Expression of murine soluble CD4 protein in baculovirus infected insect cells.

The expression of murine soluble CD4 (L3T4) protein (sCD4) by baculovirus-infected insect cells was characterized. The yield of sCD4 reached 2 mg/l culture supernatant late in infection. Nevertheless, a large amount of sCD4 remained cell-associated, presumably in the endoplasmic reticulum or an early golgi compartment, as indicated by the endo-beta-N-acetyl-D-glucosaminidase H (endo-H) sensitivity of its carbohydrate chains. The secreted form of sCD4 is modified with both endo-beta-N-acetyl-D-glucosaminidase D (endo-D) and endo-H-sensitive oligosaccharides. It was possible that the incomplete secretion indicated faulty glycosylation or improper folding of the sCD4 protein. However, inhibitor studies showed that complete carbohydrate processing is not required for secretion of sCD4 by insect cells. Moreover, maintained reactivity with a panel of monoclonal Ab as well as phase partitioning experiments suggested that secretion is apparently not caused by misfolding of the sCD4 protein. Similar results were obtained with biologically active murine interleukin-4 produced by insect cells. This indicates that an inefficient secretory pathway may be a general problem of baculovirus-infected insect cells and is not a consequence of incorrect molecular conformation.

A comparative assessment of the roles of CD8 and CD2 in the functions of activated murine CD8+ T lymphocytes.

Both CD8 and CD2 are T cell surface receptors involved in physical cell interaction and in transmembrane signalling. The present paper addresses their role in the induction of two different functions of the cloned murine cytotoxic T cell C196: target cell lysis and IFN-gamma production. These functions were induced in C196 either by stimulation with the specific stimulator/target cell P815 or, bypassing specific recognition, by the aCD3 hybridoma 145-2C11 or by solid phase aTCR antibodies. These responses were tested for their susceptibility to inhibition/enhancement by a panel of aCD8 and aCD2 mAb. In addition, CD8 deficient and CD8/CD2 double-deficient variants of C196 were transfected with the CD8 and CD2 genes and the resulting cell lines were analysed for their functional capacities. The following results were obtained: (i) CD8 is primarily important in the specific recognition process of activated CTL; (ii) transmembrane signalling of activated CTL through the TCR does not require CD8, nor is it sensitive to modification through CD8; (iii) CTL can nevertheless be directly activated through CD8; however, this is restricted to induction of cytotoxicity but does not result in IFN-gamma production; (iv) CD2 does not seem to be important in any of these responses.

Affinity enhancement and transmembrane signaling are associated with distinct epitopes on the CD8 alpha beta heterodimer.

CD8 is a heterodimeric membrane glycoprotein on MHC class I-restricted T lymphocytes that cooperates with the alpha beta CD3 TCR in the recognition of MHC class I molecules presenting antigenic peptides. Co-operation has two components: enhancement of the affinity of MHC/peptide-TCR interaction, and signal transduction through the T cell membrane. The cytolytic function of CTL is primarily dependent on the affinity-enhancement component of CD8-TCR cooperation whereas activation of resting CD8+ T cells is primarily dependent on transmembrane signaling. Using a panel of mAb, two to the alpha-chain and three to the beta-chain of CD8, we investigated the relationships between epitopes and functional regions of the CD8 molecule. Two of the antibodies, one to the alpha-chain and one to the beta-chain of CD8, inhibit the cytolytic function of CTL but not the generation of CTL from resting T cells. Another two antibodies, also one to the alpha- and one to the beta-chain, inhibited the generation of CTL while enhancing the cytolytic function of CTL. These results suggest that both the alpha- and beta-chain of CD8 possess two distinct regions, one involved in affinity enhancement and the other in transmembrane signaling. The former may be the MHC class I-binding region whereas the latter may associate with the alpha beta CD3 TCR. The data can explain the apparent functional equivalence of CD8 alpha alpha homodimers and alpha beta heterodimers.

Evidence for involvement of lymphocyte function-associated antigen 1 in T cell migration to epidermis.

Although it is well known that in various T cell-mediated skin diseases T cells migrate preferentially to epidermis, no direct evidence has been presented in which molecules on T cells are important in directing T cell traffic to epidermis. We have previously established CD4+ autoreactive cloned T cells with a special tropism for epidermis in vitro as well as in vivo. Antibody inhibition studies demonstrated that only anti-lymphocyte function associated Ag 1 (anti-LFA-1) mAb completely inhibited the in vitro migration of the T cells toward the epidermis, whereas mAb against other T cell surface molecules had little or no effect. Monovalent F(ab) fragment of the anti-LFA-1 mAb, although less efficient, also inhibited the T cell migration. The apparent dependency of the inhibition on the anti-alpha-chain mAb suggested a major role for the alpha-chain of LFA-1 in T cell migration to epidermis. The relevance of an LFA-1-dependent mechanism to the epidermotropic migration of T cells was further strengthened by the findings that the T cell migration to epidermis was inhibited by divalent cation depletion, cytochalasin B, and low temperature. These findings indicate that the LFA-1 molecule, which is thought to be primarily involved in cell-to-cell adhesions, also plays an important role in directing T cell migration to epidermis.

Loss of epidermal integrity by T cell-mediated attack induces long term local resistance to subsequent attack. II. Thymus dependency in the induction of the resistance.

Our previous study showed that in cutaneous graft-vs-host disease (GVHD) induced by intradermal injection of autoreactive T cells the epidermal structures spontaneously recovered from the destruction became resistant to the subsequent attempts to induce the cutaneous GVHD and that the resistance correlated well with a 30-fold increase in the number of Thy-1+ epidermal cells (Thy-1+EC). We show that the resistance to the cutaneous GVHD was never induced in athymic nude mice and adult thymectomy lethal radiation and bone marrow reconstitution (ATXBM) mice under the same conditions, indicating that the induction of the resistance is dependent on the presence of thymus. A great increase in the number of Thy-1+EC was similarly observed in the epidermis of the athymic nude and ATXBM mice that spontaneously recovered from the cutaneous GVHD and that remained susceptible to the induction of the cutaneous GVHD. However, the results with B10Thy-1.1----B6 radiation chimeras clearly demonstrate that the vast majority of the increased Thy-1+EC found in the "susceptible" epidermis of the ATXBM mice were of donor bone marrow origin and there was no increase in the number of host-derived Thy-1+EC, whereas in the "resistant" epidermis of the XBM mice both Thy-1+EC populations were equally increased. The overall results indicate that the expansion of Thy-1+EC that mature in the thymus is crucial to the induction of the resistance, although the migration of bone marrow-derived Thy-1+EC precursors to the epidermis occurs quite independently of the presence of thymus.

Loss of epidermal integrity by T cell-mediated attack induces long-term local resistance to subsequent attack. I. Induction of resistance correlates with increases in Thy-1+ epidermal cell numbers.

The cutaneous graft-versus-host disease (GVHD) lesions induced by intradermal injection of cloned autoreactive T cells have been shown to subside rapidly and the epidermis returns to normal 2 wk after injection. Those mice that had spontaneously recovered from the cutaneous GVHD became resistant to subsequent attempts to induce the cutaneous GVHD by the T cells while maintaining their activity to mount delayed-type hypersensitivity (DTH) responses and to induce the enlargement of the popliteal lymph nodes (PLN). The resistance appeared to be restricted to the epidermal structures of the injection sites, suggesting the involvement of locally acting suppression mechanisms. This local resistance was not specific for the clonotype used for the induction of the resistance. A loss of the epidermal integrity by an attack of T cells capable of producing cutaneous GVHD was a prerequisite for the induction of the resistance. By up to at least 8 mo after injection of the T cells, no mice became susceptible to the cutaneous GVHD again, provided that the T cells were injected into the same footpad sites that had initially received the T cells. This resistance correlated well with the great increase (20-30-fold) in Thy-1+ EC number. The great increase in the number of Thy-1+ EC following destruction of epidermal structures may be important in protecting the epidermal integrity from an additional attack by T cells.

Differential expression of lymphocyte function-associated antigen 1 (LFA-1) on epidermotropic and non-epidermotropic T-cell clones.

Various T-cell surface molecules are involved in T-cell adhesion, which is an essential requirement for epidermotropic migration of T cells. Our previous studies demonstrated that certain T-cell clones can migrate into the epidermis upon their intradermal inoculation into the footpads of recipient mice with relevant antigens, while other T-cell clones, despite their identical antigen specificities and functions, are non-epidermotropic. We therefore tested whether the differences in epidermotropism of these T cells could reside in the different levels of expression of T-cell surface molecules such as CD3, CD4, CD2, and lymphocyte function associated antigen 1 (LFA 1). The results of flow cytometric analysis showed that LFA-1 was preferentially expressed on the surface of epidermotropic T-cell clones, while non-epidermotropic T-cell clones were negative or very weakly positive for LFA-1 with one exception. After exposure to phorbol ester, epidermotropic clones with high levels of LFA-1 did not show any further up-regulation of LFA-1. In contrast, under identical conditions, significant up-regulation of LFA-1 was observed on non-epidermotropic T cells with low levels of LFA-1. However, even after exposure to phorbol ester, these T cells remained non-epidermotropic. These results suggest that the presence of high levels of LFA-1 on T cells is absolutely necessary for their epidermotropic migration, but its up-regulation is neither necessary nor sufficient to trigger the epidermotropic migration. High levels of LFA-1, regardless of cell activation, may be required to mediate stable cell adhesions leading to epidermotropic migration.

Physical association of CD4 and the T-cell receptor can be induced by anti-T-cell receptor antibodies.

CD4 can physically associate with the CD3-T-cell receptor complex as visualized in cocapping experiments. This association occurs when the T-cell receptor is cross-linked by certain anti-variable region antibodies that appear to induce a conformational change in the receptor such that it associate with CD4. Similar association has been observed in earlier studies with the same cloned helper T cell when the physiological ligand, antigen-class II major histocompatibility complex molecule, is bound by the T-cell receptor. The ability of anti-T-cell receptor antibodies to induce the T-cell receptor-CD4 association correlates with a 100-fold increase in the ability of the antibody to activate the T cell. This suggests that the complex of CD4 and the T-cell receptor act synergistically in T-cell activation, thus readily explaining the commonly observed association of CD4 expression with class II major histocompatibility complex-restricted antigen recognition. This association could also play a role in infection by human immunodeficiency virus.

Locally administered monoclonal antibodies to lymphocyte function-associated antigen 1 and to L3T4 prevent cutaneous graft-vs-host disease.

In vivo treatment with mAb directed against T cell surface molecules has been shown to prevent or reverse graft-vs-host disease (GVHD) and experimental autoimmune diseases, where T cells play a critical role. In this study we investigated the effects of in vivo administration of anti-I-A, anti-L3T4, and anti-lymphocyte function-associated Ag 1 (LFA-1) mAb on the development of murine cutaneous GVHD and delayed-type hypersensitivity responses evoked by local transfer of class II-MHC Ag-restricted, cloned autoreactive T cells. Prevention of cutaneous GVHD was achieved with local administration of either anti-L3T4 or anti-LFA-1 mAb, but not with anti-I-A mAb, while delayed-type hypersensitivity responses were inhibited by all the mAb. These results indicate that an I-A Ag-independent mechanism may be also operative in the development of the cutaneous GVHD, unlike the delayed-type hypersensitivity responses. Anti-LFA-1 mAb appeared to be more potent at inhibiting cutaneous GVHD rather than delayed-type hypersensitivity responses, whereas anti-L3T4 mAb inhibited equally both the responses. These results suggest that LFA-1 molecule may be involved in the epidermal invasion of T cells. Treatment with anti-LFA-1 mAb was found to be still, although less, effective at preventing cutaneous GVHD, even if the mAb was administered after the development of cutaneous GVHD.

Lichenoid tissue reaction induced by local transfer of Ia-reactive T-cell clones. III. Role of Ia+ keratinocytes in the epidermotropic migration of the T cells.

The role of Ia+ keratinocytes in epidermotropism of T cells was analyzed by using self-IA specific autoreactive cloned T cells with epidermotropic nature (termed BB5) and those without it (termed C10). These T-cell clones were injected into the footpads of syngeneic nude mice whose keratinocytes had been induced to express Ia by the iv injection of normal mouse serum. Ia expression by keratinocytes was associated with the increased epidermal invasion of BB5 cells, but did not render C10 cells capable of migrating into the epidermis. The migration of the T cells to epidermis was also studied in vitro using a migration assay under agar. Ia expression by keratinocytes significantly enhanced the in vitro migration of BB5 cells to the epidermis, but had no effect on the migration of C10 cells and freshly isolated unstimulated lymphocytes. We surmise from these results that Ia+ keratinocytes may facilitate the epidermal invasion of the T cells with epidermotropic nature, but not those without it. However, the possibility certainly exists that the observed preferential migration of BB5 cells to Ia+ keratinocytes may be secondary to the alteration of other factors associated with the Ia expression. The injection of normal mouse serum was accompanied by an increase in the production of epidermal cell-derived thymocyte activating factor (ETAF) indicating that the increased ETAF production may have contributed to some of the observed preferential migration of BB5 cells.

Role of Langerhans cells in epidermotropism of T cells.

Certain T lymphocytes display a specific affinity for the epidermis (epidermotropism). Recent studies have suggested that Ia+ Langerhans cells (LCs) are possible targets for the epidermotropism. A variety of self-Ia-reactive cloned T cells were tested for their ability to migrate into the epidermis following intradermal inoculation into the footpads of syngeneic mice. Clone BB5 was chosen as representative of the epidermotropic T cells. We investigated whether the depletion of Ia+ LCs from the epidermis by tape-stripping could alter the migration of BB5 cells into the epidermis. The epidermal invasion of BB5 cells was markedly impaired in those mice whose LCs were depleted by 95% after repetitive tape-stripping. Because production of epidermal-derived thymocyte activating factor (ETAF) by the epidermal cells was augmented after repetitive tape-stripping, the diminished migration of BB5 cells into tape-stripped epidermis did not result from a decrease in ETAF production which is thought to attract T cells chemotactically. These results suggest that Ia+ LCs may play an inductive role in the preferential migration of T cells into the epidermis.

The influence of valence on the functional activities of monoclonal anti-L3T4 antibodies. Discrimination of signaling from other effects.

Anti-L3T4 antibodies have different functional effects on different ligands that activate a cloned, L3T4+ helper T cell line. It is reported here that some of these effects involve positive or negative signaling induced by cross-linking L3T4 molecules, because such effects are not observed using the Fab fragment of anti-L3T4. However, the Fab fragment does inhibit responses to antigen:self class II major histocompatibility complex (MHC) and to anti-T cell receptor monoclonal antibodies directed at a particular V region epitope. The finding that the Fab fragment of anti-L3T4, which does not block binding of anti-T cell receptor V region antibodies and does not negative signal can still block activation by such antibodies suggests an intimate association of L3T4 with the T3: alpha: beta T cell receptor complex. This association may normally be induced by interaction of both structures with antigen:self class II MHC complexes. The data also support the hypothesis that cross-linking the L3T4 molecule in the absence of engagement of the T3: alpha: beta complex generates negative or inhibitory signals. Thus, L3T4 plays a central role in the process of class II MHC-restricted T cell antigen recognition and activation.

Evidence for a physical association of CD4 and the CD3:alpha:beta T-cell receptor.

CD4 is a molecule expressed on the surface of T lymphocytes which recognize foreign protein antigens in the context of class II major histocompatibility complex (MHC) molecules. Recognition of antigen:class II MHC complexes by CD4+ T cells can be inhibited by anti-CD4 (ref. 3). Nevertheless, specific recognition of the antigen:Ia complex is clearly a function of the T-cell receptor, which is composed of CD3 and the variable polypeptides alpha and beta. Thus, it has been proposed that CD4 serves an accessory function in the interaction of CD4+ T cells and Ia-bearing antigen-presenting cells by binding to non-polymorphic portions of class II MHC molecules and stabilizing the cell interaction. Based on our observation that anti-CD4 could inhibit activation of a cloned line of CD4+ T cells by antibodies directed at a particular epitope on the variable region of the T-cell receptor, we have recently proposed that CD4 is actually part of the T-cell antigen recognition complex, physically associated with CD3:alpha:beta. But numerous studies showing that CD3 and CD4 are not stably associated on the T-cell surface would appear to contradict this model. Here we show that anti-T-cell-receptor antibodies can co-modulate expression of the T-cell receptor and CD4, and that the monovalent Fab fragment of such an anti-T-cell-receptor antibody can, in conjunction with bivalent anti-CD4 antibody, generate an activating signal for the T cell. These findings provide further evidence for a physical association of the T-cell receptor complex and CD4.

The role of the murine L3T4 molecule in T cell activation: differential effects of anti-L3T4 on activation by monoclonal anti-receptor antibodies.

MHC restricted T cells can be divided into two subsets based on the mutually exclusive expression of the cell surface differentiation antigens L3T4 and Lyt-2 in the mouse. Expression of the L3T4 marker is correlated most strictly with recognition of foreign antigen in association with self class II MHC molecules, or Ia molecules. Less stringently correlated with L3T4 expression is the recognition of unmodified self or non-self Ia molecules. Finally, expression of L3T4 is also correlated with certain functional properties, although this correlation is even less stringent. The major correlation for function is between L3T4 and the ability to activate B cells. These correlations have led to the hypothesis that L3T4 recognizes Ia molecules, and plays a role in increasing the affinity of T cell:Ia bearing cell interactions. This hypothesis is bolstered by the finding that anti-L3T4 antibody blocks such interactions. Recently, we and others proposed a second effect of cross-linking L3T4 molecules, namely negative signalling. We further proposed that the natural ligand for L3T4 is Ia molecules, and that Ia-driven cross-linking of L3T4 molecules on the T cell in the absence of receptor aggregation would lead to off signalling to the T cell and separation of cell conjugates. To better understand the role of the L3T4 molecule in T cell activation, we have examined the effect of several anti-L3T4 antibodies on stimulation of a cloned line of helper T cells by a panel of monoclonal antibodies directed at what appear to be different epitopes on the T cell receptor. Unlike previous analyses of stimulation of helper T cells with anti-T cell receptor antibodies, we observe differential effects of anti-L3T4 on T cell activation by anti-receptor antibodies, the effect of anti-L3T4 depending on the characteristics of the anti-receptor antibody. This result suggests that L3T4 is intimately associated with the T cell receptor, and may thus play a critical role in T cell specificity as part of the antigen:Ia recognition complex. This proposed role is in keeping with the very strong correlation between L3T4 expression and recognition of self class II MHC molecules. While these studies do not provide definitive evidence for a physical association between L3T4 and the T cell receptor, they do place certain constraints on current models and suggest new possibilities for understanding T cell recognition and development.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct interactions between B and T lymphocytes bearing complementary receptors.

A murine cloned Th cell line specific for the antigen conalbumin in the context of self I-A molecules can be activated by low concentrations of soluble antireceptor mAb. By using an antireceptor mAb to shared antigenic determinants on T cell receptors, we have shown that the ability to be activated by soluble antireceptor mAb is an unusual, although not unique, feature of this cloned T cell line. This activation does not involve occult APC, FcR, or interaction between individual cloned T cells, as limiting-dilution analysis shows that individual cells of this clone will grow in the presence of the antireceptor antibody and IL-1 as stimulus. This cloned T cell line is highly immunogenic in vivo, giving rise to antireceptor antibodies that stimulate its growth in both mice and rats. This response is not dependent upon exogenous T cells. Rather, the clone directly interacts with complementary B cells, as shown by the production of mAb in nude mice, and by production of stimulating antireceptor antibodies by purified B cells cultured with cloned Th cells in vitro. Several features of this cloned Th cell line, most especially its ability to be activated, rather than inhibited, by antireceptor antibodies, may account for its striking ability to directly activate B cells bearing complementary receptors. The direct interaction of the cloned Th cell with B cells bearing complementary receptors may serve as a model for receptor-receptor interactions in the generation of both T and B cell repertoires.

Genetic control of B cell function. III. IgVH-controlled polymorphism in the frequencies of B cells that recognize xenogeneic red blood cells.

Previous work has shown that the primary IgM plaque-forming cell response of inbred mice to xenogeneic red blood cells (RBC) including sheep, horse and chicken RBC is under the control of two polymorphic genes or sets of genes, one linked to the Igh linkage group and the other of unknown linkage but unlinked to H-2 and a variety of other known genetic markers. Both genes together control B cell function but do not influence the function of T cells and macrophages. Thus, this system permits the study of two polymorphic loci that control B cell responsiveness. In this study we analyze the role of the Igh region in further detail. In bulk cultures and limiting dilution experiments, we confirm its exclusive influence on B cells also when analyzed separately from the background gene, i.e. in Igh-congenic strains. Moreover, we find in the majority of experiments 4-5-fold differences in sheep RBC-specific B cell precursor frequencies among lipopolysaccharide-reactive cells from 3 pairs of Igh-congenic high and low-responder strains. Similar frequency differences exist for horse RBC and chicken RBC-specific B cells but not for B cells with specificity for (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-gelatine. These differences are independent of the frequencies of B cells responding to lipopolysaccharide which are shown to be equal between Igh-congenic pairs of strains. Since the differences in RBC-specific B cell frequencies closely resemble the differences in bulk culture responses to the corresponding RBC, we conclude that the role of the Igh linkage group in controlling responsiveness to RBC lies in a selective influence on the B cell repertoire concerning precursor cells for RBC specificity. In addition, we find that the VH part of Igh is responsible for the observed frequency differences, suggesting that VH germ-line genes directly influence the composition of the mature B cell repertoire.