Cytodiagnosis of lymphoid proliferations by fine needle aspiration biopsy. Adjunctive value of flow cytometry.

OBJECTIVE: To determine the accuracy of fine needle aspiration biopsy (FNAB) complemented by flow cytometry (FC) for the diagnosis of reactive and neoplastic lymphoid proliferations and subclassification of malignant lymphomas. STUDY DESIGN: Forty-one FNABs of lymphoid lesions on which FC had been performed were evaluated retrospectively. All cases were correlated with histology or clinical follow-up. RESULTS: Twelve FNABs were diagnosed as reactive. Eleven of the 12 were confirmed as reactive on follow-up. One was a case of posttransplant lymphoproliferative disorder. Twenty-five FNABs diagnosed as lymphoma were confirmed by histology. In 22 of these 25 cases, there was 100% correlation between the subclassification given on FNAB with FC and that given on histology. Two of the remaining cases, which were correctly called follicular center cell lymphoma, showed discrepancies in grading. One case called Hodgkin's disease on FNAB was T-cell lymphoma on histology. Of four FNABs given an inconclusive diagnosis, two were lymphoma on follow-up, and two were reactive. CONCLUSION: FNAB examination, when it includes immunophenotyping by FC, is a useful technique for distinguishing reactive lymphoid proliferations from malignant lymphomas and for the subclassification of lymphomas.

Cytokine profile in acute myelofibrosis associated with aggressive large granular lymphocyte leukemia.

We report a patient with acute large granular lymphocyte (LGL) leukemia, presenting as acute myelofibrosis (AMF). The leukemic cells were immature T-cells (CD5+, CD7+, CD16-, CD56-, CD57-, and CD41-), had monosomy 7, and secreted large amounts of Transforming Growth Factor-beta 1(TGF-beta 1). The serum levels of interleukins (IL)-2, -2R, -6 and -8 were elevated, while the IL-1 beta, IL-4, and tumor necrosis factor-alpha were normal.

Evidence for a second cell cycle block at G2/M by p53.

Wild type p53 can induce cell cycle arrest at specific points in the cell cycle, in particular G1/S, an ability lost by most p53 mutants. We have previously reported that p53 mutant genes can rescue REF52 cells from ras-induced growth arrest and that over expression of wild type p53 inhibits cell growth in these cells. In this paper we examined whether p53 can also induce cell cycle arrest at the G2/M boundary of the cell cycle. To accomplish this we used the REF52 cell line and the temperature sensitive p53val135 mutant allele. Cells were enriched in the late G1 and early S phases before the temperature shift. REF52 cells expressing mutant-p53val135 alone with an activated H-ras gene arrest primarily at the G1/S and G2/M parts of the cell cycle at the restrictive temperature, as determined by flow cytometry analysis. These results suggest that the anti-proliferative activity of p53 may be involved in regulation of the cell cycle at the G2/M restriction point as well as transit through G1/S and initiation of DNA synthesis.

The antiproliferative potency of histamine antagonists correlates with inhibition of binding of [3H]-histamine to novel intracellular receptors (HIC) in microsomal and nuclear fractions of rat liver.

Previously, we identified in rat liver microsomes, low (microM) affinity histamine receptors (HIC), associated with antiestrogen binding sites (AEBS). N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), a potent AEBS ligand, is a specific HIC antagonist. Through binding HIC, newly-formed intracellular histamine mediates, and DPPE inhibits, human platelet aggregation. We now provide evidence that histamine, mobilized from cytoplasmic stores, is a mediator of the mitogenic response to concanavalin A in mouse spleen cells. DNA synthesis and intracellular histamine levels are decreased over time by the histidine decarboxylase inhibitor, alpha-fluoromethylhistidine. For DPPE, H1 and H2 antagonists, rank order of potency to inhibit [3H]-histamine binding to HIC in rat liver microsomes correlates with antiproliferative potency. DPPE also competes for [3H]-histamine binding at low and high affinity sites in rat liver nuclei (IC50 approximately 2 microM). Thus, histamine may mediate growth through two intracellular subtypes of HIC.

A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit.

Previous studies have shown that the isotype of an antibody response is selected, in part, by the inhibition of isotype-specific suppression. The antisuppressor model predicts that isotype selection is initiated through an interaction between Ag, Ig, and a T cell-derived factor within 6 h of immunization. This report characterizes some of these molecules and their contribution to isotype regulation. Cultures of murine spleen cells stimulated with the T cell-dependent Ag SRBC led to Ag-specific IgG and IgA responses that could be suppressed and then antisuppressed by a molecular complex produced by mixing purified serum Ig with the supernatant of Ag-pulsed macrophages co-cultured with T cells. The supernatants from separate cultures of Ag-pulsed macrophages and rIL-1 alpha stimulated CD4+ T cells, could be pooled and mixed with Ig to produce functional antisuppressive complexes thereby allowing the factors from the different cell types to be studied separately. Adsorption of the co-culture or the rIL-1 alpha stimulated T cell supernatants against monoclonal IgG or IgA, removed IgG and IgA binding factors, respectively, and abrogated the ability to enhance the corresponding isotype. The adherent material could be recovered and used to reconstitute enhancement by the supernatants depleted of the binding factors. When affinity purified IgG or IgA was used as the source of Ig within the antisuppressive complexes, the enhancement of the antibody response was limited to the isotype of the regulatory Ig used to form the complex. Thus, manipulation of the antisuppressive molecules has a predictable effect on isotype selection. Release of isotype-specific binding factors by CD4+ cells by rIL-1 alpha supports the hypothesis that T cell circuits play a role in initiating isotype regulation.

The isotype of an Ig-containing mediator dictates the isotype of the antibody produced: a novel mechanism of isotype regulation.

Within 6 hours after primary immunization the serum of mice contains a unique form of processed antigen which is complexed with immunoglobulin (Ig). These complexes are formed through the mediation of a T cell factor and markedly enhance in vivo the 7s antibody response. They possess potent antisuppressor activity, i.e., they reverse suppression to immunity both in vivo and in vitro. We have been able to generate in vitro complexes containing a single Ig isotype. Such complexes returned the antibody formation in a suppressed culture only for the same Ig isotype as that present in the complexes. Thus, complexes containing IgG2a, induced exclusively IgG2a antibody and those containing IgA returned only the IgA response in the suppressed culture. The selection of a particular isotype for the formation of the complexes is determined by inducer T cells that produce the factor. The spleen contains inducer T cells capable of mediating the formation of complexes with IgG2a or IgA, while the T cells from Peyer's patches can induce the formation only of IgA complexes. The formation of the complexes is the earliest response to antigen in vivo and, according to the data presented here, the type of isotypes to be synthesized during an immune response may be determined as early as 3-6 hours after immunization as a result of the formation of these isotype specific complexes.

A novel mechanism for the selection of isotype-specific antibody responses: the role of intestinal T cells in the regulation of IgA synthesis by the anti-suppressor circuit.

Within 6 hr of immunization the serum of mice contains a unique form of processed antigen, which consists of a complex of immunoglobulin (Ig) and antigen formed in the presence of a factor derived from the anti-suppressor inducer T cell. This complex binds to and activates the anti-suppressor effector T cell, which eventually leads to the inhibition of suppressor cell function. Both of these cells are present in the spleen (SPL) and play a role in the regulation of antibody responses. The purpose of these studies was to identify the anti-suppressor T cells in gut-associated lymphoid tissue and compare their function to their splenic counterparts. Inducer cells were detected in the Peyer's patches (PP), mesenteric lymph nodes but not in the intra-epithelial lymphocytes. The effector cells, which take up the complexes, were detected in PP and lamina propria lymphocytes but not in the intraepithelial or mesenteric lymph node lymphocytes. Furthermore, the uptake of the complexes correlated with the presence of T cells bearing Ia antigens. The PP and SPL anti-suppressor cells were compared for their ability to enhance the production of IgA and IgG. The data clearly showed that the product of the inducer cell, and the effector cell it activates, not only enhanced the antigen-specific responses but also selected for isotype-specific antibody responses. Cells from SPL enhanced IgG greater than IgA, whereas cells from PP selected for IgA. Thus, the presence in PP of cells in the anti-suppressor circuit and their ability to selectively promote IgA synthesis suggest that this regulatory mechanism plays a significant role in intestinal immune responses.

Inhibition of antisuppression by the acute murine graft-versus-host reaction.

Profound suppression of both humoral and cell-mediated immunity is a significant systemic effect of graft-versus-host reactions. Although no complete explanation has been advanced for this immunosuppression suppressor cells have been implicated. The data presented in this paper indicate that acute GVH reactions in (C57BL/6J X A/J) F1-hybrid mice induced by the injection of A/J cells severely disrupts the function of the antisuppressor T-cell pathway at both its induction and effector stages. Results show that within 3 weeks of induction of the reaction, Ly1+-T antisuppressor inducer cells lose their ability to generate the serum factor that mediates antisuppression. This factor is normally taken up by and activates Ly2+ T cells which then inhibit suppressor T-cell function. The data also reveal that Ly2+ T cells collected 2 weeks after induction lose their ability to be activated by the antisuppressor factor produced in normal mice. These cells are thus unable to function as antisuppressor effector cells. The uptake of the antisuppressor factor by Ly2+ T cells depends on the expression of Ia antigens on the surface of these cells. Experiments have shown that these antigens are absent from the surface of T cells derived from mice with GVH reactions. This finding may provide an explanation for the inability of these cells to function as antisuppressor effectors. Antisuppression is an important T-cell pathway that is intimately associated with the regulation of immune function. It is possible that the immunosuppression arising in mice with GVH reactions may stem, in part, from unopposed suppressor T-cell activity that results from widespread interference by the reaction with a pathway that normally inhibits suppressor cell activity.

Macrophage T cell interaction. Induction in vitro of a mediator with potent antisuppressor activity.

A simple in vitro method is described for the induction of a potent mediator that interferes with suppressor cell function. The mediator consists of three easily identifiable components, Ig, class II determinants and antigen, that form a unique complex similar to, or identical with, the complexes detected in vivo within 3-6 h after immunization. The formation of the antisuppressor mediator in vitro takes place in two steps: the first involves a macrophage-T cell interaction which generates an 'intermediate complex' containing antigen and class II determinants. In the second step the addition of immunochemically purified IgG from normal mouse serum to the macrophage-T cell supernate generates potent antisuppressor activity, which is assayed by the conversion of suppression to immunity. It is suggested that the IgG interacts with the 'intermediate complex' giving rise to the final complex identical to that found in vivo 6 h after immunization. No activity is detected when IgG is added to a supernate of antigen-fed macrophages in the absence of T cells. Furthermore, the T cell plays an additional important role in the formation of the final complexes since it restricts the source of the IgG that will generate the antisuppressor activity. In other words the antisuppressor function is detected only if the IgG matches the donor of the T cell in the Igh locus. The T cell involved in the formation of the complex is the Ly1+ subpopulation. This method should allow elucidation of the genetic, cellular and molecular mechanisms in the activation of this important T cell pathway.

The double gammopathies. Clinical and immunological studies.

A retrospective review of 1135 patients with paraproteinemias recorded 28 (2.5%) as having two M components. This group included 11 patients with myeloma, 6 with lymphoproliferative disease, 5 with a nonlymphoproliferative malignancy, and 6 with a double gammopathy of undetermined significance. In 13 cases in which the M components were measured over a period of time, three distinct patterns were observed, which may reflect the cellular and subcellular origin of the two proteins: 1) In 2 cases the minor component remained relatively stable while the dominant protein changed with time and treatment, suggesting the origin to be two cell lines--the minor arising from a quiescent clone of the monoclonal gammopathy of undetermined significance, and the major M component arising from a more rapidly proliferating plasma cell line; 2) a discordant pattern was seen in 4 patients, suggesting that the two M components arose from two separate plasma cell clones; 3) seven cases in which the proteins behaved in a concordant manner probably arose from a single plasma cell clone with incomplete class switching, producing two M components with different heavy chains.

T-cell function in B-lymphocyte-deprived mice.

Previous work has identified selective defect(s) in T cells in mice deprived of B lymphocytes by the chronic administration of anti-IgM antibody. Experiments described in the present communication revealed that anti-IgM-treated mice do not possess T cells with surface Ia and FcR, and, unlike T cells from normal animals, they also fail to bind these molecules in vitro. Functional assays disclosed that an anti-suppressor pathway which relies on Ia+ donor and acceptor T cells is interrupted in these mice at both levels. These observations may provide an insight to explain the selective failure of some T cells when B lymphocytes have been deleted.

Activation in vivo of a major antisuppressor T-cell pathway immediately after immunization. II. T-cell requirements for its expression.

Immunogens activate in vivo within 3-6 hr after injection a new and hitherto unrecognized T-cell pathway which interferes with T-cell suppression, therefore called antisuppression. An important soluble mediator with antisuppressor activity is detected in the serum of immunized animals within 3-6 hr. The mediator represents a unique form of complexes of Ig and antigen. The antisuppressor function of the complexes does not represent a direct "neutralizing" effect of the complexes on the effector T suppressor cells. The antisuppressor complexes activate an Ly2+ T cell which, with the interaction of an Ly123+ T cell, blocks completely T-suppressor-cell function. The biological significance of the T antisuppressor pathway is discussed.

Activation in vivo of a major antisuppressor T-cell pathway immediately after immunization. I. Its regulation by I-A gene products.

It has previously been demonstrated that within 6 hr after immunogenic stimulation the serum of mice contains a unique form of immunogenic antigen which represents complexes of Ig and antigen. The complexes are known to be strongly cytophilic for Ly2+ Ia+ FcR+ T cells and markedly enhance the IgG response. Anti-I-A treatment of mice suppresses the IgG antibody response and results in the generation of antigen specific T suppressor cells (Ts). Furthermore, anti-I-A treatment blocks the induction of the complexes and abolishes the enhancing effect the complexes exert on the IgG antibody response. The 6-hr cytophilic complexes were shown to block the function of Ts and allow a normal IgG response to take place; therefore, they act as mediators of a novel T-cell pathway called antisuppression. The blocking of the induction of the antisuppressor complexes by anti-I-A antibody was at least in part due to an effect on T cells. In conclusion, products of genes of the I-A subregion of the MHC control the activation early after immunization of a T-cell pathway which is called antisuppression since its major function is interference with the expression of suppression. Its early induction (within 6 hr) suggests that antisuppression may play a pivotal role in determining between immunity and unresponsiveness.

Activation in vivo of a major antisuppressor T-cell pathway immediately after immunization. III. T-cell requirements for its induction.

Immunogenic stimuli rapidly induce a potent mediator with antisuppressor activity which represents complexes of Ig and antigen. The formation of the complexes depends on the interaction of two T cells both of which bear the Ly1 phenotype. The two T cells can be separated on the basis of their sensitivity to antilymphocytic serum and dependency on the presence of thymus. T cells bearing I region coded determinants are essential for the formation of the mediator.

Liposomes as immune adjuvants: T cell dependence.

The T cell dependence of the immune adjuvant action of liposomes containing the soluble antigens bovine serum albumin (BSA) and chicken immunoglobulin (CIgG) was studied with use of a quantitative enzyme-linked immunosorbent assay to measure serum antibody levels. Normal BALB/c mice, adult thymectomized mice, and congenitally athymic (nu+/nu+) mice were intravenously inoculated with liposomes containing BSA (Lip-BSA). The high levels of serum anti-BSA antibody that were seen in the normal group were decreased in the adult thymectomized group and were almost completely abrogated in the nu+/nu+ group. Reconstitution of nu+/nu+ mice with normal thymocytes and cortisone-resistant thymocytes led to a partial restoration of the anti-BSA antibody production after Lip-BSA immunization. Examination of the class of immunoglobulin produced in normal mice, immunized with Lip-BSA, showed an early low IgM response and a sustained higher IgG response that was primarily due to the IgG1 subclass. Trypsin removal of BSA exposed on the liposome surface decreased the resulting serum anti-BSA antibody level by 30% to 50%. Animals could be primed equally with a very low dose (0.2 micrograms) of Lip-BSA or with peritoneal macrophages that had phagocytosed the same dose of Lip-BSA. The adjuvant effect of liposomes containing CIgG on the number and type of specific anti-CIgG antibody-producing cells in the spleen was an early increase in IgM-producing cells followed by a substantially higher increase in IgG-producing cells. These observations suggest that liposome encapsulation of a soluble T-dependent antigen stimulates the helper T cell, not the suppressor T cell population, and that this stimulation involves uptake by macrophages.

Pathways of T cell suppression.

The methods of detection of suppressor cells which are generated during an immune response are reviewed. The requirements and the mechanisms for their induction as well as their relationship to other functionally important cell subpopulations (e.g., helper T cells, cytotoxic T cells) are examined. Their nature and the mechanism of their action are reviewed. The control of the generation of suppressor cells by well-defined subregions of the I region of the MHC is one of the most important recent developments. Suppressor cells have been shown to play an important role in a variety of systems such as allotypic suppression, the poor response of some strains of mice to certain polypeptides, the progress of tumor growth, etc. However, their role in other phenomena such as tolerance to self antigens and thus their involvement in the development of autoimmunity is not yet clear. In the NZB/W murine lupus model loss of suppressor cells has been implicated in the progress of autoimmune disease. Soluble mediators with suppressive activity probably represent the effector molecules derived from suppressor cells. Some of them have now been shown to contain antigens coded by the I-J subregion of the MHC. The mechanism of their action and the target cell varies from system to system and is not yet well understood.

Detection in vivo 6 hours after immunization of a mediator with possible antisuppressor activity.

Immunization of mice with foreign proteins or particulate antigen induces the formation of immunoglobulin (Ig)-antigen complexes which are strongly cytophilic for T cells and have been shown recently to markedly enhance the 7 S antibody response. In this report we demonstrate that pretreatment of the animals with cyclophosphamide or anti-I-J antiserum eliminates the difference in the antibody response between the mice injected with the complexes and the controls, in other words, the enhancement. Six hours after allogeneic stimulation the serum of mice contains also a cytophilic (for T cells) Ig which most likely represents, as in the case of the foreign antigens, complexes of Ig with alloantigens. The allogeneically induced 6-h serum (6HS) contains a factor which enhances the cytotoxic T lymphocyte(s) (CTL) response in vivo. As with the 7S antibody response, pretreatment of mice with cyclophosphamide, in doses known to eliminate suppressor cell expression, "masks" the CTL enhancement of the allogeneically induced 6HS. The same result was also observed with the anti-I-J antiserum. In conclusion, the 6-h complexes in cyclophosphamide- and anti-I-J-treated mice do not produce a 7 S antibody or CTL response above that produced by the control group (no complexes). Using heat-treated allogeneic tumor cells, known to induce suppressor cells in vivo, we have shown that for a low dose of tumor cells, the allogeneically induced 6HS did not block the induction of suppressor cells, but completely blocked their function. These results suggest that the enhancement of both humoral and cell-mediated immunity by the 6HS (complexes) is likely to be due to interference with normal suppressor cell function, that is, an antisuppressor mechanism.