T lymphocytes respond to foreign antigens both by producing protein effector molecules known as lymphokines and by multiplying. Complete activation requires two signaling events, one through the antigen-specific receptor and one through the receptor for a costimulatory molecule. In the absence of the latter signal, the T cell makes only a partial response and, more importantly, enters an unresponsive state known as clonal anergy in which the T cell is incapable of producing its own growth hormone, interleukin-2, on restimulation. Our current understanding at the molecular level of this modulatory process and its relevance to T cell tolerance are reviewed.

http://science.sciencemag.org/content/sci/248/4961/1349.full.pdf

A cell culture model for T lymphocyte clonal anergy

Publisher Summary This chapter focuses on the important discovery that virus-specific cytotoxic T cells are dually specific for virus and for a self cell surface antigen encoded by the major histocompatibility complex (MHC). The initial work was carried out on the lymphocytic choriomeningitis virus system but it soon became evident that the same phenomenon applied to many other viruses. In addition, the same principle has been found to hold for other antigenic systems, such as trinitrophenyl coupled to cells, minor histocompatibility antigens, and the H-Y model. Graft rejection and the need for genetically homogeneous inbred mouse strains for cancer research led to the development of transplantation immunology and immunogenetics. The result is that the gene complex coding for major transplantation antigens is one of the better understood mammalian genetic regions. Cytotoxic T-cell specificity is comparable to serological specificity. Because quantification of specificity or cross-reactivity is difficult, and because of the technical limitations of these cytotoxic T-cell assays, results are interpreted with great reservation. MHC restriction reflects the fact that the effector function of T cells is determined by the kind of Self-H recognized together with the foreign antigen on cell surfaces: K and D are receptors for lytic signals, I determinants are receptors for cell differentiation signals that are delivered antigen-specifically by T cells.

MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness.

Elimination of CD25+ T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, leads to spontaneous development of various autoimmune diseases. These immunoregulatory CD25+CD4+ T cells are naturally unresponsive (anergic) in vitro to TCR stimulation, and, upon stimulation, suppress proliferation of CD25-CD4+ T cells and CD8+ T cells. The antigen concentration required for stimulating CD25+CD4+ T cells to exert suppression is much lower than that required for stimulating CD25-CD4+ T cells to proliferate. The suppression, which results in reduced IL-2 production by CD25-CD4+ T cells, is dependent on cellular interactions on antigen-presenting cells (and not mediated by far-reaching or long-lasting humoral factors or apoptosis-inducing signals) and antigen non-specific in its effector phase. Addition of high doses of IL-2 or anti-CD28 antibody to the in vitro T cell stimulation culture not only breaks the anergic state of CD25+CD4+ T cells, but also abrogates their suppressive activity simultaneously. Importantly, the anergic/suppressive state of CD25+CD4+ T cells appeared to be their basal default condition, since removal of IL-2 or anti-CD28 antibody from the culture milieu allows them to revert to the original anergic/suppressive state. Furthermore, transfer of such anergy/suppression-broken T cells from normal mice produces various autoimmune diseases in syngeneic athymic nude mice. These results taken together indicate that one aspect of immunologic self-tolerance is maintained by this unique CD25+CD4+ naturally anergic/suppressive T cell population and its functional abnormality directly leads to the development of autoimmune disease.

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Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state.

T cell anergy is a tolerance mechanism in which the lymphocyte is intrinsically functionally inactivated following an antigen encounter, but remains alive for an extended period of time in a hyporesponsive state. Models of T cell anergy affecting both CD4(+) and CD8(+) cells fall into two broad categories. One, clonal anergy, is principally a growth arrest state, whereas the other, adaptive tolerance or in vivo anergy, represents a more generalized inhibition of proliferation and effector functions. The former arises from incomplete T cell activation, is mostly observed in previously activated T cells, is maintained by a block in the Ras/MAP kinase pathway, can be reversed by IL-2 or anti-OX40 signaling, and usually does not result in the inhibition of effector functions. The latter is most often initiated in naive T cells in vivo by stimulation in an environment deficient in costimulation or high in coinhibition. Adaptive tolerance can be induced in the thymus or in the periphery. The cells proliferate and differentiate to varying degrees and then downregulate both functions in the face of persistent antigen. The state involves an early block in tyrosine kinase activation, which predominantly inhibits calcium mobilization, and an independent mechanism that blocks signaling through the IL-2 receptor. Adaptive tolerance reverses in the absence of antigen. Aspects of both of the anergic states are found in regulatory T cells, possibly preventing them from dominating initial immune responses to foreign antigens and shutting down such responses prematurely.

T cell anergy

We investigated the antigen specificity and presentation requirements for inactivation of T lymphocytes in vitro and in vivo. In vitro studies revealed that splenocytes treated with the crosslinker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI) and soluble antigen fragments failed to stimulate significant proliferation by normal pigeon cytochrome c-specific T cell clones, suggesting that the chemical treatment inactivated full antigen presentation function. However, T cell clones exposed to ECDI-treated splenocytes and antigen in vitro were rendered unresponsive for at least 8 d to subsequent antigen stimulation with normal presenting cells. As predicted by the in vitro results, specific T cell unresponsiveness was also induced in vivo in B10.A mice injected intravenously with B10.A, but not B10.A(4R), splenocytes coupled with pigeon cytochrome c via ECDI. The antigen and MHC specificity of the induction of this T cell unresponsiveness in vitro and in vivo was identical to that required for T cell activation. These results suggest that nonmitogenic T cell recognition of antigen/MHC on ECDI-modified APCs results in the functional inactivation of T cell clones.

https://rupress.org/jem/article-pdf/165/2/302/1096753/302.pdf

Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo.

Antigen-specific human T cell clones specific for defined peptides of influenza A hemagglutinin were found to be rendered unresponsive by incubation with moderately high concentrations of antigen. This was the case whether the synthetic peptide antigen was present for the duration of the culture or the cloned T cells were preincubated with antigen for 3-18 h at 37 degrees C, before stimulation with T-depleted irradiated sheep erythrocyte non-rosette-forming lymphocytes (E-) pulsed with the optimal dose of peptide. Tolerance could not be overcome by culture with various numbers of E- cells and antigen. The induction of unresponsiveness was antigen specific, since it depended upon incubation with the appropriate peptide recognized by that clone. In addition, the tolerant T cells remained unresponsive to stimulation with the specific peptide for at least 7 d after induction even though maintained in culture in the presence of T cell growth factor. This state of antigen-specific unresponsiveness is akin to immunological tolerance. Furthermore, the experiments reported here demonstrate that the helper T cell clone can be inhibited by the relevant peptide in the absence of any suppressor cells or their precursors. This suggests that antigen-induced unresponsiveness need not always depend on the presence of suppressor T cells. The induction of tolerance in T cell clones does not result in early T cell death, since cells that no longer proliferate in response to the specific antigen and accessory cells still proliferate in response to T cell growth factor.

https://rupress.org/jem/article-pdf/157/5/1434/1093461/1434.pdf

Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin.

Bacterial lipopolysaccharide (LPS) was demonstrated to have the capacity in mice to enhance the response to soluble bovine serum albumin (BSA) and to interfere with the induction of tolerance to human gamma-globulin (HGG) These adjuvant activities were shown to occur under conditions in which LPS could also function as a B cell mitogen This positive correlation was established by utilizing two experimental situations in which LPS was non-mitogenic for spleen cells Thus, on the one hand, it was found that LPS did not function as an adjuvant in C3H/HeJ mice, a unique strain whose spleen cells were also unresponsive to LPS-induced mitogenesis On the other hand, in strains which did respond to LPS mitogenically, LPS failed to function as an adjuvant when it was chemically altered to reduce its in vitro mitogenic activity A correlation was also observed between mitogenesis and the capacity of LPS to function as a specific immunogen i mice In contrast to the sustained and prolonged plaque-forming cell response that was observed in mice whose spleen cells were also responsive to LPS-induced mitogenesis, the response was relatively transient in the C3H/HeJ strain These results are discussed in view of the possible in vivo modes of action of LPS

Immunologic Properties of Bacterial Lipopolysaccharide (LPS): Correlation between the Mitogenic, Adjuvant, and Immunogenic Activities

The ability of bacterial lipopolysaccharides to induce lymphocyte mitogenesis and to act as an adjuvant of antibody formation was attributable to the lipid-A region of the molecule. Measured by induction of DNA synthesis, lipid A was mitogenic for bone marrow-derived lymphocytes obtained from spleens of congenitally athymic mice but not for thymocytes obtained from thymuses of normal mice. Adjuvanticity was demonstrated by the ability of lipid A to convert a tolerogenic regimen of antigen into one eliciting an immune response and by its ability to markedly enhance the antibody response to a weak antigen.

Relationship of the Structure of Bacterial Lipopolysaccharides to Its Function in Mitogenesis and Adjuvanticity

The C3H/HeJ mouse strain, previously shown to be a nonresponder to bacterial lipopolysaccharide (LPS)-induced mitogenesis in vitro, was demonstrated by the present studies to be competent to respond mitogenically to LPS, but only to LPS preparations obtained by selected extraction methods. These preparations appear to be confined to LPS isolated by mild extraction techniques, such as TCA or butanol. In contrast, those obtained by techniques utilizing phenol were only weakly stimulatory or completely nonstimulatory for spleen cells from the C3H/HeJ. All LPS preparations tested, on the other hand, were highly stimulatory for cells from another mouse strain, namely the C3H/St. The critical importance of the method of extraction of LPS on its mitogenic activity for C3H/HeJ cells was stressed by experiments in which LPS was prepared from Escherichia coli K235 using either of two procedures. In these experiments, phenol-extracted LPS, although mitogenic in the C3H/St, was completely nonstimulatory in the C3H/HeJ; whereas, butanol-extracted LPS was highly stimulatory in both strains of mice. This striking difference was attributed to a destructive effect of phenol on LPS, as demonstrated by the fact that treatment of butanol LPS with phenol resulted in a total loss of its mitogenic activity in the C3H/HeJ, but in only a partial loss in the C3H/St. In general, the mitogenic response observed with selected LPS preparations in the C3H/HeJ was quantitatively lower and more transient than that seen with the C3H/St, although qualitatively these responses appeared to be similar. This was evidenced by the observation that in both mouse strains LPS was a specific mitogen for B cells, a property which was also attributed in both strains to the same distinct structural region of the LPS molecule, that is lipid A. A preparation of LPS that failed to stimulate B cells from the C3H/HeJ nonetheless had the capacity to block activation of these B cells by a stimulatory preparation of LPS. These results strongly suggest that mitogenic stimulation of B cells by LPS is a function of the structural integrity of both the LPS molecule and putative B-cell receptors for LPS.

https://rupress.org/jem/article-pdf/142/6/1488/1087330/1488.pdf

Immunologic properties of bacterial lipopolysaccharide (LPS). II. The unresponsiveness of C3H/HeJ Mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS.

Antigen-induced proliferation of primed lymph node cells was abrogated by treatment with anti-Ly 1 serum and complement (C) but not with anti-Ly 2 serum and C. Lymph node cells from animals primed to ovalbumin were activated with antigen in vitro, followed by propagation in an antigen-free supernatant fluid obtained from lectin-induced normal spleen cells. T cells processed in this manner displayed a stepwise enrichment of helper activity for antibody production as measured in a secondary hapten-carrier response. The sequential increase in antigen-specific help was paralleled by a rise in the antigen-induced proliferative response, a phenomenon whose expression was dependent on the presence of syngeneic or semi-syngeneic irradiated filler cells.

Barry J. Skidmore

Papers

Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin.

Immunologic Properties of Bacterial Lipopolysaccharide (LPS): Correlation between the Mitogenic, Adjuvant, and Immunogenic Activities

Relationship of the Structure of Bacterial Lipopolysaccharides to Its Function in Mitogenesis and Adjuvanticity

Immunologic properties of bacterial lipopolysaccharide (LPS). II. The unresponsiveness of C3H/HeJ Mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS.

Propagation of Antigen-Specific T Cell Helper Function in vitro