Cultured Lyt-2- L3T4- T lymphocytes from normal thymus or lpr mice express a broad spectrum of cytolytic activity

T lymphocytes expressing the surface phenotype Lyt-2- L3T4- represent a minor population of immature thymocytes that appear to be the precursors of mature T cells. Cells with the same apparent surface phenotype also accumulate in vast numbers in the lymphoid tissues of the autoimmune lpr mouse. Lyt-2- L3T4- T lymphocytes from lpr lymph node (LN) or normal thymus express low to undetectable levels, respectively, of surface antigen receptor. In addition, they produce reduced amounts of lymphokines compared with normal T cells and lack precursors of alloantigen-specific cytolytic T lymphocytes. We previously showed that after culture with phorbol esters and interleukin 2, lpr Lyt-2- L3T4- T lymphocytes proliferate and differentiate, acquiring increased levels of surface antigen receptor by most cells, as well as Lyt-2 by a portion. We now show that cultured Lyt-2- L3T4- T cells from lpr LN or normal thymus are very efficiently cytolytic toward not only allogeneic tumor targets, but also natural killer (NK)-susceptible targets and syngeneic targets. Such killing was not inhibited by antibodies to H-2 or Lyt-2. In contrast, cultured mature Lyt-2+ L3T4- T cells from normal LN, thymus, or lpr LN were cytolytic only toward allogeneic targets and were dependent on Lyt-2 expression and H-2 recognition. The similarities of cultured Lyt-2- L3T4- T cells to NK and lymphokine-activated killer cells are discussed.     

CS-A therapy in MRL-lpr/lpr mice: amelioration of immunopathology despite autoantibody production

MRL-lpr/lpr mice spontaneously develop massive T cell lymphadenopathy, autoantibodies, and immune-mediated pathology. These mice are thought to be models of various human autoimmune diseases, including systemic lupus, Sjogren's syndrome, and rheumatoid arthritis. We have used cyclosporin A (CS-A) treatment as a tool by which the mechanisms of immune-mediated pathology might be dissected. CS-A was used because of its known preferential inhibition of T cell function and the marked expansion in MRL-lpr/lpr mice of an unusual L3T4-, Lyt-2-, 6B2+ T cell population. CS-A prevented lymphadenopathy and expansion of L3T4-, Lyt-2-, 6B2+ T cells in the peripheral lymph nodes, and also in the thymus. The increased expression of the c-myb and T cell receptor beta-chain genes associated with these unusual cells was also corrected. The finding of increased numbers of L3T4-, Lyt-2-, 6B2+ thymocytes in untreated mice suggests abnormal intrathymic differentiation in lpr/lpr mice, a defect that was corrected by CS-A. Treated mice had a marked decrease in arthritis and glomerulonephritis and significantly prolonged survival. These beneficial effects of CS-A occurred despite a lack of reduction in antibodies reactive with DNA, circulating immune complexes, rheumatoid factor titers, or immunoglobulin concentrations. These results demonstrate that the B cell hyperactivity of MRL-lpr/lpr mice can proceed without the T cell proliferative disease.     

Expression of the J11d marker on peripheral T lymphocytes of MRL-lpr/lpr mice

MRL-lpr/lpr (lpr) mice spontaneously develop massive lymphadenopathy resulting from the expansion of a unique population of Thy-1+ cells which are CD4- and CD8- (double negative) and the nature of which is not clear. The antibody J11d has been shown to define a differentiation Ag found on immature thymocytes but not on mature and functional peripheral CD4+ or CD8+ T cells. To analyze the possible relationship between the lpr double-negative T cells and the thymocytes, we investigated the simultaneous expression of J11d and Thy 1 Ag on the double-negative lpr lymph node cells by using two-color immunofluorescent staining technique. We observed that lpr mice at 3 to 4 weeks of age, before the onset of lymphadenopathy, did not have significant numbers (less than 4%) of J11d+ T cells in the periphery, similar to the number found in the control MRL +/+ mice. However, with increasing age of approximately 8 to 10 weeks and coinciding with the appearance of lymphadenopathy, a significant number (approximately 35%) of J11d+ Thy-1+ cells started appearing in the periphery of lpr mice and was maintained until the mice died at 20 to 24 weeks of age. The J11d+ T cells belonged to the abnormal double-negative T cell pool, inasmuch as J11d+ CD4+ or J11d+ CD8+ cells were absent in the lymph nodes of 20-wk-old lpr mice. Furthermore, 20-wk-old lpr mice demonstrated increased numbers (approximately 41%) of double-negative T cells in the thymus, a significant proportion of which were J11d+. In contrast, the 20-wk-old +/+ mice or 4-wk-old lpr mice had only 4% double-negative T cells in the thymus. The present study suggests that a significant number of peripheral double-negative T cells of lpr mice bear the immature thymic differentiation Ag J11d. The possibility that the accumulation of double-negative T cells results from abnormal peripheralization of double-negative J11d+ thymocytes, before complete differentiation into CD4+ or CD8+ T cells, is discussed.     

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.     

Growth and differentiation in vitro of the accumulating Lyt-2-/L3T4- subset in lpr mice

Mice bearing the recessive gene lpr develop an autoimmune syndrome associated with a massive lymphadenopathy, both of which are age and thymus dependent. The predominant accumulating cells in lymphoid tissue of lpr/lpr mice are Thy-1+ but express neither of the mature T cell markers, Lyt-2 or L3T4. We have purified this Lyt-2-/L3T4- subset and examined its phenotype. These cells are not actively cycling, do not express interleukin-2 (IL 2) receptors nor significant levels of antigen receptor, but do express the B cell marker B220. In vitro growth conditions were examined for the lpr Lyt-2-/L3T4- subset. By using a combination of phorbol ester and IL 2, these cells acquired transient expression of IL 2 receptors and grew in an IL 2-dependent manner. Furthermore, these proliferating cells underwent differentiation to a more mature T cell phenotype, with loss of cell surface B220 and acquisition, by a portion, of antigen receptor and Lyt-2. The possible parallels with normal T cell maturation are discussed.     

Functional analysis of T cell subsets from mice bearing the lpr gene

The autosomal recessive lpr (lymphoproliferation) gene is responsible for a thymus-dependent massive lymphoproliferation associated with the development of lupus-like autoimmune disease. Phenotypic analysis of adult lpr/lpr lymph nodes has demonstrated accumulation of a dull Lyt-1+, Thy-1+ population that expresses neither Lyt-2 nor L3T4 antigens. With the use of a depletion method based on complement-mediated lysis with an anti-Lyt-2 monoclonal antibody (31 M) and a new anti-L3T4 monoclonal antibody (RL 172.4), we have purified the Lyt-2- L3T4- subset from lymph nodes or spleens of C57BL/6-lpr/lpr mice and determined whether they are immunologically functional in vitro. Production of neither interleukin 2 nor interferon-gamma was detected by the double-negative subset after stimulation with concanavalin A and/or phorbol myristate acetate. The frequencies of allospecific cytotoxic T lymphocyte (CTL) precursors and lectin-induced antigen-nonspecific CTL precursors were diminished to almost undetectable levels, whereas the Lyt-2+ population from lpr/lpr mice had CTL-precursor frequencies comparable with that of +/+ mice. These results show that the major cell subset of adult lpr/lpr lymph nodes or spleens is composed of lymphocytes with markedly limited potential for lymphokine production or antigenic stimulation.     

Unique surface phenotype of T cells in lymphoproliferative autoimmune MRL/Mp-lpr/lpr mice

MRL/l mice, which carry the mutant gene lpr, develop massive T cell lymphoproliferation and an autoimmune syndrome. The surface antigen profile of MRL/l lymphocytes was analyzed with rat monoclonal antibodies. They included YE1/9.9, anti-transferrin receptor; YE1/7.1, which reacts with a population of proliferating immature T cells; and YE1/19.1, a newly identified antibody reactive with a surface antigen on EL-4 cells, which consists of two disulfide-bonded polypeptide chains, each of 115,000 m.w. Flow cytometric analysis of lymphocytes from the enlarged lymph nodes of MRL/1 mice showed the majority of them to be Lyt-1+, YE1/7.1+, YE1/19.1+, YE1/9.9-. In contrast, YE1/7.1 and YE1/19.1 antibodies did not significantly react with lymphocytes from the congenic MRL/n mice, which lack the lpr gene and do not develop lymphoproliferation. Lymphocytes from younger MRL/l mice, which have almost normal size lymph nodes, were also YE1/7.1-, YE1/19.1-. A small proportion of mitogen-stimulated MRL/n lymph node cells appeared to express these antigens at low densities, but it is not known whether they represent a unique cell population. Among the several lymphoid cell lines tested, only the T lymphoma line EL-4 had the Lyt-1+, YE1/7.1+, YE1/19.1+ phenotype. These results suggest that the lpr mutation induces the expansion of a unique T cell subset.     

Phenotypic analysis of thymocytes that express homing receptors for peripheral lymph nodes

Thymocytes that express high levels of homing receptors for peripheral lymph nodes can be detected with the monoclonal antibody MEL-14. We have shown that in adult mice these rare MEL-14hi thymocytes a) are cortical in location and typically constitute 1 to 3% of the total thymocyte population, b) may be a major source of thymus emigrants, and c) contain a high frequency of precursors of alloreactive cytotoxic T lymphocytes. In this study we have analyzed the phenotype of the MEL-14hi thymocyte subset. Most normal adult MEL-14hi thymocytes are midsize and express the mature phenotype typical of thymus emigrants, medullary thymocytes, and peripheral T cells: they are predominantly PNAlo, H-2K+, Thy-1+, Ly-1hi, and either Lyt-2-/L3T4+ or Lyt-2+/L3T4-. These findings argue strongly for the presence of rare MEL-14hi immunocompetent cortical thymocytes that, aside from their homing receptor expression, are phenotypically indistinguishable from medullary thymocytes. However, a minority (20 to 30%) of MEL-14hi thymocytes are large and phenotypically nonmature: they express intermediate to high levels of PNA binding sites, and are H-2K- to H-2Klo, Thy-1hi, Ly-1+, and either Lyt-2+/L3T4+ or Lyt-2-/L3T4-. Through a technique that selectively labels outer cortical cells, phenotypically nonmature MEL-14hi thymocytes have been shown to be concentrated in the subcapsular blast region of the outer cortex. Although we have no direct evidence of a precursor-product relationship, we consider it likely that the phenotypically nonmature outer cortical MEL-14hi lymphoblasts give rise to phenotypically mature MEL-14hi cells located deeper in the cortex. These results are consistent with our previous proposal that MEL-14hi thymocytes are a major source of thymus emigrants, and indicate that expression of high levels of MEL-14-defined homing receptors may be closely linked to the intrathymic selection process.     

Stimulation of murine T cells via the Ly-6C antigen: lack of proliferative response in aberrant T cells from lpr/lpr and gld/gld mice despite high Ly-6C antigen expression

Cross-linking of cell surface Ly-6C molecules with the 6C3 rat monoclonal antibody (MAb) followed by anti-rat immunoglobulin antibody acts in concert with phorbol myristate acetate (PMA) as a potent mitogenic stimulus for normal T cells. Specificity of this stimulation was demonstrated by its absence in T cells from NZB, NOD, or STb/J mice which lack the 6C3 determinant. In 6C3+ normal strains, the extent of 6C3-mediated stimulation varied, depending on the level of 6C3 antigen expression. Analysis of this stimulation in purified T cell subsets revealed that in Ly-6.1 strains (e.g., BALB/c, CBA/J), Lyt-2+ cells responded, but not L3T4+ cells, whereas in Ly-6.2 strains (e.g., C57BL/6, MRL-+/+), both subsets produced IL 2 and proliferated, although with different kinetics. Moreover, in adult MRL-+/+ mice, the minor Lyt-2-/L3T4- subset from the lymph nodes gave low responses to 6C3 cross-linking, whereas that from the thymus reacted strongly. Stimulation via Ly-6C therefore provides a pathway for differential activation of normal T cells. In contrast, the expanding population of Lyt-2-/L3T4- T cells from lpr/lpr or gld/gld mice did not proliferate in response to 6C3 antigen cross-linking plus PMA despite high levels of 6C3 antigen expression. Responsiveness of lpr/lpr T cells could not be restored with IL 1, IL 2, or both. These T cells also failed to be triggered by conjunction of PMA with either Thy-1 antigen cross-linking or concanavalin A. Moreover, they were not stimulated, in the presence of PMA, by doses of ionomycin that were optimal for normal T cells, but did respond to higher ionomycin concentrations (2 micrograms/ml), and this response was not altered by Ly-6C cross-linking. It is concluded that the Ly-6C pathway of T cell activation is not functional in the aberrant lpr/lpr (and gld/gld) T cells, and that this defect may reflect abnormalities of intracellular signaling.     

CD2 expression correlates with proliferative capacity of alpha beta + or gamma delta + CD4-CD8- T cells in lpr mice.

The T lymphocytes that accumulate in vast numbers in the lymphoid tissues of lpr/lpr (lpr) mice express a TCR-alpha beta that is polyclonally rearranged, and yet is devoid of surface CD4 or CD8 (CD4-8-) as well as CD2. lpr CD2- alpha beta + CD4-8- T cells exhibit an apparent block in signal transduction, in that when activated they produce little or no IL-2 and proliferate minimally in the absence of exogenous IL-2. In contrast to the predominant hyporesponsive alpha beta + CD4-8- T cells, we observe that a minor subset (1 to 2%) of lpr lymph node CD4-8- cells expresses a TCR-gamma delta and can proliferate upon activation with PMA and ionomycin in the absence of exogenous IL-2. Furthermore, these responsive gamma delta T cells express surface CD2. The functional and phenotypic distinctions of lpr gamma delta T cells led us to identify an analogous minor (4 to 10%) subset of alpha beta + CD4-8- cells in lpr thymus and lymph nodes that does express CD2. Similar to the gamma delta subset, these CD2+ alpha beta + CD4-8- cells are also capable of proliferation and IL-2 production. Thus the capacity for IL-2 production and proliferation by a small proportion of lpr CD4-8- T cells, either alpha beta + or gamma delta +, correlates with their expression of surface CD2. This correlation is supported by the observation that the lpr liver contains actively cycling alpha beta + CD4-8- lymphocytes that are strikingly enriched for CD2 expression. Consequently, unlike the vast proportion of abnormal lpr CD2- CD3+ CD4-8- cells, the CD2+ CD3+ CD4-8- T cells may not express the basic lpr defect, or else are not affected by its presence. These studies suggest that expression of the lpr abnormality may be restricted to a particular T cell lineage. This functional correlation with CD2 expression may be more broadly applicable to phenotypically similar subsets of normal thymocytes, and possibly peripheral tolerized T lymphocytes.     

Developmental sequence of T200 antigen modifications in murine T cells

The T200 glycoproteins of T cells were analyzed at different stages of T cell development. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Lyt-2-L3T4-, and Lyt-2+L3T4+ thymocytes had similar T200 proteins, whereas Lyt-2+L3T4- and Lyt-2-L3T4+ thymocytes expressed a distinct set of T200 molecules. This result indicated a molecular switch in regulation of T200 protein expression upon differentiation of thymocytes to mature phenotype T cells. Further modifications were evident when the T200 proteins of peripheral T cell subsets were examined. In particular L3T4+ T cells expressed T200 proteins of m.w. 220,000, 200,000, and 175,000, whereas Lyt-2+ lymph node T cells expressed an additional T200 protein of m.w. 235,000. Antigenic differences in the T200 glyco-proteins of peripheral L3T4+ and Lyt-2+ T cells were also detected. The anti-B220 monoclonal antibody, 14.8, reacted with lymph node Lyt-2+ T cells but did not react with lymph node L3T4+T cells or with Lyt-2+L3T4- thymocytes. This finding demonstrated a lineage-specific modification of the T200 protein of Lyt-2+ T cells that occurred after exit of these cells from the thymus into peripheral lymphoid organs. This modification apparently occurred on the m.w. 235,000 and 220,000 proteins since these species were precipitated by 14.8, whereas the others were not. In vitro growth and activation also resulted in further T200 antigen alterations. The monoclonal antibody, RA3, which reacts with the B220 antigen of B cells but, unlike 14.8, does not react with any peripheral T cells, showed significant reactivity with Lyt-2+ cytotoxic T cell (CTL) clones but not with L3T4+ T helper cell clones. CTL clones were also 14.8+ but T helper cell clones were not. Immunoprecipitation by 14.8 and RA3 of T200 proteins from CTL clones yielded a single protein of m.w. 240,000 that co-migrated with the B cell form of T200. Overall, the results indicate the presence of developmentally regulated mechanisms that control T200 glycoprotein expression during T cell differentiation in the thymus and in peripheral lymphoid organs.     

Molecular basis of elevated c-myb expression in the abnormal L3T4-, Lyt-2- T lymphocytes of autoimmune mice

The presence of the lpr/lpr genotype on a number of murine genetic backgrounds results in a systemic lupus erythematosus-like disease and lymphadenopathy. The T lymphocytes of these mice exhibit a variety of abnormalities; most pertinent to the present report is an abnormally high level of c-myb proto-oncogene mRNA. Since the c-myb protein is presumably the effector molecule that affects cellular functions, it is important to determine whether increased levels of this c-myb protein are produced. With the use of immunoprecipitation with an anti-v-myb reagent, we found high levels of c-myb protein in the lymph nodes of lpr mice. Detailed analysis showed that the c-myb protein is primarily expressed by an abnormal T lymphocyte population that does not express the mature T cell markers, L3T4 and Lyt-2. Analysis by two-dimensional gel electrophoresis showed that the c-myb proteins from normal thymocytes and from these L3T4-, Lyt-2-T cells are indistinguishable. DNA analysis with Southern hybridizations showed the lack of amplification, insertions, deletions, and rearrangements, which is in accord with results from the protein studies. Most interestingly, the c-myb gene in lpr L3T4-, Lyt-2- T cells is hypomethylated compared with normal controls. This suggests that a regulatory mechanism, rather than the structural alteration of the gene, is responsible for elevated expression of c-myb in these L3T4-, Lyt-2- cells.     

IL-2 receptor expression in autoimmune MRL-lpr/lpr mice. The expanded L3T4-, Lyt-2- population does not express p75 and cannot generate functional high-affinity IL-2 receptors

Autoimmune MRL-lpr/lpr mice develop an SLE-like disease characterized by a profound lymphadenopathy within an L3T4-, Lyt-2- (DN), B220+ T-cell population. Despite its immature phenotype this subset expresses mature alpha beta TCR belonging predominantly to the V beta 8 gene family and appears to be identical to an activated form of a minor T cell population present in both the thymus and periphery of normal mice. However, the mechanisms underlying the greatly increased cellularity in lpr/lpr-bearing mice are not understood. In this study, the IL-2R expression of lpr/lpr T cells was examined to assess the contribution of IL-2-mediated division to their expansion. The lpr/lpr DN T cells lacked high-affinity IL-2R, even after stimulation, suggesting that IL-2-dependent proliferation plays no role in the expansion of these cells and demonstrating the existence of this unusual T cell phenotype in vivo.     

Autoantibody in MRL/Mp-lpr/lpr mice. A monoclonal antibody specific for the abnormal T cells from mice bearing the lpr/lpr gene

We generated mAb from an unimmunized autoimmune MRL/Mp-lpr/lpr mouse. One of these mAb A108, reacted with cell surface Ag present on abnormal T cells from MRL/Mp-lpr/lpr, C3H-lpr/lpr, and C57BL/6-lpr/lpr mice. We failed to detect significant numbers of A108 bearing cells in the lymph nodes of MRL-Mp/+/+, normal C3H or normal C57BL/6 mice. Therefore, the expression of A108 correlates with the presence of the lpr/lpr gene. A108 binds to a variety of murine T cell tumor lines (e.g., EL4, BW5147, and YAC-1) and human T cell tumor lines (e.g., MOLT-3, Sup T1, and Jurkat). A108 does not bind to normal human PBL. Immunoprecipitation of surface iodinated EL-4 and BW5147 with A108 identified one major protein with a Mr of about 17.5-kDa. The significance of these findings with respect to the development of lymphoid proliferation and autoimmune disease in mice bearing the lpr/lpr gene will be discussed.     

Unique cell surface phenotypes of proliferating lymphocytes in mice homozygous for lpr and gld mutations, defined by monoclonal antibodies to MRL/Mp-lpr/lpr T cells

In mice bearing the autosomal recessive gene of either lpr or gld, generalized T-cell proliferation and autoimmunity occurs. The surface antigen profiles of these proliferating cells were analyzed using two-color flow cytometry analysis with two newly established rat monoclonal antibodies (ALP-1, ALP-2) directed to lpr cells. The Lp-1 antigen, defined by ALP-1, is expressed exclusively on approximately one-half of proliferating lpr and gld lymph node cells. The Lp-2 antigen, like B 220, is expressed on 80-90% of lpr and gld lymph node cells, the cells in B-cell lineage and a small population of Ly-2+ T cells from normal mice. Thus, the lpr and gld lymph node cells were classified into three subsets, Lp-1+/Lp-2+, Lp-1-/Lp-2+ and Lp-1-/Lp-2-. After stimulation with Con A or a combination of IL-2 and phorbol ester, a small population of T cells from normal mice became Lp-1+. The same treatment increased Lp-2+/Ly-2+ and induced Lp-2+/L3T4+ T-cell populations. Therefore, it seems likely that these phenotypically unique T cells are generated at some stage during the proliferation and differentiation of certain normal T-cell subpopulations. The aberrant T cells in mice with lpr and gld mutations may even be normal regulatory T cells, if they are not proliferating abnormally.