Alloreactive bovine T lymphocyte clones: an analysis of function, phenotype, and specificity

A bovine alloreactive cell population was subjected to complement-dependent lysis with monoclonal antibody (mAb) IL-A11. The original population and the population depleted of cells bearing the determinant recognized by mAb IL-A11 were cloned. Parent cultures and 21 clones were examined for cytolytic function and for expression of determinants recognized by mAb IL-A11 and two additional mAb, IL-A12 and IL-A17. Clones could be classified according to maximal achievable levels of cytolysis by using Theileria parva-infected bovine lymphoblastoid target cells. In this way, three groups were identified--one capable of high level cytolysis, one of intermediate levels, and one group comprising apparently noncytolytic clones. The clones in the first group reacted with mAb IL-A17; those in the second and third groups, with mAb IL-A11 and IL-A12. It was shown that cytotoxicity effected by IL-A17+ clones could be inhibited by this mAb and also by a mAb directed to MHC class I determinants on target cells. Conversely, cytotoxicity effected by IL-A11+/IL-A12+ clones could be inhibited by mAb IL-A11 and by a mAb directed to MHC class II determinants on target cells. The levels of expression of class I and class II determinants on target cells correlated with the levels of killing by clones of the IL-A17+ phenotype and clones of the IL-A11+/IL-A12+ phenotype, respectively. The results indicate that cytotoxic bovine T lymphocyte clones specific for class I MHC antigens and both cytotoxic and noncytotoxic clones specific for class II MHC antigens can be obtained. Further, their specificity for class I or class II antigens can be determined by phenotyping with mAb.     

Characterization of a subset of bovine T lymphocytes that express BoT4 by monoclonal antibodies and function: similarity to lymphocytes defined by human T4 and murine L3T4

We report the identification of a subset of bovine T cells by two monoclonal antibodies (mAb), IL-A11 and IL-A12, and some functional analyses of these cells. Both mAb precipitate two polypeptides, called BoT4, with apparent molecular mass of 52 and 55 kilodaltons. The epitopes recognized by these two mAb are different, however. BoT4 is found on approximately 70% of thymocytes and 30% of peripheral blood mononuclear cells (PBM), is not expressed by monocytes or B cells, and is found on cells in the T-dependent areas of lymph nodes. BoT4+ lymphocytes purified by a fluorescence-activated cell sorter proliferate in response to mitogenic and alloantigenic stimulation without addition of exogenous growth factors, whereas BoT4- lymphocytes do not. Monoclonal antibodies IL-A11 and IL-A12 have no effect on mitogen- (PHA and Con A) or alloantigen-induced proliferation of PBM. Monoclonal antibody IL-A12 has no inhibitory effect on the cytolytic activity of bulk populations of alloreactive T lymphocytes, and most of the cytolytic activity generated in mixed leukocyte culture is ascribable to the BoT4- population. Using cloned alloantigen-specific lymphocytes, we found that the ability of BoT4+ clones to proliferate to alloantigenic stimuli without exogenous growth factors is a characteristic of some clones, as is susceptibility to inhibition of proliferation by mAb IL-A12. These results implicate the BoT4 molecule in antigen recognition but indicate that the requirement for BoT4 is variable among clones. Our findings, together with those in the companion paper, which provides evidence that BoT4+ lymphocytes are class II restricted, indicate that BoT4+ lymphocytes are the bovine equivalent of Leu3/T4+ lymphocytes of humans and analogous lymphocytes of other species.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). II. Genuine class II specificity of Lyt-2+ CTL clones

Class II-specific allogeneic cytolytic T lymphocytes (CTL) consist of two types of cells, i.e., Lyt-2+L3T4- and Lyt-2-L3T4 T cells. The Lyt-2+L3T4- class II-specific CTL population constitutes a conspicuous exception to the general correlation observed between the class of major histocompatibility complex antigen recognized and the type of accessory molecules expressed by T cells. In order to examine the specificity of such an exceptional T cell population, CTL clones were established by limiting dilution of a bulk CTL line developed in an I region incompatible combination of mouse strains, B10.QBR anti-B10.MBR. These CTL lines showed single genetic specificity indicating their clonal nature with respect to CTL activities. Lyt-2+L3T4- (2+4-), Lyt-2-L3T4+ (2-4+) and Lyt-2-L3T4- (2-4-) clones were obtained. Among many CTL clones showing a spectrum of genetic specificities, 2+4- and 2-4+ clones with apparent I-Ak-specificity, were studied further and four lines of evidence confirmed their class II specificity: 1) genes encoding the target antigen for these CTL clones were mapped within the I-A subregion by simple genetics; 2) an I-Ak-specific monoclonal antibody readily blocked specific cytolysis by these clones; 3) the clones failed to react with cells expressing mutated I-Ak antigens; and 4) a B cell tumor transfected with alpha- and beta-chain genes of I-Ak was specifically lysed by these CTL clones. These data therefore establish the existence of Lyt-2+ CTL with genuine class II specificity. All 2-4+ CTL were sensitive to the blocking effect of an antibody to L3T4, whereas none of the 2+4- class II-specific CTL were sensitive to blocking by an anti-Lyt-2 antibody, indicating that class II-specific CTL with "wrong phenotype" is not dependent on the function of the accessory molecule. Besides true class II-specific CTL clones, 2+4- clones with a spectrum of genetic specificities were obtained, including clones recognizing a combination of an I-Ak product and the Kb molecule. Two 2-4- clones were also specific for the combination of Kb + I-Ak. These clones most likely recognize an allogeneic class II antigen in the context of a class I antigen and therefore would more appropriately be included in the class I-restricted T cell population.     

Involvement of CD45 in MHC class II-allospecific cytotoxicity

Twenty-seven different CD45 monoclonal antibodies (mAb) were assessed for their ability to block cytotoxicity of alloreactive CD4+ MHC class II-specific or CD8+ class I-specific human T cell clones (n = 3 and 5, respectively). Twelve of 27 blocked the former but only 1/27 the latter, although all 27 significantly inhibited MHC-unrestricted lysis of K562 cells by either CD4+ or CD8+ clones. MAb pretreatment of effector cells but not target cells resulted in retention of blocking. Crosslinking the CD45 with goat anti-mouse Ig serum did not result in blockade of lysis by class I-specific clones or reveal blocking of class II-specific clones not inhibited by mAb alone. These results suggest that CD45 molecules may be predominantly involved in MHC class II-specific but not class I-specific allocytotoxicity as well as MHC-unrestricted natural killer-like cytotoxicity.     

Differential recognition of tumor-derived and in vitro Epstein-Barr virus-transformed B-cell lines by fetal calf serum-specific T4-positive cytotoxic T-lymphocyte clones

Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3+, T4+, Dr+ phenotype and lysed autologous B blasts, the autologous LCL, and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 degrees C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells.     

T3 monoclonal antibody activation of nonspecific cytolysis: a mechanism of CTL inhibition

The T3 antigen is expressed on all cytotoxic T lymphocytes (CTL). Monoclonal antibodies (MAb) to the T3 antigen previously have been shown to inhibit CTL-mediated killing of cells expressing the relevant target antigens. The mechanism of T3 MAb inhibition, however, remains undefined. In this report, we describe a novel effect of the T3 MAb: the stimulation of allospecific CTL clones to kill target cells that do not express the relevant HLA antigens. The stimulation of nonspecific killing was seen only with MAb to the T3 antigen; MAb to other function-associated antigens (e.g., LFA-1, LFA-2, LFA-3, T4, T8, HLA-A,B,C, and DR) had no effect. T3 MAb stimulated nonspecific killing by CTL clones expressing both the T4+ and T8+ phenotype and by CTL clones specific for both class I and class II HLA alloantigens. Target cell susceptibility to T3 MAb stimulated killing was variable. CTL clones lysed some target cell lines very efficiently (e.g., K562, Daudi, and M124.1) but lysed other cell lines much less efficiently (e.g., 23.1, Mann, and L cells). In CTL-mediated cytotoxicity assays with target cells expressing the relevant HLA antigens, T3 MAb demonstrated the expected inhibition of cytolysis. Thus, the ability of T3 MAb to stimulate and inhibit CTL-mediated cytolysis suggests that both effects may be the result of a common mechanism of activation.     

Major histocompatibility complex-unrestricted cytolytic activity of human T cells. Analysis of precursor frequency and effector phenotype

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. These conditions were shown to expand a mean of 96% of cells cultured. All of the 198 clones generated by this method were T cells (CD2+, CD3+, CD4+ or CD2+, CD3+, CD8+) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. In addition, the activity was not inhibited by monoclonal antibodies directed against class I or class II nonpolymorphic MHC determinants. Killing, however, was inhibited by soluble monoclonal antibodies against the CD3 complex. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur or K562 was not mediated by a soluble factor secreted by the clones. Some of the clones retained their cytotoxic activity when grown in rIL-2 alone for 4 to 6 wk, whereas others exhibited markedly diminished cytotoxicity after maintenance in this manner. Clones that exhibited diminished or no cytotoxic activity after prolonged maintenance in rIL-2 could be induced to kill by stimulation with immobilized but not soluble monoclonal antibodies to CD3 in the absence of lectin. All of the clones examined expressed NKH1 and CD11b but none were CD16 positive. The degree of cytotoxicity of resting or activated clones could not be correlated with expression of these markers. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.     

OKT4+ cytotoxic T cells can lyse targets via class I molecules and can be blocked by monoclonal antibody against T4 molecules

Human cytotoxic T lymphocytes (CTL) have been shown to recognize either class I or class II major histocompatibility (MHC) products. This recognition has been correlated with the expression of OKT antigens on the surface of the CTL. Thus, OKT4+ CTL have been shown to be reactive with class II products, whereas OKT8+ effectors recognize class I molecules. In this study, responder cells were separated according to their OKT4 or OKT8 cell surface phenotype on a fluorescence-activated cell sorter (FACS). The OKT4+ subsets were stimulated with an LCL mutant that did not express DR and MB/MT but did express SB and class I antigens. After 7 days in culture, the activated subsets were tested on a panel of class I matched or mismatched targets. The cytotoxicity observed could be correlated with the presence of matched class I antigens. In addition, monoclonal antibody (MCA) W6/32, directed at a monomorphic determinant on HLA-A and -B molecules, blocked lysis. Furthermore, six OKT4+ CTL clones were derived from the OKT4+ bulk cultures; three clones were found to be directed at class I molecules whereas the other three recognized class II determinants. The ability of these clones to lyse their relevant targets was blocked by OKT4 MCA, raising questions as to the role of the T4 molecule in antigen class-specific CTL recognition.     

Studies on the bovine major histocompatibility class I and class II antigens using homozygous typing cells and antigen-specific BoT4+ blast cells

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyte reactions (MLR) with all animals in the herd carrying the w23 antigen or as antigen presenting cells to bovine T4+ cell blasts. It was shown that, within each sire line, the genes encoding the MHC class I and class II antigens were closely linked. There were no detected recombinations between the MHC class I and class II regions nor within the BoLA-D region responsible for mixed lymphocyte reactivity. MLR typing of MHC class II antigens correlated with the results from T-lymphocyte proliferation studies. Cells from these cattle, which are homozygous at the class I and II MHC loci but differ in the class II antigen expressed, could be used to type the BoLA-D of other cattle.     

T lymphocyte cloning from rejected human kidney allograft. Recognition repertoire of alloreactive T cell clones

We analyzed the recognition repertoire of 16 human alloreactive T cell clones (ATLC) derived from cells invading an irreversibly rejected kidney allograft. These clones, which specifically proliferated against the kidney donor B lymphoblastoid cell line, fell into two classes: CD4+ killers and CD8+ killers. Cytotoxic and proliferative activities of the ATLC were studied by using a panel of allogeneic cells sharing HLA specificities with kidney donor cells. Moreover, mAb recognizing monomorphic parts of HLA class I and class II molecules were used in blocking experiments of ATLC cytotoxicity. The results obtained from proliferative and cytotoxic assays were concordant. All ATLC investigated were directed against HLA molecules, and some clones were found to recognize HLA-B, -C, -DP, -DQ, or -DR products. All anti-HLA class I ATLC were CD8+, whereas both CD4+ and CD8+ ATLC were committed against HLA class II specificities. Nine of 16 ATLC were shown to react against serologically defined donor HLA determinants. These data indicate the recognition of HLA determinants in the course of an in vivo alloimmune response and particularly emphasize the role of HLA-C and DP loci products so far ignored in clinical transplantation.     

In vivo treatment of neonatal mice with anti-I-A antibodies interferes with the development of the class I, class II, and Mls-reactive proliferating T cell subset

In this study we investigated the effect of monoclonal anti-I-A Ab treatment of neonatal mice on the development of alloreactive class I-specific, class II-specific, and Mls-specific T cell proliferative responses. Responses to both class I and class II alloantigens, as well as to Mls antigens, were nearly abrogated at the end of the 2- to 3-wk in vivo treatment period in both the thymus and the spleen. Development of suppressor cells could be excluded as the cause of the observed defect. Diminished responsiveness could not be restored by the addition of IL 2-containing supernatant, suggesting that the reduced T cell proliferative response in anti-I-A-treated mice is due to defective or absent MHC-specific T cell precursors. Furthermore, generation of alloreactive class I-specific proliferative responses was dependent on self-class II recognition, thus providing an explanation for the absence of class I-specific proliferating T cells. Finally, a non-Ia-restricted T cell response, i.e., Con A-induced proliferation, was not affected by anti-I-A Ab treatment. It was previously reported that neonatal anti-Ia Ab treatment results in reduced Ia-antigen expression in the thymus, and that the development of the class I-specific CTL precursors proceeds undisturbed in these mice. The present results extend these findings and suggest that in vivo development of class II-restricted T cells is dependent on interaction with Ia-encoded products on cells either in the thymus or at other sites where T cells undergo development. Moreover, these results demonstrate that in vivo development of the alloreactive class II-specific T cell repertoire is dependent on development of self-class II recognition.     

A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones

Hybridoma H129 .19 was derived by fusion between spleen cells of a Lou / Ws1 rat immunized with an Lyt-1+,2- anti-I-Ak cytolytic T lymphocyte (CTL) clone and the nonsecreting myeloma X63-Ag8.653. The monoclonal antibody (mAb) H129 .19 (IgG2a, kappa) was selected for its capacity to inhibit the lytic potential of the immunizing clone. H129 .19 identified a monomorphic determinant on a 55 m.w. murine T cell differentiation antigen, which appeared to be homologous to the human T4 molecule in that: 1) H129 .19 reacted with 80% adult thymocytes, with a subset of splenic T cells, and with the interleukin 2 (IL 2)-producing EL4 thymoma; 2) The mAb bound to and inhibited the IL 2 production and the proliferation of various allo- or soluble antigen-reactive T cell clones that recognized restriction or activating determinants on the I-A or I-E molecules, respectively; 3) H129 .19 did not inhibit the proliferation and/or cytolysis of Lyt-2,3+ T cells specific for class I MHC antigen; and 4) Among six anti-Iak CTL clones examined in this study, the mAb H129 .19 reacted with two I-Ak-specific, Lyt-2,3- clones on which it exerted strong cytolysis inhibiting effect at the effector cell level. By contrast, two other anti-I-Ak and two anti-I-Ek CTL clones were found to express the Lyt-2,3+,T4- cell surface phenotype. The cytolytic potential of the latter clones was not inhibited by anti-Lyt-2,3 mAb. These studies strongly suggest that the mouse T4 molecule facilitates the recognition of class II MHC antigen by most but not all T cells.     

The soluble viral glycoprotein of vesicular stomatitis virus efficiently sensitizes target cells for lysis by CD4+ T lymphocytes

The soluble glycoprotein Gs of vesicular stomatitis virus (VSV), at approximately 10(4) molecules per cell, sensitized target cells for lysis by clones of CD4+ cytolytic T lymphocytes (CTL). In addition to lysis, the clones responded by proliferation and interleukin-2 release. Targets sensitized by Gs competed effectively with VSV-infected cells for recognition. Immune cytolysis by these CD4+ CTLs was restricted by class II major histocompatibility complex (MHC) antigens and was specific to VSV. The specific class II MHC antigen which was restricting for each clone remained the same whether the targets were sensitized by infection with VSV or by exogenously added soluble antigen. Sensitization by Gs appeared to require prior processing because the antigen-presenting cells that were fixed prior to exposure to Gs failed to be recognized by the CTL clones. The high efficiency of this uptake and processing was suggested by the inability of Gs at concentrations up to 10(7) per cell to block superinfection by VSV or to effect the RNA-synthetic machinery of uninfected cells. Also, Gs failed to hemolyze sheep erythrocytes when there was hemolysis by virions or an amino-terminal peptide of the VSV glycoprotein. Extrapolation of these results to viral diseases was possible because soluble viral glycoproteins were naturally synthesized during many viral infections and class II MHC antigens were inducible in cells of nonlymphoid origin. Therefore, CD4+ CTLs may be important participants in increasing virus-induced pathology, especially among adjacent uninfected cells.     

Characterization of murine T cell activation signals produced by B lymphoma cells

The activation requirements of alloreactive and antigen reactive murine T cells were examined by stimulating class II restricted T cell clones with monoclonal B lymphoma cells. One B lymphoma cell line (T27A) was found to stimulate IL 2 release from some alloreactive T cell clones without stimulating any significant T cell proliferation response. The same B lymphoma cells are capable of stimulating IL 2 release and proliferative responses from other T cell clones. Evidence is presented suggesting that B lymphoma cell stimulation of these T cell clones is largely IL 1 independent and that at least some T cell clones may require activation signals other than Ia, antigen, and IL 1. The addition of exogenous, purified IL 1 to the T cell activation assays was found to have a wide range of stimulatory effects on the proliferative responses of different T cell clones. The absence of comparable IL 1-induced stimulation of IL 2 secretion suggests that IL 1 primarily enhances antigen specific T cell proliferation through mechanisms other than acting as a co-stimulant for IL 2 release.     

Expression of H-2I region-restricted cytolytic activity by an Lyt-2+ influenza virus-specific T lymphocyte clone

The expression of Lyt-2 on T lymphocytes has been postulated to correlate closely with restriction by, or alloreactivity to, class I MHC gene products, whereas I region-restricted or alloreactive populations appear to be associated with Lyt-1 and L3T4 expression. However, exceptions to this axiom among alloreactive T cells have been shown to exist. In this report we describe a clonal population of influenza virus-specific T lymphocytes that bears the Lyt-2+, L3T4- phenotype. Notably, this clone is restricted in influenza virus recognition by class II MHC molecules and is cytolytic for virus-infected target cells expressing the appropriate class II molecules. Antibody directed to the Lyt-2 molecule does not inhibit cytolysis.     

Characterization of streptococcal antigen-specific CD8+, MHC class I-restricted, T cell clones that down-regulate in vitro antibody synthesis.

T cell clones were generated from the peripheral blood of rhesus monkeys that had been immunized with a soluble Mr 185,000 Ag (SAI/II) derived from Streptococcus mutans. The clones were CD3+ CD8+ CD4- alpha beta TCR+ and were specifically stimulated to proliferate by SAI/II. The proliferative responses of the cloned cells were class I restricted, as demonstrated by reconstitution of the cloned T cells with APC matched at various MHC class I and II loci, as well as by inhibition with anti-class I and not anti-class II mAb. The function of the CD8+ cloned cells was examined in vitro for their effect on antibody synthesis by Ag-stimulated CD4+ cells and B cells from immunized animals. Indeed, four of the five clones suppressed SAI/II-specific IgG antibody synthesis when activated with SAI/II and the appropriate MHC-matched APC. Although activation of the suppressor clones was Ag specific, the effector function of the suppression of antibody synthesis was Ag nonspecific. The latter was probably mediated by lymphokines and, indeed, the culture supernatant generated by stimulating the cloned CD8+ cells with anti-CD3 mAb suppressed both the specific and nonspecific antibody synthesis. Cytotoxicity studies showed that all five CD8+ clones showed a low level of lectin-dependent cytotoxicity. However, because four of the five clones expressed significant suppression of antibody synthesis, the suppressor activity was unlikely to be a function of the weak cytotoxicity. The results suggest that immunization of rhesus monkeys with a soluble streptococcal Ag induced CD8+ alpha beta TCR+ T cell clones that show SAI/II-specific, MHC class I-restricted proliferative responses and nonspecific down-regulatory function of in vitro antibody synthesis.     

Induction and blocking of cytolysis in CD2+, CD3- NK and CD2+, CD3+ cytotoxic T lymphocytes via CD2 50 KD sheep erythrocyte receptor

The 50 KD sheep red blood cell antigen receptor CD2 is the earliest T cell differentiation marker and is present on all blood-derived T cells, including natural killer (NK) cells. The CD2 antigen is also known to serve as an important activation site regulating various T cell functions. We report that anti-CD2 monoclonal antibodies (MAb) block MHC-restricted class I- and class II-specific cytolysis by CD2+, CD3+ clones of the relevant target cells, irrespective of whether lysis by these clones is blocked by anti-CD3 or anti-CD8 MAb. Moreover, anti-CD2 MAb (but not anti-CD3 MAb) are able to reduce MHC-nonrestricted, nonspecific cytolysis: a) by CD2+, CD3+ clones of K562 target cells; and b) by CD2+, CD3 NK clones of K562 as well as Daudi cells. Different preparations of anti-CD2 MAb vary in their capacity to inhibit cytolysis. For cloned effector cells, the percent inhibition of lysis by CLB-T11 greater than Lyt-3 MAb, whereas with "fresh" NK cells, the lysis inhibitory ability of Lyt-3 greater than CLB-T11. The antibody-dependent cellular cytotoxicity by "fresh" and cloned NK cells is not inhibited by anti-CD2 MAb. Anti-CD2 MAb also prevent the induction of lysis by cross-linked anti-CD3 MAb, e.g., by CD2+, CD3+ cloned cloned cells against (IgG-FcR+) Daudi cells. Anti-CD2 MAb can also induce cytolysis in some, but not all, CD2+, CD3- NK clones against xenogeneic P815 mouse mastocytoma cells. Anti-CD2 MAb, in combination with lectins (PHA or Con A: pretreatment of effector cells), can also induce cytolytic activity by CD2+, CD3+ clones against Daudi cells. Our data therefore support the concept that the CD2 antigen is an important activation site regulating a wide variety of T cell functions including cytolysis. Whether ligand interaction with the CD2 antigens results in augmentation or inhibition of T cell functions may very well depend on the type of CD2 antigen-ligand interaction, e.g., cross-linked ligand-receptor interaction may, in general, enhance the various T cell functions, whereas noncross-linked ligand-receptor interactions may inhibit such functions, as we and other investigators demonstrated earlier for the CD3/Ti antigen-receptor complex activation site.     

A human autoreactive T cell line specific for minor histocompatibility antigen(s) isolated from a bone marrow-grafted patient

A human autoreactive T cell line named Bur-1 has been obtained from a woman 4 mo after an allogeneic bone marrow transplantation (BMT) from one of her HLA-identical brothers. The phenotype of the cell line is 100% T11+ and over 90% T4+, and the karyotype confirms its donor (male) origin. These donor T cells proliferate specifically in the presence of donor's peripheral blood monocytes (PBM) but not recipient's cells, and they kill specifically donor's but not recipient's Epstein-Barr virus (EBV)-induced lymphoblastoid cell lines (LCL). PBM from another HLA-identical brother and from several unrelated donors also stimulate Bur-1 cells, and EBV-induced LCL from the same donors are killed in cytotoxicity assays. All of these donors share HLA-DR5 or HLA-DRw11 (the major split of HLA-DR5) with Bur-1 cells. However, some but not all of the PBM sharing HLA-DR5 with Bur-1 cells are recognized. Therefore, in contrast with the previously described autoreactive T cells, Bur-1 cells are not directed against self-MHC antigens but rather recognize autologous minor histocompatibility (mH) antigens in the context of autologous HLA class II molecules. Because both male and female cells can be recognized, the reacting minor antigen could not be the male-specific HY antigen. It is suggested that autoreactivity against mH antigens can be observed in bone marrow-grafted patients due to the education of bone marrow donor precursors in the recipient thymus not allowing tolerance to autologous (donor) mH antigens not shared by the recipient.     

Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.     

Deficient expression of class-I HLA in some cases of acute leukemia

Summary Leukemic cells from the blood and marrow of 25 cases of newly diagnosed acute leukemia were presented as target cells to alloreactive effector cells from unrelated normal donors in cell-mediated cytotoxicity assays. In three cases the leukemic targets were poorly killed relative to nonleukemic, HLA-identical target cells. The poor killing of the leukemic cells from one of these cases was shown by competitive inhibition to be due to deficient expression of normal class-I HLA antigens rather than resistance to lysis. Furthermore, the leukemic cells from these three patients were also deficient in binding monoclonal antibodies to nonpolymorphic determinants of class-I HLA and B2 microglobulin. Two additional cases were identified as having a less extensive deficit of HLA, and may be representative of a group with relatively subtle changes in these cell surface antigens. The possible significance of reduced expression of HLA in leukemic progression and in susceptibility to graft-vs-leukemia reactions after bone marrow transplantation is discussed.