Frank W. Fitch

Blocked Ras Activation in Anergic CD4+ T Cells

T cell anergy is a state of functional unresponsiveness characterized by the inability to produce interleukin-2 (IL-2) upon T cell receptor stimulation. The mitogen-activated protein kinases ERK-1 and ERK-2 and the guanosine triphosphate-binding protein p21Ras were found to remain unactivated upon stimulation of anergic murine T helper cell 1 clones. The inability to activate the Ras pathway did not result from a defect in association among Shc, Grb-2, and murine Son of Sevenless, nor from a defect in their tyrosine phosphorylation. This block in Ras activation may lead to defective transactivation at activator protein 1 sites in anergic cells and may enable T cells to shut down IL-2 production selectively during anergy.

Mouse T cell antigen receptor: Structure and organization of constant and joining gene segments encoding the β polypeptide

The germ-line joining (J) gene segments and constant (C) genes encoding the beta chain of the mouse T cell antigen receptor have been isolated on a single cosmid clone. There are two constant genes, C beta 1 and C beta 2, each associated with a cluster of J beta gene segments. The nucleotide sequences of the C beta 2 gene and of the J beta 2 cluster gene segments have been determined. The coding sequence of the C beta 2 gene is very similar to the sequence of a cDNA clone encoded by the C beta 1 gene. The C beta 2 gene has four exons; exon-intron structure does not obviously correspond to the functional domains of the protein. The J beta 2 gene segment cluster contains six functional J gene segments. We have isolated specific probes for the C beta 1, C beta 2, J beta 1, and J beta 2 regions to examine DNA rearrangements in T lymphocytes. DNA rearrangements can occur in both J beta gene segment clusters, and both C beta genes appear functional.

Regulation of T‐Cell Activation; Differences among T‐Cell Subsets

Four regulatory phenomena appear to regulate differentially the activation of TH1, TH2, and CTL clones. First, IFN-gamma selectively inhibits proliferation of TH2 but not TH1 cells; lymphokine production by TH2 cells is not affected by IFN-gamma. In addition, when fresh OVA-specific HTL clones are derived in the presence of rIL-2 TH2 cells are preferentially obtained, whereas TH1 cells predominate if cloning is performed in rIL-2 plus rIFN-gamma. These results suggest that the presence of IFN-gamma during the course of an immune response would result in the preferential expansion of HTL of the TH1 phenotype. Proliferation of CTL clones is not influenced by IFN-gamma. Second, different APC populations appear to differentially activate TH1 and TH2 clones. Purified splenic B cells stimulate optimal proliferation of TH2 but not TH1 cells, whereas macrophage/dendritic cells appear to stimulate optimal proliferation of TH1 but not TH2 cells. Since both APC types stimulate lymphokine production by each of the HTL subsets, these results suggest the existence of TH1- and TH2-specific cofactors for growth. Third, high doses of immobilized anti-CD3 mAb inhibit IL-2-dependent proliferation of TH1 but not TH2 clones. Since this effect appears to require calcium, this observation suggests that TCR-mediated signalling events might differ between the two HTL subsets. Indeed, little or no increase in [Ca++]i can be detected in TH2 clones stimulated with Con-A, while such an increase is easily discernible in TH1 cells. Although high concentrations of immobilized anti-CD3 mAb inhibit IL-2-dependent proliferation of CTL clones, proliferation of these cells in response to immobilized anti-CD3 alone reaches a plateau. Since activation with anti-CD3 is thought to mimic antigenic stimulation, these results suggest that antigen concentration may play a role in determining which predominant T-cell types proliferate in a particular immunological situation. Fourth, pretreatment of TH1 cells, but not TH2 cells or CTL, with IL-2 results in decreased lymphokine production and proliferation in response to subsequent stimulation via the TCR. This antigen-responsive state appears to involve a defect in calcium-dependent signalling, providing additional evidence for different signalling mechanisms in TH1 and TH2 clones.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloned t lymphocytes and monoclonal antibodies as probes for cell surface molecules active in t cell-mediated cytolysis.

The appearance of specific cytolytic T lymphocytes (CTL) is a prominent feature of cell-mediated immune responses to viral infections and to aliografts. CTL kill their target cells through a process consisting of at least three steps: (1) antigen recognition and celhceil adherence, (2) a Ca^-dependent lethal hit, and (3) target cell death. The highly specific nature of the antigenic recognition observed in T cell-mediated cytolysis indicates that a discriminating antigenreceptor molecule must be Involved, but attempts to identify this T cell receptor have met with only limited success. In addition to an antigen-specific receptor, other cell surface molecules appear to participate in the cytoiytic process. Antisera reactive with cell surface components have been utilized in attempts to define the molecules involved in cytolysis. Several investigators (Nakayama et al. 1979, Shinohara & Sachs 1979) demonstrated that anti-Lyl-2 alloantisera couid inhibit cytolysis mediated by T cells obtained from immunized animals or from mixed lymphocyte cultures (MLC). However, the interpretation of these results was complicated by the fact that such antisera may have contained antibodies which reacted with other antigens. In addition, it was difficult to be

Murine T Lymphocyte Clones with Distinct Immunological Functions

The study of cell-mediated immune responses has been facilitated by the development of model systems such as unidirectional mixed leukocyte culture (MLC) (Cerottini & Brunner 1974, Hayry et al. 1972, Wagner et al. 1973) which permit manipulation of the reacting cells in vitro. In most instances, the MLC is characterized by cell proliferation culminating in the development of cytolytic T lymphocytes (CTL) reactive toward the immunizing antigen. While antigenic stimulation induces cional expansion of reactive lymphocytes, the magnitude of the response is regulated by circuits of interacting T cell subsets exhibiting specific and/or non-specific helper or suppressor functions. Some of these interactions are mediated by soluble factors, others involve the expression of certain membrane proteins at the cell surface. Humoral responses are also characterized by the involvement of different cellular subsets which play diverse and highly specialized roles. Efforts to dissect the immune response and to characterize the nature of

Rejection of Renal Allografts: Specific Immunologic Suppression

Kidneys were transplanted across a major genetic barrier (Ag-B locus), from LewisxBN F1 hybrid rats into bilaterally nephrectomized Lewis rats. Survival of grafts is prolonged (indefinite?) in rats treated with a combination of (i) intravenous injection of donor spleen cells 1 day before the graft, and (ii) passive immunization with antiserum prepared in rats of the recipient strain against donor spleen and lymph-node cells. The recipients immune response to other antigens is not impaired.

Direct evidence for chromosomal inversion during T-cell receptor beta-gene rearrangements.

A germline T-cell receptor variable region (Vβ) gene segment (Vβ14) has been mapped 10 kilobases to the 3′ side of the constant region (Cβ2) gene. The Vβ14 gene segment is in an inverted transcriptional polarity relative to the diversity-region (Dβ) and joining-region (Jβ) gene segments and the Cβ genes. Analyses of a T-cell clone (J6.19), which has productively rearranged the Vβ14 gene segment, indicate that the productive Vβ-Dβ-Jβ rearrangement and its reciprocal flank recombination product are linked and located at either border of a chromosomal inversion. These data demonstrate for the first time a linkage between mammalian V and C genes and verify that a functional T-cell receptor Vβ gene can be constructed through a chromosomal inversion.

Specific Suppression of Immune Responses

The models we have discussed in detail demonstrate specific suppression of immune reactivity produced in normal adult animals by antibody and antigen. The mechanism of homeostasis of suppression in these models depends on continued exposure to antigen and on an active response by the host. The active response may include production of antibody directed against specific receptors as well as antibody directed against antigen. Thus, specific regulation of both antibody and cell mediated immunity to an antigen might be achieved by the use of only the biological agents of the response: antigen, antibody, and possibly antibody to receptors. The general implication is that these same biological agents are responsible for autoregulation of immune reactions occurring in nature. Presumably, these agents may be used to suppress or reverse immune responses for appropriate clinical objectives.

Monoclonal antibodies against purified nicotinic acetylcholine receptor.

Abstract Eleven stable monoclonal hybridoma cell lines synthesizing antibodies against purified acetylcholine receptor from Torpedo californica were produced. Spleen cells from a rat immunized with acetylcholine receptor and exhibiting experimental autoimmune myasthenia gravis were fused with a mouse myeloma cell line. Studies of the binding of these antibodies to acetylcholine receptor by use of a passive hemagglutination assay in the presence of various cholinergic ligands revealed four general categories of binding specificities: 1) blockade only by alpha bungarotoxin, 2) partial blockade by all ligands, 3) increased titer in the presence of alpha bungarotoxin and benzoquinonium chloride, 4) absence of effect by any ligand.

T-cell lines which cooperate in generation of specific cytolytic activity

THE unidirectional mixed leukocyte culture (MLC) is characterised by cell proliferation and by the development of cytolytic T lymphocytes (CTL)1–3. In murine MLC, the generation of CTL seems to involve proliferation of at least two sub-populations of responding T cells—T cells of the Ly-2+3+ phenotype, which differentiate into CTL, and cells of the Ly-1+ phenotype, which seem to ‘help’ or ‘amplify’ the CTL re-sponse4–6. The mechanism by which ‘amplifier cells’ augment the CTL response is unclear; recent reports have shown, however, that CTL precursors present in populations of long-term MLC or of immune spleen cells7,8 can respond to supernatant fluid obtained from allogeneic MLC or mitogen-stimulated spleen cell cultures in the absence of specific antigen. Several groups using such conditioned medium have reported the successful long-term culture of normal and antigen-specific immune T cells in the absence of alloantigen stimuli9–11. Whereas amplifier supernatant fluids seem to substitute functionally for a T-cell subpopulation (Ly-1+?) in the absence of alloantigen, there is only indirect evidence that amplifier cells can be activated by alloantigen to release one or several soluble factors that influence the differentiation or proliferation ofCTL12–14. We have used modifications of techniques previously described for the long-term culture of immune T cells to isolate several cloned lines of MLC-reactive T cells, including a non-cytolytic T-cell line which, when co-cultured with alloantigen, permits another cell line to proliferate and express specific cytolytic activity.