Activation of murine T lymphocytes with monoclonal antibodies: detection on Lyt-2+ cells of an antigen not associated with the T cell receptor complex but involved in T cell activation

A new T cell molecule defined by the mAb 143-4-2 has been identified that is involved in T cell activation. The expression of the 143-4-2-defined epitope is linked to the previously characterized Ly-6 locus and restricted to bone marrow cells and to a subset of peripheral Lyt-2+ cells. In comparison to other anti-Ly-6.2 mAb, the 143-4-2 mAb appears to be directed at an allogeneic determinant of the Ly-6.2C molecule. The anti-Ly-6.2C antibody can promote the lysis of antigen-non-bearing target cells by alloreactive CTL clones, and in the presence of cofactors (PMA or IL 2) induces a subset of Lyt-2+ cells to proliferate, perhaps through an autocrine pathway. Although the antibody described has antigen-like effects as described for anti-TcR complex reagents, studies performed with a recently derived anti-murine T3 mAb suggest that the Ly-6.2C molecule is not associated on the cell surface with components of the TcR complex. Nevertheless, cell surface expression of the TcR complex is required for optimal triggering of T cells via the Ly-6.2C molecule. Because Ly-6.2C determinants are expressed in bone marrow and not in the thymus, the possibility is considered that expression of this molecule identifies a distinct subset of extrathymically derived T 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.     

Role of Ly-6A/E and T cell receptor-zeta for IL-2 production. Phosphatidylinositol-anchored Ly-6A/E antagonizes T cell receptor-mediated IL-2 production by a zeta-independent pathway.

Ly-6A/E molecules were originally implicated in regulation of T cell activation because anti-Ly-6A/E mAb induce IL-2 production. More recently we have shown that anti-Ly-6A/E also inhibits IL-2 production induced by anti-CD3. In the present study we used mutant and transfected cell lines that varied in expression of Ly-6A/E or TCR-zeta to test whether the positive and negative modulations of IL-2 production by anti-Ly-6A/E occur by distinct mechanisms. Anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production for Ly-6E.1-transfected EL4J cells, but did not affect IL-2 production of the parental Ly-6A/E-negative EL4J cells. These results indicate that TCR-mediated IL-2 production can occur in the absence of Ly-6A/E expression and establish that anti-Ly-6A/E-induced inhibition of IL-2 production was the result of antibody binding to Ly-6A/E. As expected, MA5.8 (zeta-negative) or CT108 (zeta-truncated) variants of the 2B4.11 T cell hybridoma did not produce IL-2 when stimulated with anti-Thy-1 or anti-Ly-6A/E mAb. In contrast, anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production by MA5.8 and CT108. Furthermore, anti-Ly-6A/E-induced IL-2 production was restored for zeta-transfected MA5.8. Thus, although induction of IL-2 by anti-Ly-6A/E depends on zeta expression, inhibition of IL-2 by anti-Ly-6A/E occurs by a zeta-independent mechanism. Interestingly, anti-Ly-6A/E, but not anti-Thy-1, inhibited anti-CD3-induced IL-2 production by MA5.8 and Ly-6E.1-transfected EL4J. Therefore, inhibition of IL-2 production by anti-Ly-6A/E was not a general property of a mAb binding to a phosphatidylinositol-linked molecule, as has been suggested for induction of IL-2 production. Taken together these data suggest that the molecular mechanisms of induction and inhibition of IL-2 production by anti-Ly-6A/E are separable and expression of TCR-zeta is one variable that distinguishes these two pathways.     

Cellular control of abscess formation: role of T cells in the regulation of abscesses formed in response to Bacteroides fragilis

Although abscesses are a major sequela of infection, little is known about which cellular events initiate and which prevent this pathologic response. These studies are the first to indicate a role for T cells in the important pathogenic process of abscess development and also in immunity to abscesses induced by Bacteroides fragilis. We have shown that T cells initiate the formation of abscesses in mice after i.p. challenge with B. fragilis. These T cells bear both Ly-1 and Ly-2 surface markers. Nude mice (which have been shown by others to have T cell or T cell precursors) are also able to form abscesses. Cyclophosphamide-treated mice (with depressed T cell function) were not capable of developing abscesses. Reconstitution with normal or nude mouse spleen cells restored this ability. However, reconstitution with anti-Thy-1.2-treated, anti-Ly-1, or anti-Ly-2-treated spleen cells (or a mixture of the two cell populations) failed to allow abscess formation after bacterial challenge. Immunity to abscesses caused by B. fragilis requires two T cells. The first Ly-1-2+ T cell has an IJ surface marker and has been shown to release a small m.w. soluble factor (ITF) that is antigen specific. Immunity to abscesses, however, depends on the interaction of ITF with a second Ly-1-2+ T cell, demonstrated in reconstitution experiments with nude mice. The data presented document a critical role for T cells in abscess induction and suggest the existence of a suppressor-like T cell circuit in immunity to abscesses.     

Suppressor T cells and self-tolerance. Active suppression required for normal regulation of anti-erythrocyte autoantibody responses in spleen cells from nonautoimmune mice

Spleen cells from young, nonautoimmune strains of mice cultured with syngeneic E do not develop a significant anti-mouse E response in vitro, consistent with a state of self-tolerance to this Ag. In order to study the role of active suppression in regulating mouse RBC-(MRBC) specific cells in nonautoimmune cell populations, the effect of depleting T cell subsets on the generation of anti-MRBC autoantibodies by nonautoimmune spleen cells was determined. Spleen cells from young BALB/c and C57BL/6 mice were found to generate significant numbers of IgM and IgG anti-MRBC autoantibody-forming cells in culture with MRBC after depletion of Ly-2+ cells by anti-Ly-2 and C treatment. The response which develops is Ag dependent, Ag specific, and dependent upon L3T4+ Th. The magnitude and isotype of this response is similar to the anti-MRBC response generated by spleen cells from 12-mo-old, autoimmune NZB mice and young NZB mice also treated to remove Ly-2+ cells. Addition of isolated Ly-2+ T cells, but not L3T4+ or Ly-2- T cells, to spleen cells depleted of Ly-2+ cells restores apparently normal regulation of the anti-MRBC response in vitro. These data demonstrate that control of a specific autoantibody response to MRBC by nonautoimmune spleen cell populations requires active regulation by an Ly-2+ T cell subset.     

Role of Ly-1:Qa1- and Ly-1:Qa1+ inducer T cells in activation of Ly-23 effectors of suppression of antibody production in mice

Ly-2+ effectors of T cell-mediated suppression require inducing signals from antigen and a helper cell bearing the Ly-1+:Qa1+ surface phenotype. In this report, we have further examined the helper cell requirements for suppressor cell induction of antibody production in mice. By using the T cell subset education procedure in vitro, we have activated T cells to sheep red blood cells (SRBC) antigens and then purified Ly-2 cells before testing for suppressor activity in assay cultures of defined T and B cell subsets. We have confirmed our previous observations that Ly-1+:Qa1+ cells are required for activation of T suppressors, but have found that under the appropriate conditions, there is not a strict requirement for the Ly-123 subset of T cells. Furthermore, if Ly-23 cells are stimulated in the presence of Ly-1+:Qa1- T cells, effective suppressors can be obtained only if a source of Ly-1:Qa1+ inducers is added to the assay culture. If Ly-23 cells are activated by antigen in the absence of Ly-1 cells, subsequent exposure to the Ly-1+:Qa1+ subset under the conditions tested here is not sufficient to activate suppressors. These results show that effectors of suppression, like B cells and cytotoxic T lymphocytes, may respond to two helper cells.     

Characterization of responding cells in oxidative mitogen stimulation. I. Ia+ cells and Ly-1+2+ cells are required for the proliferative response

Addition of anti-Ia sera to cultures of mouse spleen cells stimulated with oxidative mitogens, neuraminidase/galactose oxidase (NaGO) or sodium periodate (NaIO4), inhibits the subsequent proliferative response 30 to 70%. Anti-H-2K or D sera were not specifically inhibitory. Similar inhibition was seen when cells were pretreated with anti-Ia sera and washed before exposure to the mitogenic enzymes. Treatment with anti-Ia serum and complement depletes greater than 89% of the NaGO and the NaIO4 responses but 2% or less of the phytohemagglutinin (PHA) response. The response to NaGO was sensitive to depletion with anti-Thy-1 serum, rabbit anti-mouse brain serum, anti-Ly-1, or anti-Ly-2 serum. Mixtures of Ly-1 and Ly-2-depleted populations did not restore responsiveness. Thus both an Ia+ cell and an Ly-1+2+ T cell are required for [3H]TdR incorporation in response to NaGO treatment.     

Investigations into the nature of Igh-V region-restricted T cell interactions by using antibodies to antigens on methylcholanthrene-induced sarcomas. I. Analysis of an Igh-V-restricted suppressor-inducer factor

We have previously demonstrated the relationship between antigens on BALB/c methylcholanthrene (MC)-induced fibrosarcomas and T cell regulatory molecules by using a variety of antisera raised to these sarcomas in BALB/c and BALB/c X C57BL/6 (CB6F1) mice. One such pool of antiserum, a CB6F1 anti-CMS 4 (Pool XIV) serum, was used to investigate the nature of the T cell regulatory structures recognized by these antibodies. Pool XIV antiserum was capable of blocking the induction of feedback suppression by Ly-1 TsiF, an SRBC-specific suppressor T cell factor secreted by Ly-1+, 2- I-J+ T cells. Ly-1 TsiF induces suppression by interacting with an Ly-1+,2+ I-J+ T cell target. Successful interaction of Ly-1 TsiF with its target cell requires genetic homology between inducer and target cells at the variable region of the immunoglobulin heavy chain gene complex (Igh-V). The addition of Pool XIV antiserum to primary in vitro anti-SRBC cultures resulted in blocking the ability of Ly-1 TsiF from Igha (BALB/c) and Ighj (CBA/J) mice to induce suppression on syngeneic cells, whereas suppression induced by Ly-1 TsiF in Ighb (B6), Ighc (DBA/2), Ighd (A/J), and Ighe (AKR) mice are unaffected by addition of the Pool XIV antiserum. The ability of Pool XIV antiserum to block Ly-1 TsiF activity is linked to the Igh region, because Pool XIV antiserum can block Ly-1 TsiF from BALB/c (H-2d, Igha) and the Igh congenic B.C9 (H-2b, Igha) while not affecting Ly-1 TsiF activity on B6 (H-2b, Ighb) or its Igh congenic C.B20 (H-2d, Ighb). In CB6F1 animals, Pool XIV antiserum could block the ability of CB6F1 Ly-1 TsiF to suppress BALB/c spleen cells but not B6 spleen cells. Conversely, Pool XIV antiserum could block the ability of BALB/c Ly-1 TsiF to suppress CB6F1 spleen cells, whereas B6 Ly-1 TsiF showed normal suppressive activity in the presence of Pool XIV antiserum. In contrast, Pool XIV was capable of blocking the ability of Ly-1 TsiF from BALB/c into CB6F1 bone marrow chimeras (BMC) to suppress both BALB/c and B6 mice, whereas the activity of Ly-1 TsiF from B6 into CB6F1 BMC on BALB/c or B6 spleen cells was unaffected by the addition of Pool XIV antiserum. We then investigated the molecular nature of the molecule recognized by Pool XIV antiserum on the Ly-1 TsiF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of T lymphocytes through the Ly-6 pathway is defective in A strain mice

The Ly-6 alloantigens have been shown to participate in the process of T cell activation based on the ability of anti-Ly-6 mAb to induce IL-2 production and proliferation of T lymphocytes. In the present investigation we have demonstrated that peripheral T lymphocytes from A strain mice exhibited abnormally low proliferative responses after stimulation through Ly-6A/E and Ly-6C molecules when compared to responses of T cells from numerous other mouse strains. The abnormal activation of the Ly-6 pathway of A strain T cells was not due to ineffective FcR cross-linking of the anti-Ly-6 mAb, to inappropriate cellular expression of the Ly-6A/E alloantigen in A strain T cells, or to an active suppressive phenomenon. T lymphocytes from A strain mice proliferated normally when the cells were activated by mAb to Thy-1 or the CD3/TCR complex suggesting that A strain mice did not exhibit a generalized T cell activation defect. Cell separation studies of T cells and accessory cells demonstrated that this defect was quantitative, rather than qualitative, and that it was complex, residing at both the T cell and accessory cell levels. These results suggest that activation of T lymphocytes via the Ly-6 molecule may involve a signaling pathway and/or cell-cell interactions distinct from those required for optimal activation via CD3/TCR.     

Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. III. In vitro studies

The alloantigenic specificity Ly-4.2 is present on a restricted population of murine lymphocytes which have previously been shown to have some of the properties generally ascribed to B lymphocytes, both with regard to distribution and function. In the study reported herein, the effect of anti-Ly-4.2 and anti-Thy-1.2 (θ) antisera have been examined in various in vitro systems. (a) T cell-mediated lysis of ⁵¹Cr-labeled P815-X2 target cells by immune allogeneic peritoneal exudate cells is inhibited by anti-Thy-1.2, but not affected by the anti-B (Ly-4.2) reagent. (b) Antibody-dependent lymphocyte-mediated lysis of ⁵¹Cr-labeled sheep red cells was only slightly inhibited by anti-Ly-4.2 and anti-Ig antisera, and not at all by anti-Thy-1.2 antisera, indicating that this type of cell lysis is mediated by neither T (Thy-l⁺) nor B (Ly-4.2⁺,Ig⁺) cells. (c) The response of lymph node lymphocytes to various mitogens was affected thus: PHA, completely inhibited by anti-Thy-1.2 but not by anti-Ly-4.2; Con A, largely inhibited by anti-Thy-1.2, and slightly by anti-Ly-4.2; PWM (pokeweed mitogen), partially inhibited by both antisera; E. coli endotoxin lipopolysaccharide, greatly inhibited by anti-Ly-4.2 but only slightly by anti-Thy-1.2. The findings demonstrate that anti-Thy-1.2 reacts predominantly with T cells and anti-Ly-4.2 with B cells.     

Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation. We show that Ly-6A/E proteins also modulated the response of normal B lymphocytes in three separate in vitro assays. First, unfractionated or small resting B cells proliferated when cultured with IFN-gamma, IL-4, and an anti-Ly-6A/E mAb. Second, this anti-Ly-6A/E mAb restored B cell proliferation responses that were inhibited when coculturing the B cells in IFN-gamma, IL-4, and anti-IgM. Third, anti-Ly-6A/E specifically up-regulated the cell surface expression of its own Ag, and this response was dependent upon co-stimulation with IFN-gamma. Mixing of T and B cells in culture suggested that T cells did not contribute substantially to the B cell proliferative response. Moreover, up-regulation of Ly-6A/E was observed for one B cell lymphoma, WEHI-231. Therefore, it appeared that modulation of B cell function by anti-Ly-6A/E was due to a direct effect of the mAb binding to the B cells. Taken together, these data suggest Ly-6A/E proteins are functional on B cells and may play a regulatory role in B cell activation.     

Selection of T cell receptor expression mutants through the functionally linked Ly-6A

Ly-6A is a glycosyl-phosphatidylinositol (GPI)-anchored molecule that participates in murine T cell activation. Activation of T cell hybridomas with anti-Ly-6A monoclonal antibody (mAb) leads to production of interleukin-2 (IL-2), but also to a paradoxical growth inhibition, which was used to select for signaling mutants. Fifteen subclones derived from two independent mutageneses and anti-Ly-6A selection were characterized. Thirteen subclones responded poorly or not at all to soluble anti-Ly-6A mAb. Although the selective pressure was exerted through Ly-6A, only one mutant did not express the Ly-6A antigen. Interestingly, 10 of the 15 subclones expressed either nondetectable or a very low level of T cell receptor/CD3 complex (TCR/CD3). Preferential expansion of TCR/CD3 expression mutants following anti-Ly-6A selection further established functional linkage between Ly-6A and TCR/CD3 complex. The mechanism of the functional coupling was investigated by analyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), one of the early events in T cell activation. We showed that PIP2 was not hydrolyzed in response to anti-Ly-6A in TCR/CD3-negative mutants. Aluminum fluoride, which activates G protein directly, did induce PIP2 hydrolysis in these cells. These data suggest that activation signals originated from Ly-6A must be transmitted first to TCR/CD3 complex, which then couples to the G protein/phospholipase C system. A similar requirement also applies to the Thy-1 protein and lectin receptors. Thus, the TCR/CD3 complex plays a central role in the integration and transmission of activation signals that originated from several T cell surface molecules.     

Down-regulation of IL-2 production by activation of T cells through Ly-6A/E

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.     

Disparate functional properties of two interleukin 1-responsive Ly-1+2- T cell clones: distinction of T cell growth factor and T cell-replacing factor activities

We describe the properties of two Ly-1+2- T cell clones (Ly-1.14 and Ly-1.21), which are maintained in long-term culture in the absence of other cell types. The clones require media containing a source of interleukin 1 as well as interleukin 2. They retain physiologic responses to interleukin 1, which is required for optimal production of T cell lymphokines by these clones in response to concanavalin A (Con A). The two Ly-1+2- T cell clones differ in their production of lymphokines after stimulation by Con A. The supernatant of clone Ly-1.21 promotes the proliferation of T cells maintained in long-term culture, induces antibody synthesis in cultures of B cells and antigen, and induces the differentiation of cytolytic cells in cultures of thymocytes and antigen; these assays define the properties of T cell growth factor (TCGF), T cell-replacing factor for B cells (TRF-B), and T cell-replacing factor for cytolytic cells (TRF-C), respectively. In contrast, the supernatant of clone Ly-1.14 contains only TCGF activity and does not promote antibody synthesis by B cells or differentiation of cytolytic cells from thymocytes. The results indicates that TCGF and TRF activities reside on independent, although perhaps related, molecules.     

Cross-linkage of Ly-6A/E induces Ca2+ translocation in the absence of phosphatidylinositol turnover and mediates proliferation of normal murine B lymphocytes

Ly-6A/E is a phosphatidylinositol (PI)-linked membrane protein whose expression is induced or upregulated on normal murine T and B cells by IFN-gamma. Cross-linkage of Ly-6A/E expressed on normal murine T cells stimulates Ca2+ translocation, and in the presence of a protein kinase C (PKC) activator, lymphokine secretion, and cellular proliferation. Utilizing an anti-Ly-6A/E mAb, we studied the effect of cross-linking Ly-6A/E on IFN-gamma-treated resting B cells, for Ca2+ translocation, PI turnover, and cellular proliferation. Since these events are known to be stimulated by cross-linkage of B cell membrane (m)Ig, we compared the changes mediated through these respective membrane proteins. We show that cross-linkage of B cell Ly-6A/E stimulates a large, rapid, and sustained increase in the concentration of intracellular free calcium ([Ca2+]i) comparable in magnitude, though somewhat delayed, relative to that observed after cross-linking of mIg. Cross-linkage of B cell Ly-6A/E does not, however, stimulate detectable PI turnover, in contrast to PI turnover induced by ligation of mIg. Both the Ly-6A/E- and mIg-mediated increase in [Ca2+]i occur through mobilization of internal Ca2+ stores as well as entry of Ca2+ into the cell from the extracellular compartment. Ly-6A/E-mediated Ca2+ translocation appears to be under the regulation of PKC in that short term pretreatment of B cells with the PKC activator, PMA, inhibits the Ly-6A/E- as well as the mIg-mediated increase in [Ca2+]i, whereas prolonged exposure to PMA, under conditions that lead to depletion of PKC, results in an augmentation in Ca2+ translocation after ligation of either Ly-6A/E or mIg. Co-capping studies indicate that Ly-6A/E and mIg cap independently in the B cell membrane, thus suggesting that the Ly-6A/E-induced effects on Ca2+ translocation are not mediated through simultaneous modulation of mIg. Anti-Ly6A/E, by itself, does not stimulate an increase in [3H]thymidine incorporation by IFN-gamma-treated resting B cells, but induces a striking increase in the presence of PMA. By contrast, anti-Ig by itself stimulates significant increases in [3H]thymidine incorporation that is inhibited by PMA. Thus, Ly-6A/E is a potent mediator of B cell activation that may use a signal transduction system in quiescent B cells that is distinct from that of the Ag receptor.     

Stimulation of human Jurkat cells by monoclonal antibody crosslinking of transfected-Ly-6A.2 (TAP) molecules.

Monoclonal antibody crosslinking of phosphatidylinositol-anchored Ly-6A.2 molecules on the surface of murine T lymphocytes leads to cell activation and secretion of IL-2. To examine the potential activity of these molecules in human T cells we transfected the Ly-6A.2 gene into Jurkat cells. Transfection of Jurkat cells with genomic Ly-6A.2 sequences results in low levels of Ly-6A.2 on the cell surface. However, linking the Ly-6A.2 sequences to the enhancer from the human CD2 gene results in greatly increased expression of Ly-6A.2. These molecules are anchored to the membrane via a phosphatidylinositol linkage. Crosslinking of Ly-6A.2 molecules with soluble mAb stimulates the transfected Jurkat cells to produce IL-2. This stimulation is abrogated by treatment with phosphatidylinositol-specific phospholipase C. The transfected human T cells displayed the same unusual crosslinking requirements for stimulation with anti-Ly-6A.2 mAbs as previously observed for murine T cells. Crosslinking of Ly-6A.2 with soluble antibodies is stimulatory, whereas immobilized antibodies are inactive. The crosslinking requirements for antiCD3 mAb stimulation display a reciprocal pattern. These data demonstrate that the Ly-6A.2 pathway for T cell activation is conserved between human and murine T cells.     

Influence of accessory cell and T cell surface antigens on mitogen-induced IL 2 receptor expression

We have assessed the inhibitory effects of various monoclonal antibodies on the expression of the IL 2 receptor. Anti-LFA-1, but not anti-Ly-2, markedly inhibited the induction of the IL 2 receptor on the Ly-2+ subset. T-depleted spleen cells, L cells, and B lymphoma cells all functioned as potent accessory cells (AC) for the induction of the IL 2 receptor on L3T4+ T cells. Anti-LFA-1 inhibited the induction of the IL 2 receptor irrespective of the type of AC used. Anti-L3T4 only inhibited the induction of IL 2 receptor expression when L cells were the source of AC. The inhibitory capacity of anti-L3T4 was not related to the expression of Ia on the AC population, because the magnitude of inhibition was comparable in cultures containing either Ia+ or Ia- L cells, whereas no inhibition was seen with either Ia+ or Ia-B lymphoma cells. We conclude from these studies that LFA-1 plays a critical role in mitogen-induced activation of both T cell subsets by promoting both T-AC and T-T interactions. Although anti-L3T4 can inhibit T cell activation in the absence of the recognition of Ia, the mechanism of inhibition and the proposed target molecule for L3T4 on the AC or the T cell have not been determined in our studies. A number of different models for the function of this cell surface antigen are discussed.     

Prevention of lymphadenopathy in MRL-lpr/lpr mice by blocking peripheral lymph node homing with Mel-14 in vivo

MRL-lpr/lpr mice develop massive lymphadenopathy and autoimmunity. There is evidence that both migration and local proliferation contribute to the accumulation of Ly-2-, L3T4-, 6B2+ T cells in the peripheral lymph node (PLN). Mel-14 is an antibody which binds to the lymphocyte lymph node homing receptor (gp90Mel-14) and can block migration of lymphocytes to the PLN. Treatment of mice from birth to 11 wk of age with Mel-14 and another rat IgG2a mAb, 6B2, resulted in reduction (10- to 20-fold) in lymphadenopathy. Mel-14, but not 6B2, preferentially reduced the percentages of Thy-1+, 6B2+ lymphocytes in the lymph node. Treatment with a third antibody, anti-Ly-1, had no effect on lymphadenopathy. Mel-14 treatment resulted in diversion of the Ly-2-, L3T4-, 6B2+, gp90Mel-14 cells to the spleen and consequently induced marked splenomegaly. Thymocytes from MRL-lpr/lpr and MRL-+/+ mice were analyzed by two-color flow cytometry analysis after depletion of Ly-2+ and L3T4+ T cells. There was no difference in the percent of Ly-2-, L3T4-, 6B2+, gp90Mel-14 positive thymocytes comparing these two strains. Mel-14 treatment did not alter Ig levels or autoantibody production. These studies suggest Mel-14 reduced lymphadenopathy by interfering with homing to PLN, whereas 6B2 may have interfered with marrow production of precursor cells or killed 6B2+ cells after they exited the marrow. The data are consistent with the idea that lymphadenopathy occurs in MRL-lpr/lpr mice due to increased homing gp90-Mel-14 T cells to the PLN and that gp90Mel-14 is a necessary receptor for the abnormal 6B2+ T cells.     

T lymphocyte responses to non-H-2 histocompatibility antigens. I. Role of Ly-1+2+ T cells as cytotoxic effectors and requirement for Ly-1+2+ T cells for optimal generation of cytotoxic effectors

Cytotoxic effector T cells specific for non-H-2 histocompatibility (H) antigens were examined for phenotypic expression of lymphocyte differentiation (Ly) antigens. Virtually all H-Y-specific cytotoxic effectors generated in mixed lymphocyte culture were Ly-1+2+ T cells. H-3-specific effectors comprised both Ly-1+2+ and Ly-1-2+ T cells. However, cytotoxic effectors specific for multiple non-H-2 H antigens were predominantly Ly-1-2+ T cells. The optimal generation of H-Y- and H-3-specific effectors required Ly-1+2+ T cells; optimal generation of multiple non-H-2 H antigen-specific effectors required an interaction between Ly-1+2- and Ly-1-2+ T cells. These observations suggest that the identity of the target H antigen in part determines the Ly type of responsive T cells. Our observations suggest that 2 alternative pathways of T cell response exist for non-H-2 H antigens. The first pathway involves an interaction between Ly-1+2- helper T cells and Ly-1-2+ cytotoxic effector precursors. The 2nd pathway simply involves the response of Ly-1+2+ T cells proliferating and generating H antigen-specific cytotoxic effectors.