D J Volkman

The production and characterization of thyroid-derived T-cell lines in Graves' disease and Hashimoto's thyroiditis

Although the thyroid gland itself is a major site of the autoimmune response, the study of T-cell function in autoimmune thyroid disease has usually relied on peripheral blood as a source of cells. In this study, we have established thyroid-derived T-cell lines from six patients with Graves disease and one patient with Hashimotos thyroiditis by culturing the thyroid lymphocytes on an autologous thyroid follicular cell monolayer in the presence of exogenous interleukin 2 (IL-2). These T-cell lines have allowed in vitro investigation of thyroid-derived T-cell function, an approach which was previously limited by the number of lymphocytes obtained from the gland. The lines were predominantly OKT3, OKT4, and HLA-DR positive but showed heterogeneous proliferative responses. Some lines gave autologous or allogeneic mixed lymphocyte reactions but other did not. Only one of the seven lines responded well to the thyroid antigens thyroglobulin and microsomes presented by autologous monocytes. However, six of the lines proliferated in the presence of live but not dead autologous thyroid follicular cells, particularly when interferon-gamma (IFN-gamma) was added. This treatment has been shown to enhance HLA-DR and -DQ antigen expression by thyroid follicular cells in vitro. Furthermore, the proliferation induced by IFN-gamma-treated thyroid follicular cells was increased when thyroglobulin was also added. Together these results support the hypothesis that the expression of Ia antigens such as HLA-DR by thyroid follicular cells in autoimmune thyroid disease may be important in enhancing the autoimmune response, conferring on these cells the ability to present thyroid autoantigens to T cells. The use of thyroid-derived T-cell lines should permit a more detailed evaluation of the disordered immuno-regulation in Graves disease and Hashimotos thyroiditis than has been possible previously.

Clonal analysis of T lymphocytes in the acquired immunodeficiency syndrome. Evidence for an abnormality affecting individual helper and suppressor T cells.

Purified helper-inducer (T4+) and suppressor-cytotoxic (T8+) lymphocytes from eight patients with acquired immunodeficiency syndrome (AIDS) and eight healthy heterosexual donors were examined by limiting dilution analysis for their ability to be clonally expanded. It was demonstrated that viable T4+ and T8+ lymphocytes from patients with AIDS had markedly reduced proportions of clonable cells compared to the healthy donors (T4 = 1:255 vs. 1:34, P = 0.06; T8 = 1:355 vs. 1:55, P = 0.01). However, the cloned T cells that were obtained from the patients with AIDS demonstrated normal proliferation in response to phytohemagglutinin and alloantigen, and normal ability to help or suppress pokeweed mitogen-driven IgG synthesis. These results strongly suggest that, in addition to a quantitative diminution of T4+ lymphocytes in AIDS, there is an intrinsic functional defect in the surviving T4+ and T8+ lymphocytes, which is reflected by a severe decrease in their potential for clonal expansion.

Activated human T cells can present denatures antigen

The requirements for activated, Ia-positive human T cells to present antigen were examined. Although activated T cells could present allo-Ia antigens, activated T cells could not present native, soluble protein antigens. We have now shown that activated T cells can present denatured protein antigens to stimulate proliferation of antigen-specific T-cell lines. Since denatured antigen may represent a processed form of antigen, the data suggest that activated T cells can present antigen but may not be able to process antigen as efficiently as other presenting cells. We have also shown that antigen-specific T-cell lines, which are also Ia positive, are able to present antigen to themselves, if the antigen is in a denatured form. Autopresentation requires a critical minimal cell number to stimulate proliferation, even with denatured antigen. The ability of activated T cells to present antigen may reflect an important amplification or feedback mechanism of immune regulation.

Activated human T cells can present alloantigens but cannot present soluble antigens

Human T cells, when activated by antigen or mitogen, express Ia antigens. We have examined the capacity of activated T cells to stimulate autologous and allogeneic T cells and their ability to present soluble antigen. Interleukin 2-dependent T-cell lines (TCL), free of accessory cells, were used for antigen-presenting cells. These activated T cells were potent stimulators in an autologous mixed lymphocyte reaction (AMLR), more so than autologous irradiated non-T mononuclear cells. Activated T cells were also able to stimulate proliferation of allogeneic T cells in the absence of any other accessory cells, and this stimulation was blocked by anti-Ia antibodies. Resting unstimulated T cells were unable to stimulate autologous or allogeneic responses. Thus, activated T cells were able to present self antigens and alloantigens. However, activated T cells could not present soluble antigens to autologous T cells or to antigen-specific TCL even if exogenous interleukin 1 was added to cultures. The ability of activated T cells to stimulate an AMLR in vitro may reflect an important immunologic amplification mechanism in vivo. The ability of activated T cells to present alloantigens but not soluble antigens suggests an inability to process antigen, and this may provide further insights into the complexities of antigen presentation.

Parasite antigen-specific human T cell lines and clones. Major histocompatibility complex restriction and B cell helper function.

The development of T lymphocyte lines and clones of defined specificity has become an important method for investigating both T cell recognition of foreign antigens as well as T cell influence on B cells. In the present study, human antigen-specific T cell lines and clones have been derived from a patient with a naturally acquired filarial infection. These T cells are of the helper phenotype (Leu 1+, Leu 2-, Leu 3+) and are independent of exogenous interleukin-2. Furthermore, these T cells have been shown to require both antigen-presenting cells and antigen for optimal proliferation. Helper function mediated by these T cells as manifested by the in vitro induction of parasite-specific antibody was antigen-dose dependent, requiring much lower antigen concentrations than those necessary to induce blastogenesis. More importantly, there is an absolute requirement of the T cell line for HLA-DR histocompatible antigen-presenting cells; clones derived from this T cell line show a more specific DR-related restriction--to only one of the two parental DR haplotypes in antigen stimulated proliferative responses. Such parasite antigen specific human helper T cell lines and clones should prove useful in exploring the fine control of the host response to naturally acquired helminth infections. In addition, these long-term T cell lines and clones can provide a potent tool for examining not only the events involved in human T cell responses to parasite antigens, but also into the associated cellular and humoral factors necessary for the B cell responses which follow.

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4+ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.

Activation of human B lymphocytes: XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

Abstract We have previously described a system for the in vitro induction of anti-sheep red blood cell (SRBC) plaque-forming cells (PFC) in human peripheral blood (PB) following polyclonal triggering with pokeweed mitogen (PWM). A soluble antigen derived from hemolyzed SRBC was added to cultures of PB mononuclear cells. Addition of this antigen to PWM-stimulated cultures led to a two- to three-fold increase in the anti-SRBC PFC response seen with PWM alone. The synergistic response was specific for soluble SRBC antigen (SRBC-SA). Depletion of adherent cells eliminated the synergy between PWM and SRBC-SA. The plaques produced were macroscopic, inhibited by the addition of puromycin, and were not seen in the absence of complement. The synergistic response was dependent on the presence of human A sera absorbed with SRBC. Thus, these studies demonstrate clear-cut synergy between SRBC-SA and PWM in the SRBC-specific response of human PB B cells indicating the potential role of nonspecific signals in the antigen-specific antibody response of human B cells.

The Effects of Interleukin 2, Gamma Interferon, and B Cell Differentiation Factor on the Differentiation of Human B Cells

It has been well known that in the human system, SAC-activated B cells proliferate and differentiate in response to helper T cell factors. Among many factors included within the broad category of helper T cell factors, three distinct factors (interleukin 2 IL-2, gamma interferon γ-IFN, and B cell differentiation factor BCDF) were reported to be involved in B cell differentiation. Recent progress in recombinant DNA technology has made highly purified IL-2 and γ-IFN available, and even though BCDF is not yet cloned by molecular biological techniques, two kinds of BCDFs derived from the supernatants of newly established cell lines have been recently reported. In the present study, employing normal B cells or EBV-transformed B cell lines, we have investigated the effects of these factors on human B cells.

Exogenous IL-2 Independent Antigen-Specific Human T Cells: Antigen-Specific Induction of Polyclonal B Cell Factors

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of the mechanisms by which T cells recognize foreign antigens and subsequently elaborate immuoregulatory lymphokines. Human antigen-specific helper T cell lines and clones have now been developed by the method of cyclic restimulation with antigen1,2 which are completely independent of exogenous interleukin-2 (IL-2) for their growth. Following a narrowly restricted stimulation signal with the appropriate antigen in the context of histocompatible antigen-presenting cells (APC), these T cells can produce several polyclonal B cell factors which act on discrete subsets of B cells depending on their state of activation or receptiveness to these lymphokines.