The Ly phenotype of suppressor T cells arising in mice subjected to a graft-versus-host reaction.

T cells with helper and suppressor functions arising during graft-versus-host reaction (B6 vs. BDF1) have been characterized with respect to their Ly surface antigens. Helper cells were found to express the phenotype Ly 1+2- and suppressor cells the phenotype Ly 1+2+. Ly 1-2+ T cells had no suppressive effect in this system. T cells of the host did not contribute to either activity.

B-cell activation by lipopolysaccharide. Distinct pathways for induction of mitosis and antibody production.

The role played by macrophages in two effects of lipopolysaccharide (LPS) on the immune system of the mouse-substitution for helper T cells and induction of B-cell mitosis-has been investigated. C3H/HeJ mice are unresponsive and do not produce (as other strains do) antibody to 2,4,6-trinitrophenol (TNP) conjugated with autologous mouse erythrocytes (MRBC-TNP) in the presence of LPS. We found that C3H/HeJ spleen cells produce antibody to MRBC-TNP when (a) LPS and macrophages from LPS-responsive C3HeB/FeJ mice or (b) tumor necrosis serum ([TNS] induced by LPS in responsive mice) are added. The mitotic response was not restored. The findings suggest that adjuvanticity and mitogenicity represent distinct pathways of B-cell activation by LPS, subject to different regulatory mechanisms.

Tumor necrosis serum induces a serologically distinct population of NK cells.

Murine spleen cells generate nonspecific cytotoxic cells, referred to as natural killer (NK) cells, within 4 d of incubation in Mishell-Dutton cultures. This NK cell type does not arise in cultures of BALB/c.nu spleen cells or in cultures of T-cell depleted C57BL/6 spleen cells, indicating that its activation depends on T cells. Another type of NK cells is induced by tumor necrosis serum in murine spleen-cell cultures. It arises within 24 h and its activation does not depend on T cells. This cell type (and its precursor) expresses the recently discovered cell-surface marker Qa5 (controlled by the Q region of chromosome 17) that distinguishes this NK cell from the NK cell that depends for its activation on thymic function. Qa5+ NK cells are also induced by interferon.

Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy.

The 4-1BB receptor (CDw137), a member of the tumor necrosis factor receptor superfamily, has been shown to costimulate the activation of T cells. Here we show that anti-mouse 4-1BB monoclonal antibodies (mAbs) inhibit thymus-dependent antibody production by B cells. Injection of anti-4-1BB mAbs into mice being immunized with cellular or soluble protein antigens induced long-term anergy of antigen-specific T cells. The immune response to the type II T cell-independent antigen trinintrophenol-conjugated Ficoll, however, was not suppressed. Inhibition of humoral immunity occurred only when anti-4-1BB mAb was given within 1 wk after immunization. Anti-4-1BB inhibition was observed in mice lacking functional CD8(+) T cells, indicating that CD8(+) T cells were not required for the induction of anergy. Analysis of the requirements for the anti-4-1BB-mediated inhibition of humoral immunity revealed that suppression could not be adoptively transferred with T cells from anti-4-1BB-treated mice. Transfer of BALB/c splenic T cells from sheep red blood cell (SRBC)-immunized and anti-4-1BB-treated mice together with normal BALB/c B cells into C.B-17 severe combined immunodeficient mice failed to generate an anti-SRBC response. However, B cells from the SRBC-immunized, anti-4-1BB-treated BALB/c mice, together with normal naive T cells, exhibited a normal humoral immune response against SRBC after transfer, demonstrating that SRBC-specific B cells were left unaffected by anti-4-1BB mAbs.

Synergism between HIV gp120 and gp120-specific antibody in blocking human T cell activation.

The human immunodeficiency virus (HIV) binds to CD4-positive cells through interaction of its envelope glycoprotein (gp120) with the CD4 molecule. CD4 is a prominent immunoregulatory molecule, and chronic exposure to antibody against CD4 (anti-CD4) has been shown to cause immunodeficiency in mice. T cell-dependent in vitro immune responses can also be inhibited by anti-CD4. Experimental findings reported here indicate that CD4-bound gp120 attracts gp120-specific antibodies derived from the blood of HIV-seropositive individuals to form a trimolecular complex with itself and CD4. Thus targeted to CD4, the gp120-specific antibody functions as an antibody to CD4; it cross-links and modulates the CD4 molecules and suppresses the activation of T cells as measured by mobilization of intracellular calcium (Ca2i+). The synergism between gp120 and anti-gp120 in blocking T cell activation occurs at low concentrations of both components. Neither gp120 nor anti-gp120 inhibits T cell activation by itself in the concentrations tested.

Applications of Mass Spectrometry Imaging to Cancer.

Pathologists play an essential role in the diagnosis and prognosis of benign and cancerous tumors. Clinicians provide tissue samples, for example, from a biopsy, which are then processed and thin sections are placed onto glass slides, followed by staining of the tissue with visible dyes. Upon processing and microscopic examination, a pathology report is provided, which relies on the pathologist's interpretation of the phenotypical presentation of the tissue. Targeted analysis of single proteins provide further insight and together with clinical data these results influence clinical decision making. Recent developments in mass spectrometry facilitate the collection of molecular information about such tissue specimens. These relatively new techniques generate label-free mass spectra across tissue sections providing nonbiased, nontargeted molecular information. At each pixel with spatial coordinates (x/y) a mass spectrum is acquired. The acquired mass spectrums can be visualized as intensity maps displaying the distribution of single m/z values of interest. Based on the sample preparation, proteins, peptides, lipids, small molecules, or glycans can be analyzed. The generated intensity maps/images allow new insights into tumor tissues. The technique has the ability to detect and characterize tumor cells and their environment in a spatial context and combined with histological staining, can be used to aid pathologists and clinicians in the diagnosis and management of cancer. Moreover, such data may help classify patients to aid therapy decisions and predict outcomes. The novel complementary mass spectrometry-based methods described in this chapter will contribute to the transformation of pathology services around the world.

Ia-restricted interaction of normal lymphoid cells and SJL lymphoma (reticulum cell sarcoma) leading to lymphokine production. II. Rapid production of antibody-enhancing factor, interleukin 2, and immune interferon.

Mixed cell cultures of syngeneic lymph node (LN), spleen, or thymus and gamma-irradiated, syngeneic lymphoma cells of transplantable reticulum cell sarcomas (gamma-RCS) produced within 24 hours high titers of interleukin 2 (IL-2) and immune interferon in their supernatant (SN). These lymphokine titers were much higher than those seen after stimulation with allogeneic cells. SN also had marked enhancing activity for antibody production by anti-T-cell serum plus complement-treated spleen cells to trinitrophenylated polyacrylamide in vitro. This activity could be removed by absorption with cells of an IL-2-dependent cytotoxic cell line. Mixtures of gamma-RCS and LN cells from SJL/J F1 hybrid mice produced these lymphokines only when the non-SJL parent contributed H-2s or H-2b, but not H-2k or H-2d, in the I-region. These I-region restrictions were similar to those observed previously with respect to the ability of T-cells from SJL F1 hybrids to give proliferative responses to gamma-RCS in vitro and of these mice to support tumor growth in vivo. gamma-RCS also induced rapid interferon production in vivo, but serum titers 24 hours after injection consisted primarily of interferon resistant to pH 2 and neutralized by antibody to virally induced interferon (IFN-alpha/beta), and the production of IFN-alpha/beta was not subject to the same genetic restrictions. Although reticulum cell sarcoma cell extracts had no detectable effect in vitro, they were capable of inducing transient IFN production in vivo.

Combination immunotherapy of cancer in a mouse model: synergism between tumor necrosis factor and other defense systems.

Bacterial lipopolysaccharide (LPS) induces the release of factors into the serum which enable mice to reject experimental tumors. One such factor is tumor necrosis factor which causes acute necrosis of syngeneic sarcoma transplants in mice. Effective therapeutic use of tumor necrosis factor is limited, however, by its toxicity. We show here that the efficacy of tumor necrosis factor can be substantially increased by combining its application with low doses of LPS. Our data suggest that LPS exerts its antitumor effects by engaging more than one defense mechanism. Characteristic for the activation of a biological system is a concomitant induction of negative feedback mechanisms which antagonize the initial stimulus. Interference with the negative feedback response may substantially increase biological reactions. We show here that the blocking of two negative feedback responses occurring as a consequence of treatment with LPS, namely the production of prostaglandin E and the generation of suppressor T-lymphocytes, increases dramatically the ability of mice to reject tumor transplants. Thus, through appropriate combination of different factors one may reduce the dose of each below toxic levels and through interference with negative feedback responses increase the efficacy of antitumor reagents. We consider our findings in the context of formulating an effective immunotherapy of malignancies and as a promising step toward it.

Anti-histone autoantibodies react specifically with the B cell surface.

In an attempt to induce an immune response against Mls-1a antigens by immunizing C57B1/6 mouse (Mls-1b) with purified B cells from DBA/2 mouse (Mls-1a), we generated a panel of monoclonal antibodies from which the 5B9.6 mAb, taken as a representative antibody, was thoroughly investigated. This antibody specifically reacts with B cells from all mouse strains studied including C57Bl/6 mice as shown by FACS analysis of double-antibody labelled spleen cells. Using enzyme immunoassays and immunoprecipitation techniques, 5B9.6 mAb was found to be specific for histones. Amino acid sequence analysis of a peptide derived from a 5B9.6-immunoprecipitated polypeptide from DBA/2 B cells showed a 100% homology with a sequence within H2B histones. Furthermore, 5B9.6 mAb was able to interact with the cell surface of 7OZ/3 cell line, known as a typical pre-B cell line. The presence of histones can be modulated on the surface of 7OZ/3 cells since this antigen was upregulated after exposure of these cells to a cocktail of IL-1 and cAMP. Finally, 5B9.6 mAb was shown to interact with freshly isolated B cells from human peripheral blood.

Deletion of active human suppressor T lymphocytes from peripheral blood by Sephadex G-10 filtration.

A method for antigen-specific generation of antibody-forming B cells in cultures of human peripheral blood mononuclear (PBM) cells based on the Mishell-Dutton system has recently been established in this laboratory. Comparing PBM cell cultures from healthy donors and from patients with advanced cancer we found the latter to be unresponsive in our assay. Passage of PBM cell suspension over Sephadex G-10 columns restored the response of patient PBM cells to normal levels. The cell population trapped on the column can be recovered and its inhibitory potential demonstrated by its graded addition to cells eluted from the column. The cell responsible for inhibition is sensitive to treatment with OKT8 antibody and complement, indicating its T cell nature. Passage of PBM cells from healthy individuals did not alter antibody responses substantially but made the activation requirements less stringent.

The toxic shock syndrome toxin-1 induces anergy in human T cells in vivo.

TSST-1 is a Staphylococcus aureus-derived superantigen which has been implicated in the pathogenesis of toxic shock syndrome. In mice, superantigen-induced proliferation is followed by deletion or anergy of reactive T cells. So far, superantigen-induced T-cell anergy has not been observed in humans. We therefore examined PBMCs derived from a 15-year-old patient suffering from severe toxic shock syndrome. Markedly elevated levels of circulating TSST-1-reactive T cells were found by cytofluorometric analysis. Upon in vitro restimulation with TSST-1, hyporesponsiveness of TSST-1-responsive V beta 2+ T cells was detected, thus confirming results obtained in the murine system.

Cytotoxic monocytes in the blood of HIV type 1-infected subjects destroy targeted T cells in a CD95-dependent fashion.

HIV-1 infection changes the functional balance of macrophages in the body; it inhibits the development of macrophages capable of costimulating T cell responses and it favors the development of macrophages that kill T cells with which they form cellular conjugates. Cytotoxic macrophages destroy CD4 T cells, which they target through CD4-reactive immune-complexed HIV-1 envelope molecules on a large scale. They also destroy T cells that they target through presented antigen or mitogen. We show here that cytotoxic macrophages destroy their cellular targets at least partially in a CD95-dependent process in which T cells first modulate expression of most of their membrane receptors and subsequently die.

Monocytes in HIV type 1-infected individuals lose expression of costimulatory B7 molecules and acquire cytotoxic activity.

Monocytes/macrophages control the function of lymphocytes through positive and negative regulation. They release immunostimulatory cytokines and initiate costimulatory signals in T cells through the expression of B7 molecules. Their negative regulatory functions include the capacity to destroy cells with which they form cellular conjugates. We show here that HIV-1 infection skews monocyte function toward negative regulation by restraining the expression of costimulatory B7 molecules and by enhancing the cytolytic monocyte function. Monocytes that express constitutively B7, a membrane component that facilitates the engagement of costimulatory signals in T cells, lose this marker after HIV-1 infection and become refractory to inducers of B7 expression. The appearance of monocytes with reduced B7 expression is associated with an increased cytolytic monocyte capacity. Monocytes from HIV-1-infected donors destroy antibody-targeted normal lymphocytes more efficiently than do normal monocytes and they destroy CD4+ T cells specifically without the exposure to an exogenous ligand. CD4-reactive HIV-1 envelope molecules, expressed on monocytes as a consequence of infection or of opsonization by antibody, may specifically target CD4+ T lymphocytes for destruction and may thereby contribute to the preferential loss of CD4 T cells in HIV-1-infected individuals.

AIDS patient monocytes target CD4 T cells for cellular conjugate formation and deletion through the membrane expression of HIV-1 envelope molecules.

The human immunodeficiency virus (HIV) causes in humans the acquired immunodeficiency syndrome (AIDS). It replicates at a high rate in lymphoid organs even before it causes clinical symptoms. It binds to CD4 cell surface markers and destroys T lymphocytes that express the receptor. The immune system replenishes CD4 T cells at a formidable rate but, unable to keep up with the losses, allows the CD4 T cell compartment to disintegrate gradually. The net loss of CD4 T cells is an indicator for disease progression. How the virus destroys CD4 T cells and whether their loss accounts for the ensuing immunodeficiency have not been fully explained. We have reported evidence, and confirm here, that HIV-infected subjects deposit on monocytes immune complexes containing the virus or its envelope molecule gp120. Armed with these immune complexes monocytes form specific cellular conjugates with CD4 T cells and kill them. The destruction of normal CD4 T cells by monocytes from AIDS patients can be blocked by soluble CD4 and by free gp120. Normal monocytes and macrophages can be armed with CD4-binding gp120, and so induced to destroy CD4 T cells, by incubating them with gp120 and gp120-specific antibody. CD4-reactive HIV-1 components have a short half-life on the phagocyte surface. Removed from the HIV-infected environment, monocytes clear their surfaces of antibody-complexed viral components within hours, which abrogates their ability to destroy CD4 T cells. Rearming the monocytes with gp120-anti-gp120 complexes restores their capacity to destroy CD4 T cells. The data imply that for uninterrupted deletion of CD4 T cells, monocytes require a continued productive HIV-1 infection of their host.

In vivo presentation of Mls-1 antigen by T and B lymphocytes.

Previous studies of minor lymphocyte stimulatory (Mls) presenting lymphoid cells had shown that B cells rather than T cells present stimulatory Mls-1 antigen in vitro whereas B as well as T cells present Mls-1 antigen in vivo. Deletion of Mls-1 reactive T cells in the thymus of newborn mice is induced by T cells rather than by B cells. Applying a recently developed method for measuring the Mls-1 response in Mls-1- mice we assessed the Mls-1 stimulatory activity of T and B cells quantitatively. B cells are significantly more effective than T cells in this process. Both Mls-1+ T and B cells are also capable of inducing Mls-1 anergy in Mls-1- mice. Remarkably few lymphoid cells from Mls-1+ animals are needed for this effect: a few thousand B cells or 10(4) to 10(5) T cells per mouse induce substantial Mls-1 anergy in Mls-1- mice. These low cellular requirements for Mls-1 anergy production correspond well to the low T cell requirements described for the induction of Mls-1 tolerance in newborn mice. However, the high efficacy of B cells in inducing peripheral Mls-1 anergy contrasts with their failure to induce neonatal tolerance in newborn animals. We attribute this discrepancy to the previous notion that stimulatory Mls-1 antigen is not delivered to the thymus and that B cells and T cells present qualitatively different Mls-1 related signals to Mls-1 reactive T cells.

Activation of human T cells by the superantigen Staphylococcus enterotoxin B: analysis on a cellular level.

Superantigens interact with and activate a sizeable fraction of T cells characterized by expression of specific V beta gene segments of their antigen receptor. The massive activation of T cells in an organism is considered responsible for clinical symptoms associated with superantigen-producing bacteria. Here we studied the in vitro activation of human T cells by the superantigen Staphylococcus Enterotoxin B on a cell by cell basis. Superantigen-reactive T cells were stained with a V beta 12-specific monoclonal antibody and analyzed in a cytofluorograph. Blast formation of SEB-reactive T cells occurs within 12 h and reaches a plateau after 24 h. Double-staining of V beta 12+ T cells with antibodies against different T cell activation or adhesion surface molecules revealed a time-dependent differential upregulation for CD2, CD11 = LFA-1, CD25, CD28, CD69, and HLA-DR. The expression of CD3, CD4 and CD5 was not influenced by the superantigen. The rapid phenotypic changes of superantigen reactive T cells in terms of marker expression and cell size could provide early tools in diagnosing diseases caused by superantigens.

Reconstitution of in vitro humoral immune function in bone marrow transplant recipients.

The process by which humoral immune function is re-established following bone marrow transplantation was investigated in 50 transplant recipients. The recovery of in vitro-specific antibody production directed at sheep red blood cells was found to proceed through three phases. B cell function and helper T cell function were undetectable in the first phase (encompassing the first 5 months after transplantation), in which only suppressor cells reached functional maturity. Suppressor cells controlled responsiveness in the second phase (encompassing the period 5 to 15 months postgrafting). During this period, B cells and helper T cells became fully responsive; their activity became measurable, however, only after removal of Sephadex G-10-adherent suppressor cells. Normal responsiveness (associated with the loss of excessive suppressor cell activity) was characteristic of the third phase (over 15 months posttransplantation). Particular attention was paid to the role of suppressor cells in the recovery of humoral immune function. Their activity was positively correlated with graft-versus-host disease (GVHD), particularly of the chronic type. Suppressor cell activity in the early posttransplant period was predominantly mediated by OKT8-positive T lymphocytes, whereas suppressor cell activity in chronic GVHD patients was predominantly mediated by peripheral blood mononuclear cells that were retained on Sephadex G-10 columns, but did not express OKT8 antigen.

Differential expression of T cell receptor variable beta genes on CD4+ and CD8+ T cells: influence by sex linked genes?

We examined the expression of seven V alpha or V beta T cell receptor (TCR) segments on human CD4+ and CD8+ T cells. Confirming previously published results, we found a preferential expression of four V segment gene products on CD4+ T cells. One of these markers (V beta 6.7) was constantly expressed on more CD4+ T cells than CD8+ T cells. None of the analyzed blood samples showed a complete deletion of T cells expressing a particular V beta gene segment. In addition, our data provide the first evidence that genes on sex chromosomes may influence the formation of the human T cell repertoire. The ratio of CD4+/CD8+ T cells expressing V beta 12 gene products was always > or = 1 in female donors, whereas approximately 30% male donors exhibited more CD8+V beta 12+ T cells than CD4+V beta 12+ T cells.

Endotoxin-induced tumor necrosis factor.

The serum of BCG-infected mice treated with endotoxin contains a substance (tumor necrosis factor, TNF) which mimics the tumor-necrotizing action of endotoxin itself. TNF is not residual endotoxin, but a factor released from host cells, probably macrophages. TNF induced in the same way in rats and rabbits also causes necrosis of transplanted murine tumors. Unlike endotoxin, TNF is toxic in vitro for neoplastic murine and human cell lines but not for mouse embryo culture. TNF has striking effects on immunologic reactions in vitro, some like those of endotoxin and others unlike those of endotoxin. TNF is a glycoprotein; its molecular weight is less than 70,000. Highly purified preparations do not contain lysosomal or nonlysosomal serum enzymes, interferon or prostaglandin E1.