Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency.

The highly regulated secretion of effector cytokines by CD4+ T cells plays a critical role in immune protection against pathogens such as cytomegalovirus. Here, we directly compare the frequency and functional characteristics of cytomegalovirus-specific CD4+ memory/effector T cells in normal and HIV+ subjects using a novel, highly efficient multiparameter flow cytometric assay that detects the rapid intracellular accumulation of cytokine(s) after short-term (6 h) in vitro antigen stimulation. Responses in this assay correlate precisely with independent measures of sensitization history (e.

Immune reconstitution with donor-derived memory/effector T cells after orthotopic liver transplantation.

Therapeutic hematopoietic stem cell transplantation has made great strides in recent years, providing curative therapy for many previously untreatable diseases. Nevertheless, the applicability and effectiveness of this procedure continues to be restricted by adverse immunoregulatory states, including graft rejection, graft vs. host disease (GvHD), and/or persistent immunodeficiency.

Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes.

By coupling intracellular staining with terminal deoxynucleotidyl transferase (TdT)-mediated labeling of internucleosomal DNA strand breaks in a flow cytometric assay, we observed a strong correlation between apoptosis-associated DNA strand breaks and immunoreactivity with the monoclonal antibody (MAb) B-F6 in activated human peripheral blood T lymphocytes (PBTs). Although MAb B-F6 has been reported to be specific for the cytokine interleukin-6, Western blot analysis of activated PBT lysates revealed that the predominant protein band detected by this MAb was 17 kd (p17), distinct from the 23-kd core protein and 26- to 30-kd mature glycosylated forms of interleukin-6. Immunoaffinity isolation and amino-terminal amino acid sequence analysis of p17 revealed identity with the histone H2B, a finding confirmed by Western blot analysis of purified histones and by similar staining of activated PBTs with an unrelated anti-histone MAb.

Neutrophil adhesion to 24-hour IL-1-stimulated endothelial cells under flow conditions.

In this study, we examine neutrophil adhesion under flow conditions to cultured HUVEC stimulated for 4 or 24 h with IL-1. Interactions are measured using videomicroscopy and a parallel-plate flow system which is capable of distinguishing primary adhesion and rolling from secondary (firm) adhesion. We find that after 24 h, E-selectin does not contribute to primary adhesion, in contrast to a significant contribution after 4 h.

Lymphocyte homing and homeostasis.

The integration and control of systemic immune responses depends on the regulated trafficking of lymphocytes. This lymphocyte "homing" process disperses the immunologic repertoire, directs lymphocyte subsets to the specialized microenvironments that control their differentiation and regulate their survival, and targets immune effector cells to sites of antigenic or microbial invasion. Recent advances reveal that the exquisite specificity of lymphocyte homing is determined by combinatorial "decision processes" involving multistep sequential engagement of adhesion and signaling receptors.

CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyte-endothelial cell primary adhesion pathway.

The extravasation of leukocytes from the blood into tissues occurs as a multistep process: an initial transient interaction ("rolling"), generally thought to be mediated by the selectin family of adhesion molecules, followed by firm adhesion, usually mediated by integrins. Using a parallel plate flow chamber designed to approximate physiologic flow in postcapillary venules, we have characterized a rolling interaction between lymphoid cells and adherent primary and cultured endothelial cells that is not selectin mediated. Studies using blocking monoclonal antibodies indicate that this novel interaction is mediated by CD44.

Primary body cavity-based AIDS-related lymphomas.

Five patients with advanced AIDS developed a unique type of high grade primary body cavity-based non-Hodgkin's lymphoma (NHL). The lymphomas were exclusively in serous effusions with no detectable mass disease in the body cavities and no lymphadenopathy or organomegaly. All of the lymphomas exhibited virtually identical morphology, which could not be precisely classified, but appeared to bridge features of large cell immunoblastic and anaplastic large cell lymphomas.

Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry.

The array of cytokines produced by T cells in effector sites is a primary means by which these cells mediate host defense. It is well recognized that cloned T cells are heterogeneous with regard to cytokine synthesis and, thus, in their ability to mediate specific immune responses, but the extent to which the patterns of cytokine secretion observed in cloned cells reflect actual populations of memory/effector T cells existing in vivo is largely unknown. Here, we report our findings using a multiparameter flow cytometric assay that allows simultaneous determination of an individual T-cell's ability to produce multiple cytokines and its phenotype after only short (4 to 8 hours) in vitro incubation with an activating stimulus and the secretion inhibitor Brefeldin A.

Expression of CD45RB and CD27 identifies subsets of CD4+ memory T cells with different capacities to induce B cell differentiation.

The capacity of four subsets of CD4+ memory T cells, defined by expression of CD45RB and CD27, to provide help for B cells was examined. Larger amounts of Ig were induced by CD45RBdimCD27- cells compared with the CD45RBdimCD27+ population, whereas CD45RBbrightCD27+ or CD27- cells were poor inducers of Ig synthesis. Mitomycin C treatment, which prevents suppressive activity, markedly enhanced Ig production supported by each subset except for CD45RBbrightCD27- cells.

Tumour dormancy and cell signalling--III: Role of hypercrosslinking of IgM and CD40 on the induction of cell cycle arrest and apoptosis in B lymphoma cells.

Polyclonal anti-IgM antibodies were more effective than monoclonal antibodies in inducing dormancy in SCID mice bearing a murine B lymphoma (BCL1). Under saturating conditions, both polyclonal and monoclonal anti-Ig antibodies induced cell cycle arrest (CCA) in both BCL1 cells and human B lymphoma cells (Daudi) but polyclonal antibodies were far more effective at inducing apoptosis. A mixture of several monoclonal antibodies specific for noncrossreactive epitopes on C mu mimicked the effects of a polyclonal anti-mu.

Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen.

The cutaneous lymphocyte-associated antigen (CLA) is the major T cell ligand for the vascular adhesion molecule E-selectin, and it has been proposed to be involved in the selective targeting of memory T cells reactive with skin-associated Ag to cutaneous inflammatory sites. To further investigate the relation of CLA and cutaneous T cell responses, we analyzed the CLA phenotype of circulating memory T cells in patients with allergic contact dermatitis and atopic dermatitis (AD) alone vs in patients manifesting bronchopulmonary atopy (asthma with or without AD) and nonallergic individuals. Significant T cell proliferative responses to Ni, a contact allergen, and to the house dust mite (HDM), an allergen to which sensitization is often observed in AD and/or asthma, was noted only in allergic and atopic individuals, respectively.

Tumor dormancy and cell signaling. II. Antibody as an agonist in inducing dormancy of a B cell lymphoma in SCID mice.

Tumor dormancy can be induced in a murine B cell lymphoma (BCL1) by immunizing BALB/c mice with the tumor immunoglobulin (Ig) before tumor cell challenge. In this report, we have investigated the immunological and cellular mechanisms underlying the induction of dormancy. BCL1 tumor cells were injected into SCID mice passively immunized with antibody against different epitopes on IgM or IgD with or without idiotype (Id)-immune T lymphocytes.

Migration of skin-homing T cells across cytokine-activated human endothelial cell layers involves interaction of the cutaneous lymphocyte-associated antigen (CLA), the very late antigen-4 (VLA-4), and the lymphocyte function-associated antigen-1 (LFA-1).

The cutaneous lymphocyte-associated Ag (CLA) is expressed by a subset of circulating memory/effector T cells and by the vast majority of skin-infiltrating T cells. CLA is thought to target skin-associated T cells to inflammatory skin sites by interacting with endothelial cell ligand E-selectin (CD62E). We have examined adhesion molecules involved in the migration of human CLA+ and CLA- memory/effector T lymphocytes through IL-1- and TNF-alpha-activated and nonactivated HUVEC layers under static (nonflow) conditions.

Bacterial superantigens induce T cell expression of the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of interleukin 12 production.

T lymphocyte infiltration is a prominent feature of the skin inflammation associated with infections by toxin (superantigen)-secreting Staphylococcus aureus or Streptococcus bacteria. The cutaneous lymphocyte-associated antigen (CLA) has been hypothesized to be a homing receptor (HR) involved in selective migration of memory/effector T cells to the skin. Since the expression of this putative skin-selective HR is known to be under strict microenvironmental control, we sought to determine the effect of staphylococcal and streptococcal toxins on T cell expression of CLA.

Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen.

The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T cells activated in vitro by the relevant food allergen.

A two-step adhesion cascade for T cell/endothelial cell interactions under flow conditions.

Neutrophil adherence to endothelial cells (ECs) under conditions of flow occurs in successive steps, including selectin-dependent primary adhesion and CD18-dependent secondary adhesion. We used a parallel-plate flow chamber to assess the steps in T cell adherence in vitro. On monolayers of L cells transfected with the EC adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1), E-selectin was capable of mediating only primary adhesion, ICAM-1 was capable of mediating only secondary adhesion, and VCAM-1 was capable of mediating both primary and secondary adhesion.

Control of lymphocyte homing.

The expression of immunity, both protective and pathologic, is critically dependent on the appropriate distribution of 'lymphoid resources' among the tissues of the body. The 'homing' mechanisms mediating this distribution have proven to have an astounding plasticity--directing, under strict microenvironmental control, the selective recruitment of specific lymphocyte subsets to the various secondary lymphoid tissues and extralymphoid immune effector sites. The past year has seen significant progress in our understanding in three areas: the molecular basis of lymphocyte interactions with endothelium, providing new insight into the complex multistep process of lymphocyte extravasation; the role of extravascular matrix and cells in retaining lymphocytes within tissues; and the mechanisms by which local microenvironments differentially regulate adhesion molecule expression and function so as to provide for site-selective lymphocyte homing.

Differential expression of lymphocyte homing receptors by human memory/effector T cells in pulmonary versus cutaneous immune effector sites.

The heterogeneous expression of lymphocyte homing receptors (HR) by the (CD45RA(low)/RO(high)) memory/effector T cell population in the human is thought to define subsets with tissue-selective recirculatory potential. To investigate further the localization characteristics of these T cells, we used multiparameter flow cytometry to quantitate T cell subsets defined by expression of the skin-selective HR called the cutaneous lymphocyte-associated antigen (CLA), the peripheral lymph node (PLN) HR L-selectin, the mucosal-associated HR alpha 4 beta 7-integrin, and the mucosal-associated adhesion molecule alpha e beta 7-integrin in either cutaneous or pulmonary immune effector sites and corresponding peripheral blood. Compared to peripheral blood, skin T cells were highly enriched for the CLA+/L-selectin+/alpha e beta 7-integrin- memory/effector subset, whereas lung memory/effector T cells were predominantly CLA-to low L-selectin-, and almost half were alpha e beta 7-integrin+.

Expression of monoclonal antibody HECA-452-defined E-selectin ligands on Langerhans cells in normal and diseased skin.

The cutaneous lymphocyte-associated antigen recognized by the monoclonal antibody HECA-452 has been thought to play a major role in the homing of memory T-cell subsets to the skin by virtue of its ability to bind to E-selectin of dermal microvascular endothelial cells. Considering that the homing of different leukocyte populations to the skin may involve similar mechanisms, we studied the expression of HECA-452-reactive molecules on CD1a+ epidermal Langerhans cells. Immunofluorescence double-labeling of cryostat sections and epidermal sheets of normal skin revealed HECA-452 immunoreactivity on a subpopulation of dermal and epidermal CD1a+ cells, whereas upon flow-cytometric analysis of epidermal single cell suspensions virtually all CD1a+ cells bound HECA-452 antibodies.

Lyn tyrosine kinase signals cell cycle arrest but not apoptosis in B-lineage lymphoma cells.

Signal transduction initiated by binding of antibodies to cell surface molecules can have an important impact on the growth of tumor cells. The malignant behavior of the murine lymphoma BCL1 can be suppressed and the neoplastic cells can be induced to enter a dormant state by in vivo ligation of membrane immunoglobulin. Anti-CD19 antibodies can prolong the survival of SCID mice challenged with the human Burkitt lymphoma cell line, Daudi.

Anti-CD19 inhibits the growth of human B-cell tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest.

In this report, we extend our previous findings that IgG or F(ab')2 fragments of HD37 anti-CD19 antibody (Ab) in combination with the immunotoxin (IT), RFB4-anti-CD22-deglycosylated ricin A chain (dgA) (but neither reagent alone), prolonged the survival of SCID mice with disseminated human Daudi lymphoma (SCID/Daudi mice) to 1 year at which time they still remained tumor-free. We explored the mechanisms by which the HD37 Ab exerts antitumor activity in vivo by studying its activity in vitro. We found that it has antiproliferative activity (IC50 = 5.

Skin disease-related T cells bind to endothelial selectins: expression of cutaneous lymphocyte antigen (CLA) predicts E-selectin but not P-selectin binding.

Cutaneous lymphocyte antigen (CLA), defined by the HECA-452 antibody, is a cell surface glycoprotein found on a subset of T cells in peripheral blood that binds specifically to E-selectin. This marker is present on the majority of T cells at sites of cutaneous inflammation and immune responses. Based upon such evidence, an association between T cell CLA expression and skin homing has been proposed.

The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing.

The extravasation of blood-borne lymphocytes into organized lymphoid tissues and sites of chronic inflammation is directed in part by interactions of lymphocyte surface adhesion molecules, known as homing receptors, with tissue-selective endothelial ligands called vascular addressins. In mice and humans, lymphocyte L-selectin and the peripheral lymph node addressin (PNAd) form a homing receptor-endothelial ligand pair involved in lymphocyte traffic to peripheral lymph node (PLN). We have examined the tissue distribution and function of human PNAd, using monoclonal antibody MECA-79 and in vitro assays of L-selectin-dependent lymphocyte binding.

Regulation of tissue-selective T-lymphocyte homing receptors during the virgin to memory/effector cell transition in human secondary lymphoid tissues.

Conventional virgin T cells efficiently and homogeneously recirculate through all secondary lymphoid tissues, but not "extralymphoid" effector sites. In contrast, memory/effector populations are composed of distinct subsets with differential, often tissue-selective, migratory capability to both secondary lymphoid tissues and effector sites. In keeping with these observations, CD45RA(high)/RO(low) virgin T cells in human peripheral blood uniformly express the peripheral lymph node (PLN) homing receptor (HR) L-selectin, and lack the skin-selective HR CLA, whereas among the CD45RA(low)/RO(high) "memory/effector" population, differential expression of these HR yields three predominant subsets: L-selectin+/CLA+, L-selectin+/CLA-, L-selectin-/CLA-.

Coordinate expression of beta 1 and beta 2 integrin "activation" epitopes during T cell responses in secondary lymphoid tissue.

The monoclonal antibodies (mAb) 15/7 and 24 recognize unique activation-dependent, conformational epitopes on beta 1 and beta 2-integrins, respectively. The expression of both of these epitopes closely correlates with the ligand binding ability of their respective integrins, and thus serves as indicators of functional integrin "activation". Here, we have used six-parameter flow cytometry to examine the expression of these epitopes and conventional beta 1- and beta 2-integrin epitopes during human T cell activation in secondary lymphoid tissues in vivo, focusing particularly on the virgin to memory/effector cell transition.