Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

The expression of theta-like antigen by rat peripheral lymphocytes: serologic and functional studies.

AKR/Cum mice (Thy-1b = thetaC3H) immunized with nucleated cells from WF rat thymus, Peyer's patches, peritoneal exudate, mesenteric lymph nodes, blood, bone marrow, or spleen produced antibodies cytotoxic for ADR/J (Thy-1a = thetaAKR) but not for AKR/Cum thymocytes. The specificity of these antibodies for the Thy-1.1 (theta-AKR) antigen was confirmed by tests using thymocytes from backcross mice segregating at the Thy-1 locus. This result suggested that the rat lymphocyte antgen cross-reactive with Thy-1.1 was expressed by at least some members of each of the rat lymphoid cell populations tested. AKR/Cum mice immunized with killed rat cells also produced anti-Thy-1.1 antibodies; thus indicating that further differentiation of the injected cells was not a prerequisite for the anti-Thy-1.1 response. Unexpectedly, about 9% of unimmunized adult AKR/Cum males were found to be producing antibodies against Thy-1.1. To our knowledge, natural antibodies of this specificity have not been previously reported. Finally, it was found that peritoneal exudate cells taken from WF rats previously immunized with EL-4 mouse leukemia cells were neither killed nor functionally inactivated by treatment with anti-Thy-1.1 antibodies and complement.     

A Thy-1.1-specific monoclonal alloantibody activates both mouse and rat T cells

In an attempt to further evaluate the role of Thy-1 in the antigen-independent triggering of mouse T cells, we have examined the activating properties of two Thy-1.1-specific mouse monoclonal antibodies (mAb). These reagents were established from an (A.TH X A.TL)F1 hybrid mouse (Thy-1b) immunized with IL-2 producing (BALB/c (Thy-1b) X BW5147 (Thy-1a)) T hybridoma cells. Although both mAb recognized the same Thy-1.1 determinant, one mAb of the gamma 3,kappa class (H171-146) was found to induce several T hybridoma cells to produce IL-2, and AKR thymocytes or cloned helper T cells to proliferate, whereas another mAb of the gamma 1,kappa class (H171-112) failed to do so even in the presence of phorbol myristic acetate (PMA). Increased IL-2 responses of T hybridoma cells were observed when the cell bound Thy-1.1-specific mAb were crosslinked by goat anti-mouse Ig (GaMIg) antibodies. Both a T-cell activating rat anti-Thy-1.2 mAb and the anti-Thy-1.1 mAb H171-146, although directed at distinct cell surface molecules, synergistically stimulated IL-2 production by T hybridoma cells. In addition, the mouse mAb H171-146 was found to stimulate LOU/M rat thymocytes to proliferate in the presence of exogenous IL-2. These data demonstrate that T cells can use Thy-1 as a signal-transducing molecule in both mouse and rat species, and support the notion that the activating properties of Thy-1.1-specific mAb are influenced by their heavy chain isotypes.     

Effect ofH-2 incompatibility between recipient and donor on the magnitude of response to Thy-1.1 antigen

Mice were immunized intravenously with 4 × 10⁷ thymocytes from Thy-1 disparate, eitherH-2-compatible orH- 2-incompatible donors. The magnitude of the anti-Thy-1.1 response was measured by determining the number of PFC in spleens of animals 6 days after immunization. Regardless of the origin of immunizing and target thymocytes, the assay employed detected exclusively PFC-producing antibodies to the Thy-1.1 antigen. In almost all instances,H-2-compatible thymocytes elicited a significantly higher response than didH-2-incompatible thymocytes, although the latter occasionally evoked a high response. TheH-2 incompatibility between donor and recipient appeared to be responsible for the differences in responsiveness of the standard inbred mice and theirH-2 mutants immunized with thymocytes compatible with standard inbred strains. The phenomenon observed appears to have several features in common with antigenic competition. We propose that the requirement forH-2 compatibility in the anti-Thy-1.1 response may be the expression of a general requirement of T cells to recognize an antigen in the context of the H-2 molecule.     

Alterations in expression and glycosylation pattern of the Thy-1 glycoprotein during maturation and transformation of mouse T lymphocytes

The mouse Thy-1 glycoprotein of normal and transformed lymphoid cells was studied with regard to amount per cell, apparent m.w., and glycosylation characteristics. Thy-1 was measured by a solid-phase radioimmunoassay calibrated with pure mouse brain Thy-1. Thymocytes were shown to contain five times the amount of Thy-1 found in lymph node cells (1 X 10(6) vs 2 X 10(5) molecules per cell), whereas the T cell lymphomas studied (P52-127-166, RBL-5, YWA, Y191, Y274, YAC-1, RL male 1, and BW5147) varied in their Thy-1 content. The apparent m.w. of Thy-1, as determined by SDS-PAGE, was in all cases 25,000 to 30,000. However, the appearance of the Thy-1 bands revealed a size heterogeneity that was less pronounced with material from lymph node cells than from thymocytes. This band broadening seemed to be inversely correlated to the affinity for lentil lectin. Whereas half the Thy-1 molecules from thymocytes were bound to the lectin, lymph nodes Thy-1 showed 75% binding. All T lymphomas but one (BW5147) contained Thy-1 also heterogeneous in lentil lectin binding. The charge, previously shown to be dependent on the sialic acid content, was shifted to more acidic forms for lymph node Thy-1 compared to thymocytes. The T lymphomas possessed Thy-1 with charge properties similar to those of the thymocytes; the only exception was BW5147, which showed more basic forms. These results show that the expression and the glycosylation of Thy-1 is altered when thymocytes mature into immunocompetent cells and after malignant transformation of lymphocytes.     

Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in detergents

1. A radioactive binding assay for Thy-1.1 alloantigen which functions in the presence of detergents was established by using glutaraldehyde-fixed thymocytes as target cells. Thy-1.1 activity in detergent extracts was then assayed by measuring inhibition of the binding assay. 2. Solubilization of Thy-1.1 from whole thymocytes, and their membranes by a large number of non-ionic detergents and deoxycholate was studied. In the same extracts Ag-B(4) histocompatibility antigenic activities were measured. With the exception of Nonidet P-40, the detergents did not affect the antigenicity of Thy-1.1, but only Lubrol-PX and deoxycholate gave effective solubilization as measured by activity remaining in the supernatant after centrifugation at 200000g for 40min. With Ag-B(4) antigen, Triton X-100, Triton X-67 and Nonidet P-40 gave effective solubilization as well as Lubrol-PX and deoxycholate. Solubilization of Thy-1.1 activity from leukaemia cells and a brain homogenate was also studied, but none of the non-ionic detergents gave satisfactory results with these tissues. 3. Extracts from thymocyte membranes were further examined by gel filtration and sucrose gradient centrifugation. The Thy-1.1 activity behaved as a single component in deoxycholate with a density similar to that of a globular protein, but in Lubrol-PX the antigen was contained in a low-density complex. In Lubrol-PX extracts Ag-B(4) was also found in aggregates not observed in deoxycholate. 4. The s(20,w) values for Thy-1.1 and Ag-B(4) antigens in deoxycholate were 2.4 and 4.4, and v values were 0.70 and 0.75 respectively. The Stokes radius observed for Thy-1.1 was 3.1nm and for Ag-B(4) 5.3nm. By using these values the molecular weights for the antigen-detergent complexes were calculated to be 28000 for Thy-1.1 and 100000 for Ag-B(4).     

High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells.

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T-lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.     

Preferential repopulation of the small intestine by gut-derived T cell precursors in the murine system.

The potential of intestinal intraepithelial lymphocyte (IEL) precursors to repopulate the lymphoid components of lethally-irradiated mice was evaluated. Mice injected with total IEL, or IEL depleted of mature T cells, died within 2 weeks post-irradiation. Injection of T cell-depleted Thy-1.1 IEL and Thy-1.2 bone marrow (BM) into lethally-irradiated Thy-1.2 mice resulted in survival rates greater than 90%. The vast majority of thymocytes analysed at 2, 6, and 10 weeks post-treatment were Thy-1.2+. The Thy-1.1+ and Thy-1.2+ cells were detected in the spleen 2 and 6 weeks post-reconstitution. After 10 weeks, the majority of splenic T cells were Thy-1.2+. The majority of Thy-1+ IEL were of the Thy-1.1 subtype at 2 and 6 weeks after reconstitution. After 10 weeks, Thy-1.2+ IEL became the predominant subtype. Flow cytometry (FCM) analyses of Thy-1.1+ IEL showed that Thy-1.1 was co-expressed with CD3, CD4, CD5, CD8, TCR alpha beta and TCR gamma delta T cell markers. These findings indicate that IEL precursors home preferentially to gut epithelia and generate complex IEL phenotypic subsets.     

A monoclonal antibody detecting unusual Thy-1 determinants

20-10-5S is a monoclonal antibody produced by the fusion of C3H anti-C3H.SW splenocytes with the SP2/0 cell line. The antibody appears to react with Thy-1 determinants by several criteria including cytotoxicity patterns, functional assays, genetic analyses, and competitive binding experiments. However, the antibody and the determinants it detects are unusual in that: 1) 20-10-5S is autoreactive; 2) the antibody shows allospecificity for Thy-1.2 vs Thy-1.1 antigens only on peripheral lymphocytes and not on thymocytes; and 3) the antibody reacts only with determinants on murine T cells and not with antigens on brain tissue or on rat thymocytes. It therefore seems that 20-10-5S reacts with murine T cell-specific Thy-1 determinants that are lost or modified during maturation of the cells on which they are expressed.     

Serologic relatedness between Thy-1.2 and actin revealed by monoclonal antibody

Monoclonal antibodies with affinity for Thy-1.2 on thymocytes also can bind to actin within marsupial, murine, and human cells. A similar cross-reactivity between Thy-1.1 and vimentin was revealed by Dulbecco and co-workers employing monoclonals. A computer-assisted analysis of the amino acid composition provided suggestive evidence for the occurrence of sequence homology between Thy-1.1 or Thy-1.2, actin, and vimentin that likely accounts for the serologic relatedness detected by hybridoma antibodies.     

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.     

Anti-thymocyte autoimmunity in BALB/C mice accompanying immunization to a syngeneic lymphoma.

As immunization of BALB/c mice to the syngeneic P1798 lymphoma is effected by administration of iodoacetamide-modified P1798 cells, serum antibodies appear which are reactive with P1798 and normal BALB/c thymocytes, splenocytes, and peripheral blood lymphocytes. Anti-P1798 serum also cross-reacted with thymocytes from AKR, DBA/2, and C3H mice as well as the allogeneic lymphoma 6C3HED. Anti-P1798 serum was unreactive with the Thy-1 deficient L1210 lymphoma. Multiple absorptions of anti-P1798 serum with normal BALB/c thymocytes or brain or P1798 removed antibodies to P1798 and thymocytes commensurately. Normal BALB/c liver and kidney did not absorb antibody activity. Treatment of a BALB/c splenocyte suspension with anti-Thy 1.2 serum and complement removed the population of spleen cells which were capable of reaction with anti-P1798 serum. The data suggest that antibodies to P1798 and thymocytes are the same and that specificity may be directed toward a Thy-1 related structure but without distinguishing Thy-1.1 and Thy-1.2.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

A role for the thymic epithelium in the selection of pre-T cells from murine bone marrow

A rat thymic epithelial cell line IT45-R1 has been previously described as secreting soluble molecules that in vitro chemoattract rat hemopoietic precursor cells. The development of such an in vitro migration assay was based on the ability of cells to migrate across polycarbonate filters in Boyden chambers. In the present paper, by using the same strategy, we studied murine bone marrow cells capable of migrating in vitro toward IT45-R1 conditioned medium. The responding cells were shown to represent a minor bone marrow subpopulation characterized by a low capacity to incorporate tritiated thymidine in vitro (less than 10% of control). Moreover, this cell subset was considerably impoverished with respect to granulocyte-macrophage CFU (less than 7% of control) and pluripotent hemopoietic stem cells (less than 12% of control). Potential generation of T cells of donor-type in the lymphoid organs of irradiated recipients was measured by using C57BL/Ka Thy-1.1 and Thy-1.2 congenic mice. Thy-1.1 irradiated mice were injected intrathymically or intravenously with the selectively migrated cell subset of Thy-1.2 donor-type bone marrow cells. The use of an i.v. transfer route allowed us to show that these cells possess thymus-homing and colonization abilities. In a time-course study after intrathymic cell transfer, these migrated cells were able to generate Thy-1.2+ donor-type thymocytes represented by all cortical and medullary cell subsets in a single wave of repopulation from day 20 to day 30 after transfer, with a peak around days 23 to 25. The degree of repopulation closely resembled that seen with unfractionated bone marrow cells in terms of absolute numbers of donor cells per thymus (82% of control, 22 x 10(6) Thy-1.2+ cells) as well as in percent donor cells per thymus (105% of control). Thy-1.2+ cells were also detected in the lymph nodes and the spleens of reconstituted recipient mice. Taken together, these results support the idea that the supernatant of the established thymic epithelium IT45-R1 induces the migration of a murine bone marrow subset that contains hemopoietic stem cells already committed to the lymphoid lineage (i.e., pre-T cells).     

Thy-1: more than a mouse pan-T cell marker

Thy-1 (CD90) is a small GPI-anchored protein that is particularly abundant on the surface of mouse thymocytes and peripheral T cells. T cell proliferation and cytokine synthesis in response to Thy-1 cross-linking by specific mAb suggests a role for Thy-1 in mouse T lymphocyte activation. However, a physiological ligand or counterreceptor for murine Thy-1 in the lymphoid compartment has not yet been identified. Thy-1 cross-linking, in the context of strong costimulatory signaling through CD28, results in an activating signal that can at least partially substitute for TCR signaling during mouse T cell activation. Remarkably, Thy-1 cross-linking also results in the potent costimulation of T cells activated through the TCR. This novel dual signaling capacity suggests a possible role for Thy-1 in the maintenance of T cell homeostasis in the absence of TCR triggering, as well as potentiating Ag-induced T cell responses.     

Effect of gene dosage on cell-surface expression of Thy-1 antigen in somatic cell hybrids between Thy-1− abelson-leukemia-virus induced lymphomas and Thy-1+ mouse lymphomas

Hybrids between pseudodiploid Thy-1.1⁺ lymphomas and Thy 1.2^– pseudodiploid Abelson-leukemia-virus-induced (ALV-induced) lymphomas express Thy-1 glycoprotein on their cell surface. These Thy-1⁺ hybrids invariably express the Thy 1.1 allelic form of the glycoprotein and may be either Thy 1.2⁺ or Thy 1.2^–. Sublines expressing both Thy 1.1 and Thy 1.2 can be isolated from Thy 1.1⁺, Thy 1.2^– hybrids by cell sorting. In contrast to hybrids with pseudodiploid ALV-induced lymphomas, hybrids between Thy 1.1⁺ lymphomas and pseudotetraploid Thy 1.2^– Abelson-leukemia-virus-induced lymphomas do not express Thy-1 glycoprotein on their cell surface and Thy-1 glycoprotein cannot be detected in detergent extracts of these cells. Thy-1⁺ revertants were isolated from one of the Thy-1^– hybrids by cell sorting. — These results demonstrate a gene dosage effect for the expression of the Thy-1 glycoprotein in somatic cell hybrids. They are consistent with the idea that diffusable gene products regulate Thy-1-glycoprotein expression in these hybrids. They also suggest that there may be additional, apparentlycis-active, regulatory mechanisms which determine the ability of theThy-1 structural genes of the Abelson-leukemia-virus-induced lymphoma parent to be expressed in somatic cell hybrids.     

Modulation of Thy-1 alloantibody responses: donor cell-associated H-2 inhibition and augmentation without recipient Ir gene control

In this study we have found that in vivo PFC responses to Thy-1 are strongly modulated by H-2 gene products in at least 2 ways. First, a profound inhibition of primary PFC responses occurs when foreign H-2 antigens are expressed on Thy-1 incompatible donor cells. This interference effect does not reflect a requirement for H-2-restricted antigen presentation by donor cells, since it is also seen using semi-syngeneic antigenic cells that share a full H-2 haplotype with the recipient. Interference acts more profoundly on slower primary responses than on more rapid secondary responses and requires associative recognition of the H-2 and Thy-1 antigens. By contrast, strong augmentation of the anti-Thy-1 response was obtained when foreign H-2 antigens were expressed in the recipient, as shown by a poor response of an H-2k/k recipient to Thy-1.1 on AKR cells (H-2k) compared with high responses of H-2k/b recipients. A gene controlling this phenotype was mapped to the H-2IA or H-2K regions. However, subsequent experiments revealed that donor recognition of recipient H-2 antigens was required for these high responses; thus, an Ir-gene effect was excluded and an 'intimate form' of an allogeneic effect was postulated. Thus, the immune response to Thy-1 is regulated by at least 3 factors acting at the level of the donor cell, including non-H-2 helper alloantigens, H-2 interference, and H-2-associated allogeneic effects.     

Cellular mechanism of primary anti-Thy-1 antibody responses in vitro induced by uniquely immunogenic thymocyte antigens

Thy-1 antigens are the only cell membrane antigens known to be able to induce primary antibody responses in vitro. We have shown that antigens from the thymocytes of mice and rats were highly immunogenic in cultures of murine spleen cells for the induction of Thy-1.1-specific plaque-forming cell responses, whereas antigens from other tissues, including brains and bone marrow, were poorly immunogenic, if at all. The thymocyte-specific Thy-1 immunogenicity was carried by disrupted cell membranes, and the specific activity for inducing responses was closely linked to Thy-1. We then tried to determine the mechanism of anti-Thy-1 antibody responses in vitro that were induced by the uniquely immunogenic thymocyte antigens. The thymocyte Thy-1 antigens behaved as T cell-independent class 2 (TI-2) antigens: they induced responses in athymic nude mice but not in CBA/N mice with a B cell defect. The apparent TI-2 responses to thymocyte Thy-1 did, however, require Thy-1+ cells in the responder, similar to anti-DNP-Ficoll responses. The full development of the anti-Thy-1 responses required the participation of splenic adherent cells (SAC). Nevertheless, the mechanism of the SAC dependency of anti-Thy-1 responses did not involve antigen presentation to lymphocytes by antigen-pulsed SAC, which contrasted with the finding that the presentation of antigen by live SAC to lymphocytes was indispensable for responses to DNP-Ficoll. The poor Thy-1 responsiveness of SAC-depleted spleen cells was fully restored by the addition of soluble factors (IL 1-like molecules) released from SAC into the culture, which did not replace the SAC-requirement of responses to DNP-Ficoll. It was concluded from these results that Thy-1 or Thy-1-linked structures on thymocyte membranes have an intrinsic activity to directly signal either TI-2 B cells or immature T cells, or both, for activation in the presence of soluble factors released from adherent accessory cells. This conclusion is discussed in relation to a hypothetical view that the thymocyte Thy-1 would physiologically mediate cell-to-cell interactions among special subsets of lymphocytes under thymic influence.