Selective abrogation of antigen-specific human B cell responses by antigen-ricin conjugates.

The feasibility of selectively eliminating human antigen-specific B cell responses by treating cells in vitro with antigen covalently linked to a cell toxin was examined. Tetanus toxoid (TT) was conjugated to the toxin ricin via a thioether linkage. Peripheral blood mononuclear cells from recently immunized subjects were preincubated for 2 h with TT-ricin in the presence of lactose. Antigen was then removed, and the cells from recently immunized subjects were preincubated for 2 h with TT-ricin in the presence of lactose. Antigen was then removed, and the cells were stimulated with pokeweed mitogen to induce antibody production. TT-specific antibody production was completely abrogated by preincubation with TT-ricin but not by TT alone or a mixture of TT and ricin. In contrast, polyclonal immunoglobulin production was not diminished by TT-ricin. This selective abrogation was also seen when B cells alone were preincubated with TT-ricin and a source of T cell help was later provided. T cell blastogenic responses to TT remained intact after TT-ricin exposure. Thus, antigen-toxin conjugates are capable of selectively eliminating specific antibody-producing B cell clones, while leaving intact the remainder of the B cell repertoire.

In vitro antigen-induced, antigen-specific antibody production in man. Specific and polyclonal components, kinetics, and cellular requirements.

A highly specific and reproducible antigen-induced, antigen-specific culture and assay system for antibody production by human peripheral blood B lymphocytes has been developed. The system is clearly T cell and monocyte dependent and is independent of exogenous mitogens. The major factors in our ability to trigger specific antibody production with antigen alone have been the use of extremely low concentrations of antigen in vitro (doses several orders of magnitude below those inducing a peak blastogenic response), careful attention to in vitro cell density and culture vessel geometry, and appreciation of the kinetics of the circulating antigen-inducible B cell repertoire. A dichotomy and overlap between antigen-induced, antigen-specific and antigen-induced, polyclonal responses was observed in the study of doubly immunized individuals. Whereas antibody responses highly specific for the antigen in culture were observed under one set of culture conditions (flat-bottomed vessels, 1.5 x 10(6) cells), switching to another culture system (round-bottomed vessels, 5 x 10(5) cells) resulted in polyclonal responses to antigen. Despite these culture condition-related differences in the induction of antibody synthesis, the suppression of specific antibody production that occurred at high concentrations of antigen was specific only for the antigen in culture. The capability to easily and reproducibly look at truly antigen-induced, antigen specific antibody production should be a major tool in furthering the understanding of human B cell activation and immunoregulation.

Human T cell leukemia/lymphoma virus I infection and subsequent cloning of normal human B cells. Direct responsiveness of cloned cells to recombinant interleukin 2 by differentiation in the absence of enhanced proliferation.

A human T cell leukemia/lymphoma virus (HTLV)-I-infected B cell clone expressed Tac antigen on its cell surface and responded to recombinant interleukin 2 (IL-2) by increased production of IgM without any increase in proliferation. Anti-Tac antibody completely inhibited the IL-2-induced differentiation of this HTLV-I-infected B cell clone. This study demonstrates that HTLV-I can directly infect normal mature human B cells, and that the Tac antigen, which may be induced by infection with HTLV-I, is the functional receptor for IL-2-induced B cell differentiation. The availability of such cell lines and clones should provide useful tools to delineate precisely the differentiation step in the human B cell cycle.

Interleukin 2 receptors on human B cells. Implications for the role of interleukin 2 in human B cell function.

In the present study, we examined the expression of interleukin 2 (IL-2) receptors on normal human B cells as well as established B cell lines. Anti-Tac monoclonal antibody did not bind to freshly separated normal human B cells. Unexpectedly, with the appropriate activation of the normal B cells by anti-mu antibody, phorbol myristate acetate, or Staphylococcus aureus Cowan I (SAC), Tac antigen was induced on the activated B cells. Anti-Tac antibody showed consistent reactivity with two B cell lines that were infected by human T cell leukemia virus (HTLV) and some reactivity with two out of eight Epstein-Barr virus-transformed B cell lines established from normal adult donors. Immunoprecipitation analysis revealed that antigens of similar size with a molecular weight of 50,000-60,000 can be precipitated with anti-Tac antibody from phytohemagglutinin-stimulated normal T cell blasts and normal activated B cells, as well as a cloned B cell line. Binding assays of IL-2 on normal activated B cells and on the cloned B cell (HS1) revealed that B cells have significantly fewer sites and lower-affinity IL-2 receptors compared with phytohemagglutinin-stimulated normal T cell blasts. Finally, biological properties of the IL-2 receptor on B cells were examined by incubating B cells with recombinant IL-2. It was found that moderate concentrations of IL-2 induce significant enhancement of proliferation and differentiation in SAC-activated normal B cells. These results suggest that normal B cells may express functional IL-2 receptors or closely related proteins and thus IL-2 may play a significant role in the modulation of B cell function.

Alteration of T-cell functions by infection with HTLV-I or HTLV-II.

Two functionally different types of human T-cell clones, one with helper function and two with specific activity, were infected with different isolates of HTLV-I and HLTV-II. Both types of human T cells showed alterations in specific function after infection with either of the HTLV subgroups. Before HTLV infection, the T-cell clone with helper function proliferates and provides help to B cells only in the presence of both a specific soluble antigen (keyhole limpet hemocyanin) and histocompatible antigen-presenting cells. After HTLV infection, these cells respond with increased proliferation and indiscriminant stimulation of polyclonal immunoglobulin production by B cells, regardless of the histocompatibility of the antigen-presenting cells or the presence of the soluble antigen. Infection of the normal cytotoxic T-cell clones led to a dimunition or loss of the cytotoxic function. The results of these studies suggest some possible mechanisms for induction of immune deficiency and of polyclonal B-cell activation by viruses of the HTLV family.

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.

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.

The in vitro regulation of human thyrocyte HLA-DR antigen expression.

The ability of endocrine organs to express human immune response-associated antigens (Ia), such as HLA-DR, is a subject of intense current interest. In this study, the effects of various potential modulators of thyroid follicular cell HLA-DR expression were examined using in vitro cultures. A culture supernatant containing T-cell-derived lymphokines caused DR antigen expression on 13-18% of thyroid cells; more consistent effects were produced by recombinant gamma-interferon, which led to 46-100% of the thyroid cells becoming HLA-DR positive after 3 days in culture. This effect was both time and concentration dependent and occurred in thyroid cells derived from patients with Graves' disease (n = 7) and Hashimoto's thyroiditis (n = 2) as well as from three subjects with no autoimmune thyroid disease. Thyroid cells stained with the monoclonal antibodies 4F2 and 5E9, which recognize cell activation antigens, regardless of whether they were treated with gamma-interferon. The lectin phytohemagglutinin also induced HLA-DR antigen expression (21-91% of cells positive). This response was dependent on T cell contamination of thyroid cell suspensions, since the effect was inhibited by cyclosporin A. HLA-DQ antigen expression, identified by the Leu-10 monoclonal antibody, was also induced on thyroid cells by gamma-interferon and phytohemagglutinin. In contrast, neither recombinant alpha-interferon nor interleukin-2 induced HLA-DR antigens. Irradiation reduced the response of thyroid cells to gamma-interferon, but two of the known inhibitors of macrophage Ia expression, prostaglandin E2 and (Bu)2cAMP, did not affect gamma-interferon-induced thyroid cell HLA-DR expression. We were unable to detect interleukin-1 production by thyroid cells. These results suggest that 1) under normal circumstances, thyroid cells are 4F2 and 5E9 positive, but are incapable of expressing Ia antigens and, thus, of activating T cells to initiate autoimmune thyroiditis; and 2) once activated, for example by a virus, T cells could release gamma-interferon and induce thyroid cell HLA-DR and -DQ antigen expression; these Ia-positive thyroid cells could then have a role in maintaining or enhancing the autoimmune response.

Central nervous system abnormalities in Lyme neuroborreliosis.

Intrathecal production of anti-Borrelia burgdorferi antibody occurs frequently in CNS Lyme, yet reliable diagnosis of neuroborreliosis is still considered difficult and controversial. Therefore, we assessed the utility of this measurement in 103 Lyme patients. Among 15 patients with Lyme meningoradiculitis and 41 controls, diagnostic specificity was 93% and sensitivity 87%. Application of this method permits the identification of a rare B burgdorferi-associated multifocal encephalitis (brain infection) and its differentiation from a milder encephalopathy, or confusional state; the latter may not require CNS bacterial invasion. The encephalitis involves white matter more often than gray; severity varies widely. Of six patients with this antibiotic-responsive encephalitis, five were positive for HLA DQw3(DQw7). We conclude that (1) measurement of intrathecal antibody production is a reliable indicator of CNS infection, (2) North American neuroborreliosis includes the same spectrum of neurologic dysfunction as described in Europe, and (3) HLA typing may be useful in furthering our understanding of severe CNS involvement.

Lyme borreliosis-associated encephalopathy.

Borrelia burgdorferi infection (Lyme disease) is frequently accompanied by CNS dysfunction. Particularly common is a mild confusional state, the mechanism of which is unknown. Since CNS infection with B burgdorferi is usually accompanied by intrathecal synthesis of specific antibody, we studied CSF in 73 patients referred for presumed CNS Lyme, manifested primarily as this confusional state. Of 30 seropositive patients evaluated, only 5 had intrathecal antibody production. Seven seronegative patients had positive cell-mediated immune responses to B burgdorferi in the peripheral blood; none had antibody production in the CSF. Of the remaining 36 patients referred with this diagnosis despite negative serologic studies, none had compelling evidence of CNS infection by this criterion. We conclude that CNS infection with B burgdorferi does occur in a small proportion of seropositive patients with this confusional state but is extremely uncommon among seronegative individuals with this clinical presentation.

Lyme neuroborreliosis: central nervous system manifestations.

We evaluated 85 patients with serologic evidence of Borrelia burgdorferi infection. Manifestations included encephalopathy (41), neuropathy (27), meningitis (2), multiple sclerosis (MS) (6), and psychiatric disorders (3). We performed lumbar punctures in 53, brain MRI in 33, and evoked potentials (EPs) in 33. Only patients with an MS-like illness had abnormal EPs, elevated IgG index, and oligoclonal bands in the cerebrospinal fluid. Twelve of 18 patients with encephalopathy, meningitis, or focal CNS disease had evidence of intrathecal synthesis of anti-B burgdorferi antibody, compared with no patients with either MS-like or psychiatric illnesses, and only 2/24 patients with neuropathy. MRIs were abnormal in 7/17 patients with encephalopathy, 5/6 patients with an MS-like illness, and no others. We conclude that (1) intrathecal concentration of specific antibody is a useful marker of CNS B burgdorferi infection; (2) Lyme disease causes an encephalopathy, probably due to infection of the CNS; (3) MS patients with serum immunoreactivity against B burgdorferi lack evidence of CNS infection with this organism.

Immunohistochemical characterization of intrathyroid lymphocytes in Graves' disease. Interstitial and intraepithelial populations.

The phenotypic cell surface markers of the lymphocytes present in thyroid tissue from four patients with Graves' disease were quantitatively analyzed using the avidin-biotin immunoperoxidase technique. As control specimens, normal perinodular tissues from three patients who had benign thyroid nodules resected were also studied. In contrast to normal thyroid tissue, which contained very few T cells and no B cells, thyroid tissue of all four patients with Graves' disease contained a lymphocytic infiltrate, and this could be divided into two populations of lymphocytes. The first population was located in the follicular epithelium and expressed a cytotoxic-suppressor T cell marker (Leu2a). On the average, these cells were 4.2 times as numerous in Graves' tissues as in normal tissues (p less than 0.05). Most of these cells did not express Leu1, a pan-T cell marker. The second population was found in the interstitial tissues, often within lymphoid aggregates, and 70 to 83 percent of the cells expressed Leu1. The majority of these cells expressed a helper-inducer T cell marker, Leu3a; Leu3a/Leu2a ratios within aggregates ranged from 1.9 to 2.1. The number of B cells present was small, ranging from 5.8 to 12.1 percent of the interstitial lymphocytes. These findings are consistent with the involvement of both helper-inducer and suppressor-cytotoxic T cells in a localized autoimmune reaction directed, at least in part, against the thyroid follicular epithelial cells.

Activated human T cells can present denatured 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.

New chemotherapeutic approaches in the treatment of Lyme borreliosis.

1. It was demonstrated that while B. burgdorferi may be sensitive to relatively small concentrations of penicillin and ceftriaxone, the organism is killed slowly. This implies that, as in syphilis, prolonged blood levels of these drugs may be necessary in order to ensure cure. In contrast, the activity of tetracycline is more rapid in its action but is more dependent on drug concentration achieved. Unfortunately, the MIC and MBC for some strains are at or above the peak level achieved under optimal conditions. 2. Increasing the concentrations of penicillin or ceftriaxone above the MIC for the organism has little effect on the rate of killing. In contrast, the killing by tetracycline can be augmented by increasing concentrations of the drug. 3. Ceftriaxone is more active than penicillin, as measured by MIC, against the five strains of B. burgdorferi tested. 4. Ceftriaxone was efficacious in the treatment of Lyme borreliosis, which was recalcitrant to penicillin therapy. In a randomized trial comparing ceftriaxone to high-dose penicillin therapy, ceftriaxone was significantly more efficacious than penicillin in the treatment of the late complications of Lyme borreliosis.