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.     

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.     

Isolation of monoclonal antibodies to antigen Lyt-3.2

A hybridoma producing monoclonal antibodies (McAb) NATF9.9 (F9) was obtained from fusion of murine myeloma X63 and splenocytes of AKR mice immunized with a single intravenous injection of 5 X 10(7) thymocytes of CBA mice. F9 McAb were cytotoxic for 80% thymocytes, 10% splenocytes, 20% lymph node cells, 85% cortical and 32% medullary thymocytes of CBA, C57BL/6, BALB/c, DBA/2 and SJL but not for the cells of C58 and AKR mice. F9 McAb reacted only with T cells and did not react with B cells and EL4 thymoma cells (Thy-1.2+, Lyt-1+2-3-). The proportion of F9+ cells accounts for about 40% among T lymphocytes of the lymph nodes and spleen as tested by flow-type cytometry. Lymph node cells treated with F9 McAb plus complement completely lost their reactivity with rat anti-Lyt-2 McAb and only partly (by 30%) with anti-Lyt-1 McAb. The reactivity pattern of F9 McAb attests to their specificity for Lyt-3.2 antigen.     

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.     

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.     

Cell-mediated immune responses to syngeneic tumors. I. Identification of two distinct CTL effector pathways which differ in antigen specificity, genetic regulation, and cell surface phenotype

It has been demonstrated previously that draining lymph nodes (DLN) from tumor-immunized mice contain a population of lymphoid cells that are capable of differentiating into functional antitumor cytotoxic T lymphocytes (CTL) during in vitro culture. In the present studies, it was observed that DLN cells from either C57BL/10 (B10) or C3H mice that had been footpad-immunized with syngeneic tumor cells differentiated into CTL during a 4-day in vitro culture in the absence of added antigen. The specificity patterns of the CTL thus generated, however, were quite different in the two strains. DLN from B10 mice immunized with ultraviolet light-induced fibrosarcoma cells of B10 origin differentiated into CTL which were only capable of lysing target cells from the tumor used for immunization. Thus, the antitumor CTL which differentiate from B10 DLN appeared to be specific for the tumor-specific antigen (TSA) expressed by these tumor cells. In contrast, DLN from C3H mice immunized with a syngeneic ultraviolet light-induced fibrosarcoma differentiated into CTL which effectively lysed not only target cells from the immunizing tumor, but several other fibrosarcomas of both B10 and C3H origin, and which did not lyse normal nontumor targets. These C3H effectors thus appeared to be specific for a tumor-associated antigen (TAA) which is widely shared by a number of tumors. Cold target-blocking studies demonstrated that the CTL generated by C3H DLN cells contained a subpopulation of TSA-specific cells in addition to cross-reactive TAA-specific effectors. (B6 X C3H)F1 (B6C3F1) mice generated cross-reactive TAA-specific CTL in response to in vivo challenge with either B10 or C3H tumors, indicating that the ability to generate a TAA-specific CTL response behaves as a dominant trait of the responding mouse strain and not as a function of the tumor used for immunization. TSA-specific CTL and cross-reactive TAA-specific CTL were distinguishable on the basis of their cell surface phenotypes, because the TSA-specific CTL generated by B10 DLN cells were Thy-1.2+ Lyt-2.2+, whereas TAA-specific B6C3F1 CTL were Thy-1.2+ Lyt-2.2-; alloantigen-specific CTL generated from the same B6C3F1 lymph nodes were Thy-1.2+ Lyt-2.2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunoregulation in experimental disseminated histoplasmosis: flow microfluorometry (FMF) studies of the Thy and Lyt phenotypes of T lymphocytes from infected mice

Previous studies have shown that mice infected i.v. with 6 X 10(5) yeast phase Histoplasma capsulatum (Hc) develop suppressed immune responses during weeks 1 to 4 of infection but that by weeks 8 to 12 of infection these responses return to normal. In this study total and differential cell counts showed that as early as the third day of infection there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymus of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 28 both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric (FMF) studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (slg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 5 and 7 the thymocytes from infected mice displayed a higher relative fluorescence intensity (RFI) of the Thy-1.2 marker than normal thymocytes, whereas at day 10, the RFI was less than that of normal thymic lymphocytes. Between days 7 and 10 of infection the RFI of the Lyt-2 marker was less on thymocytes from Hc-infected mice; however, there was no change in the Lyt-1 marker. Examination of these lymphocyte markers in blood, spleen, and mesenteric lymph nodes showed that there were decreases in the RFI of both the Thy-1.2 and Lyt-2 between days 5 and 10 of infection. No changes were observed in the Lyt-1 or slg markers. By day 28 there were no differences between the normal and infected mice with respect to any surface marker in any of the organs studied. In other experiments, the effect of adrenalectomy before infection on these surface markers was studied. Absolute numbers of Thy-1.2+, Lyt-1+, and Lyt-2+ cells were significantly increased in the spleen and significantly decreased in the thymus and peripheral blood of infected mice relative to normal controls. These studies suggest that there is a migration of cells from the thymus, blood, and bone marrow to the spleens of mice with disseminated Hc infection.     

Fractionation of T cell subsets on Ig anti-Ig columns: isolation of helper T cells from nonresponder mice, demonstration of antigen-specific T suppressor cells, and selection of CD-3 negative variants of Jurkat T cells

In the present experiments we have explored the possibilities of a modified immunoadsorbent technique to select for (1) mutagenized T cell receptor (Tcr) negative variants of Jurkat T lymphoma cells and (2) purified CD-4+ or CD-8+ T lymphocytes. The basic principle was to make large numbers of immunoglobulin (Ig) negative T cells Ig+ by T cell subset-specific monoclonal antibodies (mAb), and to select such cells on Ig anti-Ig columns. Our results demonstrated that Thy-1+, Fc receptor positive, antigen-specific T cells regulate the immune response in mice nonresponders to pork insulin, and the "autologous" mixed lymphocyte reaction. In addition, the immunoadsorbent method very efficiently selects Tcr/CD-3- variants from mutagenized Jurkat cell populations incubated with anti-CD3 mAb. The described method is easy and quick and can fractionate large numbers of cells; it is the "poor-man's cell sorter." The most important finding is the demonstration of antigen-specific Thy-1+, CD-8+, and Fc receptor+ T suppressor cell that apparently react with antigen in a non-major histocompatibility complex-restricted manner.     

Thymectomized, irradiated, and bone marrow-reconstituted chimeras have normal cytolytic T lymphocyte precursors but a defect in lymphokine production

A model system has been developed to study extrathymic T cell differentiation; mice have been thymectomized, lethally irradiated, and reconstituted with bone marrow cells depleted of Thy-1+ cells. After 8 wk, the spleen cells of these athymic, bone marrow-reconstituted chimeras contain Thy-1+ precytolytic T lymphocytes (CTL) that are able to respond to antigen only if supernatant from Con A-activated T cells is added to culture. The phenotype of these pre-CTL is similar to that of thymocytes, suggesting that they may be immature T cells. Initial evaluation of the CTL repertoire of these athymic mice demonstrated that the CTL generated to trinitrophenyl-modified syngeneic cells are H-2-restricted, and that the CTL generated to alloantigens have many of the cross-reactivities observed in normal mice but not in nude mice. In this report, we demonstrate a helper T cell defect in these thymectomized chimeras. These chimeras lack an Ly-1+ helper cell required for thymocytes to differentiate to CTL. Further studies revealed that when spleen cells from these thymectomized chimeras were stimulated with Con A, they produced normal levels of interleukin 2. However, these splenocytes were defective in the production of another factor needed for CTL differentiation.     

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.     

Phenotype and distribution of T lymphocytes in Peyer's patches of athymic mice

The distribution and phenotype of T (Thy-1.2+) cells was examined in Peyer's patches of 8 and 16 week old athymic mice by peroxidase and two-color-fluorescence immunohistochemistry. Despite the generally recognized T cell deficiency of nude mice, some T cells consistently occurred in Peyer's patch domes in all mice. However, many Thy-1.2+ lymphocytes lacked cell surface markers for either helper T cells (L3T4) or cytotoxic/suppressor T cells (Lyt-2), indicating that these cells may be an immature subset of T cells. These cells may represent a population of resident T cell precursors delayed in maturation or T cells newly immigrated to Peyer's patches.     

Murine lymphokine-activated killer (LAK) cells: phenotypic characterization of the precursor and effector cells

Murine and human lymphocytes incubated in recombinant interleukin 2 (RIL 2) generate a population of cytotoxic cells (lymphokine-activated killer cells [LAK]), which are able to lyse a wide array of fresh tumor cells but do not lyse fresh normal cells. Intravenous administration of these cells with the concomitant administration of RIL 2 can eliminate established pulmonary and hepatic metastases in mice. To characterize the cell that has in vitro LAK activity, we subdivided murine lymphocytes by lysing select subpopulations with the use of complement and antibodies against lymphocyte surface markers or by fluorescence-activated cell sorting. Thy-1.2-negative splenocytes were found to generate near normal amounts of LAK activity after RIL 2 incubation. Small and inconsistent LAK cell activity was generated from Thy-1.2-positive splenocytes. Ia-positive and surface immunoglobulin-positive splenocytes had little or no LAK precursor capability and did not appear to be necessary for LAK activation. Treatment of splenocytes with anti-asialo GM1 (anti-ASGM1) heterosera and complement markedly decreased their ability to generate LAK activity. At the effector stage, cytotoxic cells were of the Thy-1.2-positive, Ia-negative phenotype. Ia-depleted cells were separated into subpopulations bearing or not bearing the gamma Fc receptor (gamma FcR). The majority of cytotoxicity resided in gamma FcR-positive cells. Thus the precursors of murine LAK cells are "null" lymphocytes bearing neither T nor B cell surface markers but develop the Thy-1.2 cell surface marker in vitro, in association with the development of lytic activity for fresh tumor cells after stimulation by RIL 2.     

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.     

Origin of autoreactive T helper cells. I. Characterization of Thy-1+, Lyt-, L3T4- precursors in the spleen of normal mice

A new population of dull Thy-1+, Ly-1-, Lyt-2-, L3T4- PNA- cells, resistant to a double cytotoxic treatment by monoclonal antibodies to these T cell markers plus complement, has been isolated from the spleen of normal adult BALB/c and DBA/2 mice (Tkr cells). These cells exhibit no spontaneous autoreactivity or alloreactivity but can be activated with concanavalin A (Con A). Once activated, they differentiate into bright Thy-1+, Ly-1+, Lyt-2-, L3T4+ PNA- T lymphocytes. Con A-activated Tkr cells also strongly proliferate in the presence of allogeneic or syngeneic dendritic cells in secondary cultures. Moreover, contrary to other Con A-stimulated T cell populations, they induce B lymphocytes to proliferate and to differentiate into Ig-secreting cells at a very high level. Con A-activated Tkr cells are therefore very potent polyclonal B cell activators. Restimulated of Tkr cells by syngeneic dendritic cells can be inhibited by anti-L3T4 or anti-class II monoclonal antibodies. The results suggest that Tkr cells are the precursors of class II-specific autoreactive T helper cells. Tkr cells are absent in the spleen of B6 animals. This indicates that their expression might be genetically controlled. It also suggests that Tkr cells may not be the unique splenic precursors of autoreactive T cells. Con A activation of Tkr cells in Click's medium is 2-mercaptoethanol dependent and highly sensitive to pCO2, like the response of thymocytes. Tkr cells are also absent in the spleen of nude mice. We conclude that Tkr cells represent splenic precursors of autoreactive T helper cells equivalent to Thy-1+, Ly-2-, L3T4- PNA- cortical thymocytes.     

Accessory cells in the in vitro generation of type C virus-specific T killer lymphocytes. II. Demonstration of a T helper cell in the spleen of in vivo primed mice

The existence of a helper T cell cooperating with cytolytic T lymphocytes (CTL) in cell-mediated anti-tumor responses specific for the virus-induced FMR antigens can be demonstrated by using unprimed thymocytes as CTL precursors and in vivo primed irradiated spleen cells as helper. The helper T cells express Thy-1.2 and Lyt-1.2 antigens at their surface, but not Lyt-2.2. The helper function required the presence of macrophages to be detected, is antigen specific, and appears unusually radiosensitive compared with previously described helper T cell function.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with 1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; 2) monoclonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and 3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: 1) the majority of cortical thymocytes are strongly Thy-1+ (positive), strongly T-200+, variable in Lyt-1 expression, and strongly Lyt-2+; 2) the majority of medullary thymocytes are weakly Thy-1+, strongly T-200+, strongly Lyt-1+, and Lyt-2- (negative); 3) a minority of medullary cells are weakly Thy-1+, T-200+, strongly Lyt-1+, and strongly Lyt-2+; 4) a small subpopulation of subcapsular lymphoblasts is Thy-1+, T-200+, and negative for the expression of Lyt-1 and Lyt-2 antigens; 5) a small subpopulation of subcapsular lymphoblasts is only Thy-1+ but T-200- and Lyt-; and 6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.     

Phenotype and distribution of T lymphocytes in Peyer's patches of athymic mice

Summary The distribution and phenotype of T (Thy-1.2^–) cells was examined in Peyer's patches of 8 and 16 week old athymic mice by peroxidase and two-color-fluorescence immunohistochemistry. Despite the generally recognized T cell deficiency of nude mice, some T cells consistently occurred in Peyer's patch domes in all mice. However, many Thy-1.2⁺ lymphocytes lacked cell surface markers for either helper T cells (L3T4) or cytotoxic/suppressor T cells (Lyt-2), indicating that these cells may be an immature subset of T cells. These cells may represent a population of resident T cell precursors delayed in maturation or T cells newly immigrated to Peyer's patches.     

Surface molecules and cell interactions

Many of the cell surface molecules of lymphocytes or their precursors are expressed in an unpredictable way on a limited set of other cell types. This often seems to involve expression on lymphoid and brain cells. The Thy-1 antigen is in this category, being a major glycoprotein of murine neuronal cells, fibroblasts and thymocytes. Structural studies show that this molecule is homologous with immunoglobulin domains which are the structural sub-units of all immunoglobulin polypeptides. Thy-1 is the size of one immunoglobulin domain and its sequence is most homologous with variable regions of immunoglobulins.It is suggested that Thy-1 is one of a set of surface molecules concerned with triggering interactions between cells and that this is the primitive function of the immunoglobulin domain. Cell interactions could be mediated by domain-like structures and receptors for them in a way which parallels the triggering of immunological effector reactions by the interaction of receptors with immunoglobulin constant regions. If this is so then the structure seen in the immunoglobulin domain would have evolved along with the evolution of cell organisation. The genes specifying the cell interaction molecules could then have provided the genetic material for the evolution of antibody and histocompatibility antigen at the time of vertebrate emergence.     

T-cell-activating monoclonal antibodies, reacting with both leukocytes and erythrocytes, recognize the guinea pig Thy-1 differentiation antigen: characterization and cloning of guinea pig CD90

A glycophosphatidylinositol (GPI)-linked differentiation antigen expressed on guinea pig T and B lymphocytes was identified by several monoclonal antibodies; it has been shown previously that this membrane protein induced strong polyclonal T cell proliferation upon antibody binding and costimulation by PMA. Purification by immunoadsorption and microsequencing revealed that this T-cell-activating protein is the homologue of Thy-1 or CD90. In contrast to the Thy-1 antigen of most other species, guinea pig Thy-1 has a much higher molecular weight, which is due to a more extensive N-linked glycosylation, bringing the molecular weight of the total antigen up to 36 kDa. Molecular cloning of guinea pig Thy-1 indicated that the deduced molecular weight of the protein backbone is 12,777 after removal of an N-terminal 19-amino-acid leader peptide and cleavage of the 31 amino acids for GPI anchoring the C-terminal end. Sequence comparison showed that guinea pig Thy-1 has an 82% homology to human and a 72% homology to mouse Thy-1 on the amino acid level. Immunohistological staining of cryostat sections revealed intensive staining with the monoclonal antibody H154 on fibroblasts, fibrocytes, Kupffer cells, alveolar macrophages, and mesangial cells. As observed in the human, mouse, and rat, Thy-1 is abundant in the guinea pig brain. Unlike Thy-1 expression in other species, guinea pig Thy-1 is strongly expressed on most resting, nonactivated B cells and, to a lesser extent, on erythrocytes. While treatment of erythrocytes and lymphocytes with GPI-specific phospholipase C largely decreased reactivity with mAb H154, T cells retained the proliferative response to antibody and phorbol esters.