble analogue (SEA-X or SEA-HB) present in the plasma. The anti-X response, unique to NZB mice, is directed to a normally exposed murine erythrocyte autoantigen, whereas the anti-HB response is directed to a cryptic erythrocyte autoantigen exposed by limited enzymatic cleavage of the membrane. The anti-I response also is directed to a cryptic but distinct autoantigen, and anti-HOL autoantibodies react with an erythrocyte autoantigen located at the cytoplasmic surface of the membrane. Analysis of the predominant immunoglobulin class of each of the autoantibodies has demonstrated that anti-HB and anti-I antibodies are predominantly of IgM class, whereas anti-X and anti-HOL antibodies are IgG immunoblobulins. Only anti-X and anti-HB autoantibodies are recovered from Coombs' positive erythrocytes from NZB mice and erythrocytes with surface C3 are detected only in NZB mice greater than 9 months of age. These data suggest that only the anti-X and anti-HB responses are pathogenetically implicated in the autoimmune hemolytic anemia of NZB mice.     

Evidence that autoimmunity in NZB mice is caused by a defect in immune specificity rather than a generalized defect in tolerance of self-antigens

NZB mice which were already producing anti-erythrocyte autoantibodies were not able to respond to their own liver F antigen, thus providing evidence that their autoimmunity is not caused by a generalized breakdown in self-tolerance mechanisms. The specificity of autoantibodies produced in the spontaneous hemolytic anemia was different from that of antierythrocyte antibodies induced in normal mice and in young NZB mice by injections of rat erythrocytes. This indicates that the B-cell clones which can be triggered by heterologous antigen are different from those responsible for the NZB disease; the latter clones may not exist in normal mice.     

Natural thymocytotoxic autoantibodies in autoimmune and normal mice.

Natural thymocytotoxic autoantibodies (NTA) were found in all mouse strains. Among those strains that show autoimmune syndromes resembling human systemic lupus erythematosus (SLE), the NZB and NZBxNZW had high levels of NTA, the BXSB had moderate levels, and the MRL/1 and MRL/n had very low levels. In addition, some normal strains had high levels, sometimes even higher than the autoimmune strains. The NTA were mostly IgM and were present, but not concentrated, in the cryoprecipitates of teh autoimmune mouse strains. In most strains, they were directed toward an antigen shared by thymocytes and brain. The failure to find high levels of NTA in all autoimmune mouse strains, as well as the finding of very high levels in some normal strains, make it unlikely that such auto-antibodies are a fundametnal etiologic factor in all murine SLE.     

Parasite specific antibodies in three strains of mice after infection with Trypanosoma cruzi

Three inbred strains of mice (BALB/cJ, C3H/HeJ and NZB/BInJ) were infected with trypomastigotes of Trypanosoma cruzi. Sera were taken at different times after infection and radioimmunoprecipitation assays were used to detect antibodies against individual T. cruzi epimastigote and trypomastigote antigens. The mouse strains differed in regard to the spectrum of antibodies and the time after infection when the various epimastigote specific antibody species appeared. NZB mice had antibodies against at least 25 polypeptides ranging in molecular weight from 20,000 to 90,000 D at 3 wk after infection, and these persisted until at least 10 wk post-infection. C3H and BALB/c had antibodies against fewer than 5 antigens at 3 wk after infection; whereas by week 10, antibodies against at least 25 polypeptides were detected. C3H mice that were most susceptible to infection (but not NZB or BALB/c mice) had antibodies against a 25,000 D molecular weight epimastigote antigen. The antibody response against trypomastigote polypeptides was more uniform. Sera from all mouse strains at 3 wk after infection precipitated the same polypeptides and the radioimmunoprecipitation patterns did not change as a function of time after infection.     

IgG anti-DNA autoantibodies within an individual autoimmune mouse are the products of clonal selection

Although mice from almost all inbred strains produce IgM anti-DNA antibody in response to B cell mitogens, only (NZB x NZW)F1 mice and mice from other strains that are genetically predisposed to autoimmunity spontaneously produce anti-DNA antibody of the IgG isotype. Because (NZB x NZW)F1 mice display marked B cell hyperactivity, anti-DNA antibody production in these mice has been thought to result from spontaneous, polyclonal B cell activation. Although this may be true for IgM anti-DNA antibodies, our results demonstrate that IgG anti-DNA antibodies are not polyclonal. Rather, IgG anti-DNA autoantibodies within an individual autoimmune mouse are oligoclonal and somatically mutated. These results demonstrate that IgG anti-DNA autoantibodies are the products of clonally selective B cell stimulation and exhibit the same characteristics as secondary immune antibodies to conventional immunogens: they are IgG, they are clonally restricted, and they are somatically mutated.     

Tissue localization and biochemical characteristics of a new thymic antigen recognized by a monoclonal thymocytotoxic autoantibody from New Zealand black mice

Naturally occurring thymocytotoxic autoantibodies (NTA) have been described in both humans and mice with SLE. To define further the role of anti-thymic autoantibodies in murine lupus, we studied the cellular and molecular specificity of a spontaneous monoclonal NTA, designated TC-17, derived from a 4-mo-old New Zealand Black mouse. TC-17, an IgM autoantibody, has been shown previously to be unreactive with Lyt-1, Lyt-2, and L3T4 (T helper) antigens. We have shown further that it is also unreactive with Thy-1. TC-17 recognizes a new thymic antigen that appears to mark a distinct subpopulation of cortisol-sensitive cortical thymocytes. The antigen consists of a single glycoprotein chain with an apparent m.w. of 88,000. TC-17 shows reduced binding to thymocytes treated with tunicamycin, indicating either that glycosylation of TC-17 antigen is necessary for TC-17 to bind to it or that glycosylation is required for expression of the antigen on the cell surface. TC-17 uniquely reacts with two of 17 murine lymphoid tumor cell lines of intermediate cellular maturity. The thymocytotoxic activity of TC-17 is absorbed by single cell suspensions of murine stomach, small intestine, large intestine, kidney, and thymus. Moreover, the specific binding of TC-17 to gut tissue of normal and germfree mice can be demonstrated by indirect immunofluorescence, suggesting antigenic cross-reactions between thymic and gut tissue. TC-17 reacts with rat thymocytes as well as it does with murine cells, indicating moderate evolutionary conservation of the TC-17 antigen. The expression of this glycoprotein by a discrete thymocyte subset may prove to be a valuable probe for the study of murine T cell differentiation.     

Culture and characterization of thymic epithelium from autoimmune NZB and NZB/W mice

Autoimmune NZB and NZB/W mice display early abnormalities in thymus histology, T cell development, and mature T cell function. Abnormalities in the subcapsular/medullary thymic epithelium (TE) can also be inferred from the early disappearance of thymulin from NZB. It has also been reported that NZB thymic epithelial cells do not grow in culture conditions that support the growth of these cells from other strains of mice. In order to study the contribution of TE to the abnormal T cell development and function in NZB and NZB/W mice, we have devised a culture system which supports the growth of TE cells from these mice. The method involves the use of culture vessels coated with extracellular matrix produced by a rat thymic epithelial cell line. TEA3A1, and selective low-calcium, low-serum medium. In addition TEA3A1 cells have been used as an antigen to generate monoclonal antibodies specific for subcapsular/medullary TE. These antibodies, as well as others already available, have been used to show that the culture conditions described here select for cells displaying subcapsular/medullary TE markers, whereas markers for cortical TE and macrophages are absent.     

Studies of congenitally immunologically mutant New Zealand mice. IX. Age-related microenvironmental effects on autoantibody production in NZB and NZB.Xid mice studied by transplantation

The mechanism of polyclonal expansion of B cells and subsequent autoantibody production in New Zealand mice remains a critical question. We have been studying the requirements for autoantibody production both in NZB mice as well as NZB mice congenic with the Xid gene of CBA/N mice. In this study, we have attempted to alter the immunologic phenotype of NZB.Xid mice by transfer of cells from young and old NZB mice. There was little difficulty in restoring normal levels of serum IgM, IgG3, splenic Lyb-5 cells, and response to DNP-Ficoll in young NZB.Xid mice that were injected with young NZB bone marrow cells. Although such animals had an almost immediate change in their immune profile to values characteristic of NZB mice, they required, much like unmanipulated NZB mice, a latency period of an additional 6 mo before autoantibodies were detected. In contrast, adult NZB.Xid mice, who likewise developed an immune profile similar to NZB after transfer of bone marrow cells from young NZB mice, began to express autoantibodies immediately without any latency period. NZB.Xid mice who were recipients of adult NZB bone marrow cells did not show sustained autoantibody production, reflecting the limited state of B cell precursors in adult NZB mice. Thus, the age of both donor cells and the age of recipient mice are critical factors for determining the latency period and the age at which autoantibodies will appear. Similarly we attempted to alter the production of autoantibodies in NZB mice that were irradiated and injected with bone marrow cells from NZB.Xid animals. NZB mice had a major amelioration of disease when they received cell transfers from young NZB.Xid mice. This amelioration, which included the acquisition of the immune profile of NZB.Xid animals, was not seen in adult NZB mice that were recipient of young NZB cells. We suggest that although Lyb-5 cells may be the effective mechanism for autoantibody production, there are other interacting influences that may selectively turn on or turn off autoantibodies and that are required and are responsible for the latency period.     

Specific antigen-binding and antibody-secreting lymphocytes associated with the erythrocyte autoantibody responses of NZB and genetically unrelated mice.

The receptor characteristics as well as incidence of antigen-binding lymphocytes (ABL) or B and T cell classes with membrane receptors specific for the exposed (X) and cryptic (HB) murine erythrocyte autoantigens were examined in NZB and nine control strains of mice. Whereas only NZB and NZB hybrid mice synthesize anti-X autoantibody pathogenetically implicated in the genetically determined autoimmune hemolytic anemia, the NZB as well as control strains synthesize the ubiquitous anti-HB anti-erythrocyte autoantibody. By utilizing immunocytoadherence assays, maximum numbers of specific ABL of both B and T lymphocyte classes were optimally demonstrated at erythrocyte:lymphocyte ratios of 20:1 and after lymphocyte fixation at 56 degrees C for 20 min. Surface membrane receptor specificity was established by inhibition with semi-purified soluble X or HB autoantigen. Inhibition of immunocyto-adherence with class specific antisera to mouse immuno-globulins demonstrated that the receptors on both B and T cells were of IgM class. Specific receptors regenerated in vitro after trypsinization which excluded the role of cytophilic antibody in the immunocytoadherence reactions. B lymphocyte ABL reactive with the X autoantigen were demonstrable in NZB, NZB hybrid, and control mice. Only in NZB and NZB hybrid mice, strains that uniformly synthesize anti-X autoantibody, were X ABL of T lymphocyte class demonstrated. The presence and incidence of T lymphocyte X ABL is compatible with the expression of a single dominant gene carried by the NAB strain. The incidence of B lymphocyte X ABL increased with age, suggesting proliferation of this cell population. HB ABL of both B and T lymphocyte classes were observed in all strains, concordant with the ubiquitous presence of humoral anti-HB autoantibodies. Differentiation of precursor B cells are evaluated by PFC assay of cells secreting specific autoantibodies. Anti-X PFC were observed only in NZB and NZB hybrid mice; and the observed frequency suggested that less than 3.5% of the specific ABL were differentiated for the secretion of anti-X autoantibody. Anti-HB PFC were observed in all strains and represented as high as 11.8% of specific ABL. Genetic determination of the anti-X anti-erythrocyte autoantibody response does not prescribe the presence of precursors of the antibody-forming cell, but rather appears to influence regulation of the differentiation of these cells. These data suggest that circumvention of immunologic tolerance to this specific erythrocyte autoantigen may occur at the level of the T lymphocyte; or alternatively, that T lymphocytes as well as B lymphocytes, are induced to proliferate and differentiate in the NZB strain.     

The regulatory role of NZB T lymphocytes in the production of anti-DNA antibodies in vitro

Murine lupus is characterized by the production of numerous autoantibodies and immune complex glomerulonephritis. Anti-DNA antibodies are the hallmark of this disorder and may be associated pathogenetically with the glomerulonephritis. The cellular mechanisms underlying the regulation of the production of anti-DNA antibodies may prove to be the fundamental abnormalities responsible for the lupus syndrome seen in these mice. By using a system of spontaneous anti-DNA antibody production in vitro, we have determined that such production is characteristic of autoimmune NZB and MRL-lpr/lpr mice but not of the nonautoimmune control strains. Additional examination of the cellular mechanisms involved in the regulation of this response in NZB mice revealed: 1) this response is markedly T cell dependent, 2) NZB T cells are essential for maximal production of this autoantibody, and 3) NZB T cells actively interfere with normal immune regulatory mechanisms that lead to the production of anti-DNA antibodies spontaneously in vitro by nonautoimmune syngeneic B lymphocytes. Although these studies of anti-DNA antibody production in vitro disagree with previous work by others they successfully reproduce the results obtained earlier in experiments performed in vivo.     

Isolation and characterization of novel suppressor T cell clones from murine fetal thymus

Murine fetal thymus from C57BL/6J (B6) and DBA/2J contains a cell population that suppresses CTL responses to alloantigens. This suppressor cell population was found to exist in high frequency in murine fetal thymus at the 14th day of gestation. The activity of this cell in the thymus declined rapidly with increasing time of gestation, and suppressor activity in the thymus was undetectable by the time of birth. On the other hand, suppressor activity could be detected in organ cultures of 14-day fetal thymus even after the organs were cultured for 14 or 21 days. Fetal thymocytes from B6 or DBA/2J mice were grown as long-term lines in interleukin 2 (IL 2)-containing medium. Clones of suppressor cells were derived from long-term cultures by micromanipulation. The clones had an average doubling time of 13 to 16 hr and were dependent on IL 2 for growth. The clones were 10- to 100-fold more efficient in suppressing CTL responses to alloantigens than day 15 fetal thymocytes. Analyses of cell surface molecules with the use of monoclonal antibodies and conventional anti-H-2 sera by radioactive binding assays showed that cloned suppressor cells from B6 fetal thymus were Thy-1 and Lyt-2+, and expressed little or no L3T4, Lyt-1, H-2K, H-2D, and class II molecules. The suppressor clones lacked the cytolytic activity of conventional CTL and also served as very poor target cells in CTL-mediated cytolysis. The suppressor function of the cloned cells was radiation-resistant, and this suppression could not be reversed by the addition of excess exogenous IL 2. The cloned cells suppressed CTL responses only when they were added within the first 48 hr of a 5-day culture period. Analyses of the antigen specificity of the suppressor cells showed that they suppressed CTL responses in a nonantigen-specific manner.     

Autoantibody-secreting Plaque Forming Cells in Spleen and Thymus of NZB and Normal Mice

NZB mice develop several manifestations of autoimmune disease and, in particular, Coombs positive haemolytic anaemia¹ which can be accompanied by hepatosplenomegaly, glomerulonephritis and the appearance of antinuclear antibodies. The anaemia is associated with erythrocyte autoantibodies detected by direct and indirect antiglobulin tests and which from 3–4 months of age appear in an increasing proportion of mice until virtually all are involved by 9–12 months. Although there has been a good deal of work on the various disorders in NZB mice, the aetiology of the disease remains obscure². The thymus has been suggested as the site of origin of the disease³ and recently a viral cause for the autoimmune state was proposed⁴, but this evidence for these hypotheses has been equivocal^5–7. Although the spleen has been implicated as the chief site of autoantibody production⁸, no direct demonstration of the autoantibody secreting cells in NZB mice has been reported.     

Glycolipid markers of murine lymphocyte subpopulations.

We have shown previously that purified antibodies to ganglioside GM1 react with peripheral T cells and most thymocytes in several strains of mice, independent of Thy-1 phenotype. GM1 and the Thy-1.2 antigen cap independently on C3H thymocytes, which provides additional evidence that GM1 is not the Thy-1.2 antigen. In C3H and nude mice antibodies to GM1 also react with a population of cells, comprising about 25% of lymphocytes from lymph nodes or spleen, that bear surface immunoglobulin. After removal of immunoglobulin from these cells by digestion with proteolytic enzyme, the GM1+ cells regenerate their surface immunoglobulin during 18 hr in culture, which indicates that these double-labeled cells synthesize their surface immunoglobulin. Protease treatment of lymphocytes reveals receptors for antibodies to GM1 on most cells. These data indicate that T and B cells differ in the accessibility of GM1 to antibody, and not necessarily in their content of GM1. Purified antibodies to asialo GM1 react with mature T cells in all strains of mice tested. In contrast to anti-GM1, these antibodies do not react with most thymocytes, with immunoglobulin-bearing lymphocytes of C3H or nude mice, nor with pronase-treated B cells.     

Absence of cross-reactivity between murine Ly-6C and Ly-6G.

BACKGROUND: The Ly-6 family has many members, including Ly-6C and Ly-6G. A previous study suggested that the anti-Ly-6G antibody, RB6-8C5, may react with Ly-6Chi murine bone marrow (BM) cells. This finding has been interpreted as cross-reactivity of RB6-8C5 with the Ly-6C antigen, and has been generalized to many hematopoietic cell types, using the terminology Ly-6G/C. The present study was undertaken to determine whether anti-Ly-6G antibodies truly cross-react with the Ly-6C antigen on multiple hematopoietic cell types. METHODS: Splenocytes, thymocytes, and BM cells obtained from Ly-6.1 and Ly-6.2 strains of mice were stained with a variety of antibodies to Ly-6C and Ly-6G. Flow cytometric analysis was performed on these populations. RESULTS: Evaluation of anti-Ly-6C and anti-Ly-6G staining showed only Ly-6C expression and no Ly-6G expression on subsets of splenic T and B cells and thymocytes from Ly-6.1 and Ly-6.2 mice. Bone marrow cells were identified that express both Ly-6G and Ly-6C; no Ly-6G+Ly-6C- populations were seen. CONCLUSIONS: Multiple Ly-6C+ hematopoietic cell populations were identified that do not stain with anti-Ly-6G antibodies. This calls into question the use of the Ly-6G/C nomenclature and suggests that epitopes recognized by anti-Ly-6G antibodies should simply be designated Ly-6G.     

CD4+ T cell lines with selective patterns of autoreactivity as well as CD4- CD8- T helper cell lines augment the production of idiotypes shared by pathogenic anti-DNA autoantibodies in the NZB x SWR model of lupus nephritis

The (NZB x SWR)F1 hybrid mice (SNF1) uniformly develop lethal glomerulonephritis in marked contrast to their parents and produce nephritogenic autoantibodies that consist of highly cationic, IgG anti-DNA antibodies that share distinct cross-reactive idiotypes called IdLNF1 (idiotypes-lupus nephritis-SNF1). Herein we found that spleen cells of SNF1 mice at the late prenephritic stage, contained CD4+/CD8- and CD4-/CD8- Th that not only induced their B cells in vitro to produce highly cationic IgG autoantibodies to DNA but IdLNF1-positive IgG antibodies as well. The double-negative Th were unexpected in the SNF1 mice because they lack the lpr (lymphoproliferation) gene. We also derived IL-2-dependent CD4+/CD8- as well as CD4-/CD8- T cell lines from nephritic SNF1 mice, that could simultaneously induce IdLNF1-positive and cationic anti-DNA antibodies of IgG class. The CD4+ T cell lines consisted of "autoreactive" T cells, but not all of the lines were equal in autoantibody-inducing capability. Remarkably, the T cell lines that preferentially responded to F1-hybrid-MHC determinants, had a significantly greater ability to augment the production of pathogenic autoantibodies. The SNF1-Th could also augment autoantibody production by the NZB or SWR parent's B cells; however, IdLNF1-positive and cationic anti-DNA autoantibodies of IgG class were not induced, suggesting that the SNF1 mice possess a select population of inducible (susceptible) B cells that are committed to produce nephritogenic autoantibodies and the parental strains are deficient in such B cells. Thus, production of nephritogenic autoantibodies with IdLNF1 markers in the SNF1 mice could result from an interaction between a select population of B cells and CD4+ Th that preferentially recognize unique F1-hybrid-MHC determinants, as well as double-negative auxiliary Th.