'Autoreactivity' of some hybridomas may be caused by a three-cell interaction

We have produced hybridomas by the fusion of BALB/c (H-2d) bone marrow cells, bone marrow cells from BALB/c-nu/nu mice, BALB/c fetal liver cells, and BALB/c fetal thymocytes with the AKR (H-2k) thymoma BW5147. The hybridomas were selected for the expression of the Thy-1.2 antigen of the normal cell donor and for their ability to produce IL-2 upon co-culture with irradiated normal spleen cells. The hybridomas produce IL-2 when co-cultured with H-2k, H-2u, H-2j, or H-2v cells of some strains but not in mixtures with H-2p, H-2s, H-2f, H-2b, H-2q, or H-2d cells. An investigation into the nature of these differences revealed a novel type of interaction between hybridomas, mature T lymphocytes and class II-positive spleen or lymph node cells. The experiments described in this communication suggest that irradiated L3T4+, Ly-1 High T cells recognize syngeneic class II-positive spleen or lymph node cells, but only in some strains. The ability to recognize the syngeneic cells depends both upon the H-2 complex and on the non-H-2 genes. The recognition leads, in the absence of proliferation, to the secretion of an as yet unidentified and largely hypothetical factor which then acts on the hybridoma cells. Upon stimulation with the T lymphocyte-derived factor, the hybridoma cells begin to secrete IL-2, which can then be measured by the proliferation of the IL-2-dependent CTLL line. The IL-2 production by the hybridoma cells is independent of their proliferation. The described interaction apparently does not involve the T-cell receptor of the hybridoma cells. The interaction resembles the autoreactivity of thymocyte hybridomas described by other investigators, and therefore it is possible that this 'autoreactivity' may in fact be generated by a similar mechanism to the one described here.     

B cell participation in the recursive selection of T cell repertoires

Normal BALB/c mice produce 2,4,6-trinitrophenyl (TNP)-I-Ad specific T helper (Th) cells expressing a receptor heterodimer which share with anti-TNP antibodies an idiotope defined by the F6(51) anti-idiotypic antibody. Expression of this Th idiotype is controlled by major histocompatibility complex and immunoglobulin heavy chain-linked genes and results from antibody-dependent selection of T cell repertoires (Martinez-A. et al., Eur. J. Immunol. 1986. 16: 417). We now present evidence for the recursive nature of T----B cell repertoire selection and suggest that perinatal B cells, present in adult peritoneal cavity, operate in the early phases of this process. Thus, the Th idiotype is absent in BALB/c mice which are either suppressed from birth with anti-mu antibodies, or reconstituted with autologous bone marrow after lethal irradiation as adults. Supplementation of bone marrow reconstitution with syngeneic Thy-1-, Ly-1+ peritoneal B cells, however, selects Th cell repertoires that are undistinguishable from normal mice as to expression of the F6(51) clonotype. This effect is lost after depletion of Ly-1+ cells in the reconstituting Thy-1- peritoneal cell population. Interestingly, large in vivo "naturally" activated Ly-1- splenic B cells can also reconstitute Th idiotype expression if they are isolated from normal, but not from athymic, nude donors. However, transfer of normal large splenic T cells to adult nude mice "educates" the splenic "large B cell" compartment in these animals such that they acquire the ability to recursively select, upon transfer to bone marrow reconstituted recipients, the Th clonotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow microfluorometric analysis of alloantigen expression during T cell development

The fluorescence-activated cell sorter (FACS) was used to examine the expression of several alloantigens on T cells: Thy-1, Lyt-1.1, Lyt-2.1, Ly-5, Ly-6 and Ly-7. Antibodies secreted by monoclonal cell lines were used to characterize the Thy-1.2, Lyt-1.1 and Lyt-2.1 antigenic determinants, whereas Ly-5.1, Ly-6.2 and Ly-7.2 determinants were identified with alloantisera raised by conventional hyperimmunization. Using indirect immunofluorescence with fluorescein isothiocyanate-conjugated reagents, the profiles obtained with the FACS demonstrated that: (a) the expression of Thy-1 is greater on normal thymocytes than on cortisone-resistant thymocytes (CRT), lymph node and spleen cells; (b) in contrast to Thy-1, the expression of Ly-1 on normal thymocytes is less than on CRT, lymph node and spleen cells; (c) about 90% of normal thymocytes but < 50% of CRT, spleen and lymph node T cells are Lyt-2⁺ and (d) although weakly expressed on normal thymocytes, the amount of Ly-6 and Ly-7 present on peripheral T cells was considerably increased. The significance of these findings and the potential for further analysis of T cell developmental pathways is discussed.     

Characterization of V beta-bearing cells in athymic (nu/nu) mice suggests an extrathymic pathway for T cell differentiation

In the present article, the expression of the T cell receptor (TcR) beta chain and other T cell molecules was evaluated in surface immunoglobulin-negative spleen cell populations of young and old BALB/c and C57BL/6 nude mice, using a panel of monoclonal antibodies. The results obtained show that in young nude mice, most Thy-1high cells do not express other T cell markers. These mice have, however, a sizable population of Thy-1low cells with the same phenotype of alpha/beta+, CD4-CD8- thymocytes or MRL/lpr peripheral T cells, expressing predominantly genes of the V beta 8 family. The evolution of alpha/beta+ cells in aging nudes is strongly suggestive of an extrathymic pathway of differentiation of these cells since (a) the acquisition of high density TcR and CD3, as well as Thy-1 or CD4CD8 antigens at the cell surface of nude V beta+ T cells is not simultaneous; (b) alpha/beta+ cells in nude mice co-express other T cell markers at random and, even in old mice, they never completely resemble to the predominant high Thy-1+ CD3+ TcR alpha/beta+, CD4+CD8+ cells of euthymic controls; and (c) BALB/c nude T cells express V beta 11 genes, that are deleted in euthymic BALB/c mice. This latter finding may also indicate differences in the mechanisms of selection of T cells specificities in the thymus vs. the peripheral pools.     

Restitution of the thymus in lethally irradiated mice after transplantation of syngeneic or allogeneic bone marrow

The early restitution of the thymus of bone marrow chimeras was investigated by the immunoperoxidase technique using monoclonal antibodies against Thy-1 and Lyt-1, Lyt-2, Lyt-3. Within two weeks, normal thymus histology was restored in mice which received untreated syngeneic BM or syngeneic or allogeneic BM pretreated with SAL (specificed antilymphocytic serum). Irradiation depleted the thymic cortex of small Thy-1+, Lyt-1+2+3+ cells but did not affect a medullary population of medium sized weakly stained Thy-1+, strongly stained Lyt-1+ cells. Preceded by the appearance of an increasing number of large Thy-1+, Lyt-1- blasts (days 2 and 4), the thymic cortex was repopulated (beginning on day 6) by smaller Thy-1+ cells which acquired Lyt-1, Lyt-2 and Lyt-3 though, obviously not in a strictly sequential manner. Simultaneously, the medullary radioresistant cells disappeared, nd the medulla was subsequently repopulated (beginning on day 8) by thymocytes of a mature phenotype. Early restitution of the thymus in radiation control mice was similar to the bone marrow chimeras. The results indicate that the histological restitution of the thymus originates substantially from radioresistant precursors of host origin. Graft-versus-host reaction induced by untreated allogeneic bone marrow cells prevented normal thymic restitution. A delayed localized cortical repopulation with small Thy-1+, Lyt-1+2+3+ cells, progressive destruction of thymic architecture and almost no restoration of the medullary immunocompetent thymocytes were noted. T cell differentiation obviously was seriously affected by the injuries to the thymic microenvironment due to alloreactive T cells.     

Analysis of a murine B cell lymphoma, CH44, with an associated non-neoplastic T cell population. I. Proliferation of normal T lymphocytes is induced by a secreted product of the malignant B cells.

A non-neoplastic T cell population associated with a murine monoclonal B cell malignancy, CH44, was analyzed. Immunofluorescence on cell suspensions and immunoperoxidase staining on tissue sections using monoclonal antibodies to the antigens Thy1.2, Ly-1, L3T4, and Lyt-2 confirmed the presence of both TH (Ly-1/L3T4+, Lyt-2-) and Tc/s (Ly-1/L3T4-, Lyt-2+) T cell subpopulations. The non-neoplastic T cells were present in both a 0.6 and 2.1 g CH44-bearing spleen. T cells, not normally in liver in significant numbers, were found in liver tissue when the CH44 tumor cells were present. These data implied an active proliferation of the T cell populations within tissues containing the malignant B cells. Supernatant from an in-vitro-adapted cell line of CH44 (CH44.LX) was tested for its ability to induce proliferation of normal murine splenocytes and thymocytes. As assayed by tritiated thymidine incorporation, both spleen and thymus cells proliferated in the presence of CH44.LX supernatant. Although supernatant from two of nine other B cell lines was able to stimulate the proliferation of spleen cells, only CH44.LX could induce proliferation of thymus cells. Supernatant from the seven other B cell lines and three hybridomas had no measurable effect on either splenocytes or thymocytes in this assay. It is hypothesized that the presence of a non-neoplastic proliferating T cell population associated with a neoplastic B cell lymphoma during in vivo passaging of the tumor is the result of effects derived from a secreted product of the malignant B cells. Whether the T cells have any effect on the growth of the malignant B cells is not known.     

The role of hematogenous and intrinsic precursor cells in lymphocyte production in murine bone marrow and thymus

It is well recognized that the bone marrow contains cells that can repopulate a depleted thymus as well as cells that can be induced to express phenotypic markers characteristic of T cells. It is not known, however, to what extent thymocytopoiesis in the normal thymus relies on immigrant, bone marrow-derived cells, nor whether some T cell precursors have entered the bone marrow from the circulation. We used the parabiotic system to test whether thymocytopoiesis relies on progenitors intrinsic to the thymus or on cells that enter the organ from the circulation. In the same system, we have also investigated whether Thy-1^? bone marrow lymphocytes that respond to phytohemagglutinin (PHA) by proliferation and Thy-1 expression are produced by my-elogenous or hematogenous progenitors. Syngeneic CBA/HT6 and CBA/CaJ mice were joined in parabiotic union at 4–6 weeks of age. Cross circulation between the two partners was verified by the equilibration of Evans' blue dye injected into one partner and by the equilibration of PHA-responsive T cells in the spleen of the parabionts. Chromosome spreads were prepared from the PHA-stimulated T cell-depleted bone marrow and from spontaneously proliferating thymocytes as well as from thymocytes stimulated by PHA or Concanavalin A (Con A). The exchange of spleen colony-forming units (CFU-S) in the femoral marrow was assessed by karyotyping individual spleen colonies. Regardless of the length of parabiotic union, ranging from 4 to 20 weeks, Thy-1^?, PHA-responsive bone marrow lymphocytes remained predominantly of the host type with only 3% being derived from the opposite partner. The same held true for CFU-S in the femur; only around 5% of this cell population were of the nonhost type. Thus, although some Thy-1^?, PHA-responsive lymphocytes in the bone marrow may be derived from hematogenous stem cells, the majority of them are generated by precursors resident in the bone marrow. Likewise, regardless of the length of parabiotic union, at least 95% of spontaneously proliferating cells in the thymus of each partner possessed the karyotype of the host, and this held true also for PHA- or Con A-stimulated thymocytes, indicating that the small population of spontaneously proliferating immigrant cells cannot account for the production of the large number of postmitotic (mitogen-responsive) thymocytes. Our findings, therefore, demonstrate a high degree of self-maintenance for Thy-1^?, PHA-reponsive lymphocytes in the bone marrow and also for intrathymic T cell precursors. In the unperturbed, postnatal thymus, thymoctye production does not rely on cell input from the circulation; the vast majority of thymoctyes are generated by an intrathymic precursor pool that is independent of immigrant myelogenous T cell precursors.     

The peritoneal Ly-1 (CD5) B cell repertoire is unique among murine B cell repertoires.

Ly-1 (CD5) B cells and conventional B cells represent two distinct lineages of murine B cells which are distinguishable by expression of surface molecules, organ location, ontogeny and development and antibody production in vivo. In order to assess whether the different developmental pathways of Ly-1 B cells and conventional B cells result in different antibody repertoires, we have used limiting dilution analyses to determine frequencies of B cells making antibodies capable of binding to a range of antigens including haptens, proteins, bacterial polysaccharides and bromelain-treated mouse red blood cells. Starting populations of B cells were purified from spleen, peritoneum and bone marrow of adult BALB/c mice or from spleens of newborn mice by use of the fluorescence-activated cell sorter. The peritoneal Ly-1 B cell repertoire was found to be different from that of conventional B cells, with between 5- and 100-fold higher frequencies of clones producing IgM antibodies capable of binding to the antigens tested. However, when tested, the majority of Ly-1 B cell anti-haptenic antibodies did not show the high affinity binding or fine specificity characteristics of specific antibodies elicited in immune responses in vivo. The high frequencies of antigen-reactive antibodies within the Ly-1 B repertoire are most likely explained by the presence of clones secreting low-affinity or multireactive antibodies. The Ly-1 B cell repertoire is not mirrored in repertoires from either newborn B cells or virgin B cells in adult bone marrow. Therefore, either Ly-1 B cells develop from distinct precursors with intrinsically different mechanisms of V gene usage and recombination, or newly formed Ly-1 B are heavily selected on specificity for entry into this peritoneal lineage. If the second alternative is true, bacterial antigens in the gut are not required for selection of this unique repertoire, as Ly-1 B cells in germ-free mice also show the multireactive repertoire characteristic of this B cell lineage in normal mice.     

Rat bone marrow cells undergo thymopoiesis in mouse fetal thymic organ culture

We have developed an in vitro differentiation assay allowing the study of thymopoiesis from rat bone marrow cells. In this assay, Wistar rat bone marrow cells repopulated fetal Swiss mouse thymic lobes depleted in endogeneous lymphoid cells by deoxyguanosine treatment. Due to the xenogeneic situation, repopulating rat cells from any hemopoietic lineage could be easily recognized by anti-rat monoclonal antibodies such as anti-Thy-1.1 that did not react with Swiss mouse thymocytes. After 15 days in vitro, 80% of the developing rat cells were Thy-1.1+ lymphoid cells and about 70% of the Thy-1.1+ cells expressed CD5, CD2 and leukosialin. The percentages of cells expressing pre-B cell, B cell and myeloid determinants were less than 20%. The developing thymocytes comprised CD4-CD8- T cell receptor (TcR) alpha/beta-, CD4-CD8+TcR alpha/beta low and CD4+CD8+TcR alpha/beta low cells, indicating that the early stages of rat thymopoiesis occurred within mouse thymic lobes. Limiting dilution assays showed that 50% of positive assays were obtained with 3000 nucleated bone marrow cells, which is in good agreement with recent estimates derived from in vivo reconstitution after intrathymical transfer. Moreover the limiting dilution assays proved to be sensitive enough to evidence a tenfold enrichment of pre-T cell activity in the low-density fraction of rat bone marrow. This xenogeneic system might greatly facilitate studies on prethymic and intrathymic stages of rat T cell development and permit new in vitro approaches of the colonizing bone marrow T cell precursor properties.     

Genetics and strain distribution of concanavalin A-reactive Ly-2-, L3T4- peripheral precursors of autoreactive T cells

Cytotoxic treatment of BALB/c cells from different peripheral lymphoid tissues by a cocktail of monoclonal antibodies against Thy-1, Ly-1, L3T4 and Ly-2 differentiation markers (anti-T cocktail) plus complement eliminates all mature T lymphocytes. Yet a population of dull Thy-1+, Ly-1-, L3T4-, Ly-2-, corresponding to about 1% of the initial population, can be detected by flow cytometry which proliferate under concanavalin A stimulation. These anti-T killing-resistant cells (TKR) were previously shown to be capable of differentiating in culture into class II-restricted autoreactive T helper cells. We demonstrate here that such cells can be detected in mice of BALB/c and DBA/2 genetic background but are absent in C57BL/6 and B10 animals. The presence of TKR cells is dominant in (BALB/c x C57BL/6)F1 hybrids and genetically controlled by two genes which are neither H-2 nor Igh linked. TKR cells are also detected in young NZB mice but disappear with the development of the systemic autoimmune disease in old animals. Thy-1+, L3T4-, Ly-2- cells from MRL lpr/lpr mice also respond to concanavalin A but are removed by the anti-T treatment. Altogether, arguments are presented suggesting that TKR cells represent a particular subset of double-negative peripheral T cells which may correspond to autoreactive T cell recursors that would escape the thymic selection. We postulate that these cells are present in all mouse strains but their susceptibility to killing by anti-Thy-1 antibodies differs depending on background genes.     

Monocyte IgG-Fc receptors in myotonic dystrophy.

The effect of affinity purified natural thymocytotoxic autoantibodies from New Zealand Black (NZB) mice (NTA-1 and NTA-2) against pre-T cells was studied. Pre-treatment of spleen cells from nu/nu mice or bone marrow cells from DBA/2 mice with NTA-1 and complement (C) or with NTA-2 and C markedly inhibited the induction of T cell markers with a thymic extract in vitro. The effect of NTA-2 and C was abolished by absorption of NTA-2 with neuraminidase treated thymocytes, whereas its effect was not abolished by absorption with intact thymocytes. The effect of NTA-1 and C was abolished by absorption of NTA-1 with either neuraminidase treated thymocytes or intact thymocytes. The effect of NTA-2 was inhibited by lactose whereas the effect of NTA-1 was not. These results suggest that NTA-2 specifically recognizes the cell surface characteristics of pre-T cells and blocks the differentiation pathway of T cells.     

An analysis of T lymphocyte subsets in tumour-transplanted mice on the basis of Lyt antigenic markers and functions

Small lymphocyte subsets were characterized radioautographically on the basis of several surface markers, viz. surface Ig (S-Ig), Thy-1 and Lyt (Ly-1, Ly-2 and 3) antigens in host lymphoid organs (thymus, spleen and blood) as well as at the tumour site at various stages of subcutaneous growth of two different syngeneic tumours—MPC-11 plasmacytoma and WEHI-164 fibrosarcoma in BALB/c mice. In both tumour-host combinations there was a rise in the levels of null (S-Ig^-, Thy-1^-) small lymphocytes as well as the Ly-23⁺ subset of T small lymphocytes at all the sites examined. The absolute number of these two subsets also increased excepting the case of null cell rise in the thymus which was relative. The functional potentials of Lyt subsets were explored by employing in vitro and in vivo assays. While no appreciable levels of anti-tumour cytotoxic T cells (T_c) were detectable by a ⁵¹Cr release assay in the host spleen or the tumour-draining lymph nodes at any stage of growth of MPC-11 tumour, such T_c was generated in vitro by a co-cultivation of unprimed spleen cells with irradiated MPC-11 cells. These T_c were Thy-1⁺ and Ly-12⁺, as noted from antibody+C′ mediated abrogation of cytotoxicity. These results suggested that the generation of anti-tumour T_c in vivo was suppressed in tumour-bearing hosts. The possibility of a cell-mediated suppression was tested by an adoptive transfer of thymocytes or splenocytes from tumour-bearing mice into naive or pre-immunized recipients which then received fresh tumour transplants. This procedure caused a specific enhancement of tumour growth in three tumour-host combinations: MPC-11 or WEHI-164 tumour in BALB/c mice and W-1 fibrosarcoma in CBA mice. The suppressor lineage lymphocytes appearing in vivo were found to be Thy-1⁺ and Ly-1^-, 2⁺, as noted from antibody +C′ mediated abrogation of their tumour-growth promoting ability. They appeared earlier (7 days) in the thymus and later (>2 weeks) in the spleen and then persisted during the tumour lifetime. The parallel kinetics of the increase in the overall level of Ly-23⁺ cells and the appearance of Ly-2(3)⁺ suppressor lineage T cells in tumour-bearing hosts may indicate that studies of T-cell surface markers may be useful in predicting changes in the functional lymphocyte subsets.     

Rapid production of monoclonal antibodies to T lymphocyte functional antigens

Brief immunization of rats with mouse lymphoid cells was combined with the rat/mouse hybridoma technology and functional hybridoma screening to yield a rapid method for the production of monoclonal antibodies (MAb) against functionally important T lymphocyte cell surface antigens. Two protocols were used. In one, rats were immunized once with mouse thymocytes followed by fusion and screening of the hybridomas for interference with the thymocyte co-stimulator (interleukin 1) assay. The resultant hybridomas included producers of MAbs against the L3T4-antigen (inhibitory), the Ly-1-antigen (stimulatory), and the Thy-1-antigen (inhibitory?). In the second protocol, rats were immunized twice with a T cell hybridoma. The resultant hybridomas were screened for inhibition of polyclonal T cell activation, induced by an anti-Thy-1 (MAb G7). A panel of MAbs against the Thy-1 antigen with different reactivity profiles was generated by this procedure. Most of the MAbs were of the IgM class. Short-term immunization may lead to less selection of response to highly immunogenic determinants than a protocol involving several boosters. Thus, this alternative may be useful for producing MAbs against rare or weakly immunogenic cell surface molecules, as suggested by the ease with which we were able to make MAbs against the L3T4-molecule.     

A monoclonal antibody recognizing human Thy-1: distribution on human and non-human primate haematopoietic cells

A monoclonal antibody designated 'antibody 390' (Ab 390) with anti-human Thy-1 reactivity was prepared by the hybridoma technique from the splenocytes of BALB/c mice immunized with human fetal brain. This antibody was shown to have anti-human Thy-1 reactivity because (1) it precipitated a molecule with a molecular weight of about 24,000 daltons, (2) it had a pattern of reactivity similar to that of previously described anti-human Thy-1 antibodies and (3) purified human Thy-1 antigen specifically inhibited binding of Ab 390 to a known antigen-positive cell line. It was the intent of this study to investigate the distribution of Thy-1 on normal and malignant haematopoietic cells in humans and non-human primates. We show here that Ab 390 did not react with human peripheral blood leucocytes, bone marrow cells or splenocytes by immunofluorescence but did react with subcapsular and cortical fetal thymocytes by peroxidase-antiperoxidase immunohistology. A section of fetal spleen demonstrated staining of connective tissue and blood vessels and rare reactive lymphocytes. Adult spleen contained Thy-1-positive cells surrounding the white pulp and in the marginal zone, but single-cell suspensions of splenocytes did not react with Ab 390. Ab 390 was tested against a variety of fresh human leukaemia cells and human cell lines and was shown to react with only the acute lymphoblastic leukaemia T cell lines RPMI 8402 and HPB-MLT. Non-human primate studies revealed reactivity with a number of T cell lines from New World primates (cotton-topped and red-bellied marmosets) and peripheral blood granulocytes (owl monkey). Our studies support previous findings that suggest that human Thy-1 may be a marker for early T lymphocytes in man, and its distribution on non-human primate T cell lines suggests the same for certain species of non-human primates. Not consistent with the distribution on human cells was the demonstration of Ab 390 reactivity with owl monkey granulocytes.     

Quantitative analysis of tissue distribution of Ly10-like murine alloantigen(s) with antisera and monoclonal antibodies

Tissue distribution of non-Lyt1.1 ("Ly10-like") antigen or antigens encoded by short chromosomal segment differentiating B6-Ly-1a congenic strain from B6 strain of mice was studied by quantitative absorption of (BALB/c X B6)F1 anti B6-Ly-1a antiserum and by direct cytotoxicity of Ly-10-132-12-26 monoclonal antibody on lymphoid cell populations. Identical strain but not tissue distribution pattern does not allow to conclude whether antiserum and monoclonal antibody detect the same or closely linked antigens. Absorption experiments revealed the highest antigen content in the brain tissue, lower in testis and kidney, still lower in lymphoid organs and the lowest in liver and lung. Among lymphoid cells, bone marrow cells had highest absorbing capacity, followed by thymus, spleen and lymph nodes. Monoclonal antibody lysed almost 100% of thymocytes, 30% bone marrow cells and 10-20% of spleen and lymph node cells (both T-cell and B-cell enriched populations contained the same proportion of positive cells). Cortisone resistant thymocytes showed the same sensitivity as cortical thymocytes to Ly-10-132-12-26 antibody which is distinguishable characteristics of medullary thymocytes from peripheral T cells. Mitogen activated lymphocytes exhibited significantly higher expression of Ly10-like antigen than resting peripheral lymphocytes.     

Isolation of specific antigen-reactive T-cell clones from nude (nu/nu) mice infected with Mesocestoides corti.

Infection of nude mice with Mesocestoides corti results in spleen enlargement, with a decrease in percentage positive, but overall increase in Thy-1 positive cells. A low frequency of both parasite antigen-specific and alloantigen-reactive T-cell clones were isolated from the spleen, mesenteric lymph nodes, peritoneal exudate and Peyer's patches. The T-cell clones from spleen were found to express the antigens Thy-1, Lyt-1 and Lyt-2; furthermore, after stimulation with antigen in the absence of exogenous T cell growth factor (IL-2), they produced normal levels of haemopoietic growth factor (IL-3), bone marrow proliferation activity and eosinophil differentiation activity. A higher proportion produced IL-2 compared to clones from normal mice. The isolation of antigen-specific T-cell clones showing normal T-cell characteristics from young nude mice adds weight to suggestions that exposure to antigen results in significant extra-thymic T-cell maturation.     

Analysis of the Thy-1 pathway of T cell hybridoma activation using 17 rat monoclonal antibodies reactive with distinct Thy-1 epitopes

Seventeen monoclonal anti-Thy-1 antibodies (mAb) derived from LOU/M rats immunized with mouse T cell clones were used to study the role of Thy-1 in antigen-independent T cell activation. These mAb identified Thy-1.2 or monomorphic determinants and immunoprecipitated a molecule of 25-28 kDa from detergent-solubilized, 125I-labeled T cell surface proteins. Competitive cross-inhibition binding assays demonstrated that these reagents defined 3 epitope groups including either Thy-1.2 (group A) or Thy-1 monomorphic (groups B and C) determinants. Experiments using high titered culture supernatants revealed that all 6 IgG mAb defining the epitope group C, and one IgG2c mAb directed at a determinant in group A were capable of stimulating the terpolymer-L-glutamic acid60-L-alanine33-Ltyrosine10 (GAT) plus I-Ad-reactive BALB/c T cell hybridoma T14-117.9 to produce interleukin 2 (IL2) in the absence of accessory cells. Cross-linking of cell-bound rat mAb by a BALB/c anti-rat kappa chain mAb, or the presence of B cell lymphomas in the culture resulted in an increase of the Thy-1-mediated IL2 responses of this hybridoma. Some mAb from group B required antibody doses exceeding 80 micrograms/ml in order to activate T cells, while others remained nonstimulatory at any dose tested. Striking synergy in mAb-mediated T cell activation was observed when nonmitogenic doses of mAb group groups A and C were mixed in the same culture. Analysis of a panel of GAT plus I-Ad-specific T cell hybridomas revealed that these cells markedly differed in the magnitude of their IL2 responses induced by a given amount of stimulating anti-Thy-1 mAb. Such reagents also stimulated normal thymocytes to express IL2 receptor on their surface. These studies show that the epitopic specificity and the amount of anti-Thy-1 mAb, and the susceptibility of the T cell examined represent important parameters for the triggering of the Thy-1 pathway of T cell activation.     

Dissociation of signal transduction via Thy-1 and CD3 antigens in murine T cells

To understand the proliferation/differentiation of immature thymocytes which have not express T cell antigen receptor (TCR), we studied whether Thy-1 has signal-transducing capacity. Thy-1+ CD3-TCR- cells including thymocytes from BALB/c embryos and SCID mice and nude mouse splenic cells did not show proliferative responses in the culture with anti-Thy-1 (G7) plus phorbol myristate acetate (PMA), whereas Thy-1+ CD3+ cells from normal thymus or spleen did show a response to them. Since Thy-1-mediated activation is suggested to require co-expression of the CD3-TCR complex, we compared the T cell proliferative response in mature T cells stimulated with anti-Thy-1 (G7) and anti-CD3-epsilon (2C11). Under the presence of PMA or IL-2, accessory cell-depleted splenic T cells were cultured with G7 or 2C11. PMA augmented the proliferative response of splenic T cells cultured with G7 much more than that with 2C11. IL-2, however, showed reciprocal effect on the proliferation of G7 and 2C11-treated splenic T cells. These data suggest that signals triggered via Thy-1 and CD3-epsilon may provide a distinct intracellular pathway for T cell activation.     

Many cells in rat bone marrow have cell-surface Thy-1 antigen.

Thy-1.1 and Thy-1 xenoantigenic determinants were detected at the cell surface of many rat bone marrow cells. The absorptive capacity of bone marrow cells was 6-10% of that of thymocytes for Thy-1 antigenic determinants, and 30-45% of rat bone marrow cells were specifically labeled with anti-Thy-1 antibody as detected by autoradiography. Thus, while mice and rats are similar in having large amounts of Thy-1 in brain and thymocytes, they differ in that the rat lacks the antigen in most peripheral T cells and expresses it in a large number of bone marrow cells; the opposite is true in the mouse.