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.

Dexamethasone inhibits CD4 T cell deletion mediated by macrophages from human immunodeficiency virus-infected persons.

Prednisolone slows the loss of CD4 T cells in individuals with human immunodeficiency virus (HIV) disease and inhibits antigen-induced apoptosis of recently HIV-infected CD4 cells in vitro. This study investigated whether dexamethasone inhibits the ability of macrophages to delete CD4 T cells via anti-CD4 antibody or immune-complexed HIV envelope protein gp120. Peripheral blood mononuclear cells from HIV-negative persons were incubated with CD4-reactive ch412 monoclonal antibody or with gp120/IgG immune complexes and resident macrophages, with and without dexamethasone. Dexamethasone inhibited CD4 cell deletion in a dose-dependent manner. The deletion of normal CD4 cells by macrophages from HIV-infected patients also was inhibited by dexamethasone. Furthermore, up-regulation of CD95 expression on T cells exposed to anti-CD4 and gp120/IgG, which predisposes T cells to CD95-mediated apoptosis, is inhibited by dexamethasone in a dose-dependent fashion. Dexamethasone inhibits the macrophage-mediated deletion of CD4 lymphocytes in HIV-infected persons.

Interleukin-10 induces macrophage apoptosis and expression of CD16 (FcgammaRIII) whose engagement blocks the cell death programme and facilitates differentiation.

The development of monocytes into macrophages is regulated by helper T cells (Th) cells and, vice versa, the differentiation of Th cells into Th1 and Th2 is regulated by macrophages. Herein we examined the role of the Th2 cytokine, interleukin-10 (IL-10), on the development of macrophages. IL-10 is known to block the expression of antigen-presenting major histocompatibility complex (MHC) II and of costimulatory B7 molecules but it induces the expression of FcRs, especially the FcgammaRIII (CD16). The expression of CD16 enables the macrophage to carry out antibody-dependent cell-mediated cytotoxicity (ADCC) functions. However, this differentiation step is largely undercut by the capacity of IL-10 to induce macrophage apoptosis before the process of differentiation ensues. We found that the negative effect of IL-10 on the survival of macrophages is reversed in an environment that contains immunoglobulin G (IgG). IgG, especially when immune complexed with antigen, stimulates CD16 to transmit survival signals in macrophages which enable them to complete the differentiation process into CD16+ cells. Thus, IL-10 suppresses macrophage accumulation in healthy tissues where IgG is absent, and facilitates macrophage accumulation and differentiation in tissues that contain IgG, for example inflamed tissues or tissues that present autoreactive antibodies.

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.

Polyspecific self-reactive antibodies in individuals infected with human immunodeficiency virus facilitate T cell deletion and inhibit costimulatory accessory cell function.

Self-reactive polyspecific IgG antibodies (PSAs) arise in human immunodeficiency virus (HIV)-seropositive subjects before they develop AIDS. Self-reactive PSA levels correlate with the destruction of CD8 T cells in HIV-infected individuals and mediate the antibody-dependent cellular toxicity-based destruction of human T cells in tissue culture. PSAs react across the species barrier and bind to T cell antigens in mice. Such reactivity with mouse lymphocytes was not detected in normal human serum. Injection of human PSA IgG causes massive T cell depletion in the spleen, lymph nodes, and thymus in mice: evidence that PSA IgG facilitates T cell destruction in vivo. In addition to facilitating macrophage cytotoxicity, self-reactive PSA IgG inhibits the macrophage-mediated activation of T cells with antigen receptor-specific monoclonal antibody or with antigen. Exogenous costimulatory stimuli or interleukin (IL)-12 can reverse the inhibition. In contrast, exogenous IL-10 mimics this inhibition. These data implicate PSA IgG as a pathogenic factor in the development of HIV disease.

MHC class I molecules on CD4 T cells regulate receptor-mediated activation signals.

Three T cell populations can be distinguished based on their response to antigen receptor engagement. A sizable fraction dies within hours of TCR ligation, a smaller fraction enters the mitotic cycle, and the remaining T cells merely upregulate the expression of certain cell surface markers. An MHC-I-controlled regulatory mechanism has been identified. MHC I MAbs, or Fab fragments, prevent T cells from mounting a proliferative mitogen response but do not inhibit the mitogen-induced deletion of T cells. IFN-gamma enlarges the fraction of T cells which proliferate in response to mitogen stimulation but, in the presence of MHC I MAb, these cells fail to clonally expand and enter the deletion pathway. Phenotypically, MHC I MAb Fab fragments induce T cells to upregulate the expression of the apoptosis marker CD95, even in the absence of TCR ligand, and prevent the upregulation of costimulatory CD28 molecule expression.

Activation or destruction of T cells via macrophages.

Macrophages (MPhi) affect the T cell response in two mutually exclusive ways: activation or deletion. A MPhi type with T cell activating functions (M1) is able to express and upregulate receptors of the B7 family. IFN-gamma favours this MPhi differentiation pathway via upregulation of CD80 (B7-1) and CD86 (B7-2). The treatment of MPhi with IFN-gamma enhances the alphaCD3-mediated T cell blast transformation and reduces the fraction of deleted T cells. This MPhi type may prevent antibody-mediated T cell destruction by the expression of costimulatory receptors. An IL-10-induced MPhi type (M2) fails to express costimulatory molecules of the B7 family but is an effective cell for T cell destruction. Forming cellular conjugates with T cells through antibodies or immune complexes, M2-MPhi preferentially delete targeted cells in vitro and in vivo.

Elevated major histocompatibility complex class I expression protects T cells from antibody- and macrophage-mediated deletion.

Macrophages are capable of destroying T cells with which they form cellular conjugates. The deletion can be prevented by the simultaneous transmission of costimulatory signals. We show here that T cells with elevated major histocompatibility complex (MHC) class I expression are resistant against macrophage-mediated cytotoxicity. T cells that express the CD45RO isotype, considered memory T cells, exhibit MHC class I antigen at higher density than naive CD45RA T cells and upregulate MHC class I expression promptly when they form cellular conjugates with macrophages. We confirm previous observations that CD45RA T cells are more susceptible to antibody- and macrophage-mediated deletion than memory CD45RO T cells. When MHC class I molecules are masked by specific monoclonal antibody or antibody Fab fragments, CD45RA T cells and CD45RO T cells exhibit equal susceptibility to macrophage cytotoxicity, demonstrating that the difference between CD45RA and CD45RO T cells in their sensitivity to macrophage cytotoxicity is determined by their MHC I expression. Separation of CD4 T cells from CD8 T cells deprives memory CD4 T cells of their resistance against macrophage cytotoxicity, suggesting that memory T cells' resistance against destruction by macrophages is controlled by regulatory T cells.

Macrophages may activate or destroy T cells with which they form antigen- or coreceptor-mediated cellular conjugates.

The formation of antigen- or mitogen-mediated cellular conjugates with T cells enables macrophages to trigger in T cells costimulatory signals and to facilitate T cell clonal expansion and differentiation. The present study describes T cell death as an alternative consequence of T cell interaction with macrophages. Macrophages initiate the deletion of T cells which they target for conjugate formation through CD4 coreceptors. After suboptimal engagement, the TCR mediates a deletion program. Optimal TCR stimulation induces a rescue program which overrides the deletion program induced by suboptimal antigen receptor ligation or by coreceptor engagement. Evidence is presented suggesting that receptor clustering favors the transmission of activation signals, whereas ligation of nonclustered receptors facilitates T cell deletion.

Staphylococcal enterotoxin B-induced T-cell anergy is mediated by regulatory T cells.

Naive T cells mount a vigorous proliferative response to superantigen (SAg) stimulation in vivo. The proliferative response is followed by a partial deletion of responder T cells. Part of the deletion process has recently been attributed to the action of regulatory cytotoxic T cells that recognize major histocompatibility complex (MHC) class I-associated antigen receptor determinants on the target cell surface. Responder T cells that survived the SAg response were found to be incapable of generating a secondary proliferative response to a SAg challenge. We show here that this 'anergy' is enforced by CD8-positive regulatory suppressive T cells. These regulatory cells inhibit cell division of preactivated T cells but not the Sag response of naive T cells. Regulatory T cells are not generated in the presence of cyclosporin A and, once activated, become inactivated or deleted when restimulated in the presence of this immunosuppressive drug.

Lymphocyte-reactive autoantibodies in human immunodeficiency virus type 1-infected persons facilitate the deletion of CD8 T cells by macrophages.

The number of peripheral blood CD8 T cells declines in advanced stages of human immunodeficiency virus (HIV) infection coinciding with the transition from a clinically asymptomatic state of infection to AIDS. Although blood monocytes/macrophages exhibit cytotoxicity for CD4 T cells soon after HIV infection, cytotoxicity against CD8 T cells occurs at the time when HIV infection advances. The cytotoxic reaction is mediated by immunoglobulins that bind to T cells and which can be eluted from them. The immunoglobulins enable macrophages from noninfected persons to destroy healthy T cells in tissue culture. Lymphocyte-reactive autoantibodies (LRAs) occur physiologically as a result of chronic allo- or self-antigen stimulation. Lymphopenic, autoimmune lupus erythematosus patients exhibit LRAs that facilitate the deletion of T cells by macrophages. It is proposed that LRAs represent an immunoregulatory cytotoxic mechanism that is activated after chronic immune stimulation and is engaged by HIV to deplete host lymphocytes.

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.

Two distinct pathways of human macrophage differentiation are mediated by interferon-gamma and interleukin-10.

Forming cellular conjugates with T cells, macrophages can help their targets to mount an immune response or they can destroy the targeted T cell. The two functions are performed by two distinct macrophage subsets that can be distinguished by cell surface marker phenotypes, B7+ CD16- and B7- CD16+. Interferon-gamma (IFN-gamma) induces the former, interleukin-10 (IL-10) induces the latter phenotype. The two macrophage differentiation pathways are mutually exclusive; each cytokine inhibits the effect of the other cytokine. The second messenger cAMP enhances the macrophage B7 expression and suppresses the macrophage CD16 expression. However, together with IL-10, cAMP blocks the generation of both macrophage phenotypes. In the chimpanzee we noted deviations from this differentiation pattern that are suggestive of an enhanced IL-10 presence in the primate environment.

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.

Normal development of mice lacking metablastin (P19), a phosphoprotein implicated in cell cycle regulation.

Metablastin, also called P19, stathmin, prosolin, Lap18, and oncoprotein18, is a highly conserved cytosolic protein that undergoes extracellular factor- and cell cycle-regulated serine phosphorylation and developmentally regulated expression in mammals. It has been implicated in a variety of cellular functions including growth and differentiation, and recent evidence suggests an involvement in cell cycle control. To explore its potential role in mammalian development, we have disrupted the gene encoding metablastin by gene targeting in mice. The metablastin null mutants have no overt phenotype regarding development, growth rate, behavior, T cell maturation, or fertility and do not exhibit an increased predisposition to tumors. SCG10, a protein closely related in structure to metablastin, shows no compensatory up-regulation in metablastin-/- mice. Although the data suggest that metablastin is not essential for mammalian development, the knockout mice should prove valuable in exploring the role of this protein in cell cycle regulation.

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.

The superantigen Staphylococcus enterotoxin B induces a strong and accelerated secondary T-cell response rather than anergy.

The primary and secondary immune response of V beta 8+ T cells to the bacterial superantigen Staphylococcus enterotoxin B was compared in BALB/c mice. Secondary responder T cells were found to up-regulate the expression of the adhesion molecule LFA-1 faster, and to enter the cell cycle earlier than primary responder T cells. Both, primary and secondary responder T cells upregulate the expression of CD2 and CD25 and turn into blast cells with superimposable time kinetics. Secondary responder T cells terminate DNA synthesis, blast formation and the upregulation of CD25 and CD2 expression earlier than primary responder T cells and become more rapidly deleted. Two days after superantigen challenge, when primary responder T cells reach peak activity in terms of DNA synthesis and blast formation, secondary responder T cells have returned to the size of microblasts and ceased to replicate their DNA. Whereas our results are consistent with the observations leading to the concept of superantigen-induced T-cell anergy, they demonstrate, by revealing the accelerated vigorous secondary T-cell response to the superantigen, that this concept requires reconsideration.