Noel L. Warner

Ionizing radiation and the immune response.

Publisher Summary This chapter illustrates several ways in which radiation may be employed to dissect several individual cellular components of the immune response. The chapter considers the interrelationships between radiation and the various components of the immune response from three perspectives: (1) the effect of irradiation on normal lymphoid tissues and on isolated lymphocytes, (2) the effect of irradiation on antibody production, transplantation immunity, and other forms of cellular immunity, and (3) the effect of irradiation upon tolerance with specific references to putative autoimmune consequences after radiation-induced alterations in normal immunological homeostasis. A wide variety of approaches are available to characterize and define distinct populations of lymphocytes. These include biophysical and functional methods and characterization of antigenic and cell surface receptor components. The mechanisms of radiation effects in biological systems, particularly in humans, have been derived from experiments utilizing cells exposed in vitro and maintained in tissue culture. Such cells can be examined for: (1) loss of viability, (2) alterations in biophysical structure, (3) loss of functional capabilities, (4) biochemical changes, and (5) evidence of injury to subcellular components.

Membrane Immunoglobulins and Antigen Receptors on B and T Lymphocytes

Publisher Summary This chapter describes membrane immunoglobulins and antigen receptors on B and T lymphocytes. There are several qualitative and quantitative distinctions between T-cell and B-cell M-Ig that may explain many of the apparent differences in reactivity of these cells without requiring the existence of a different set of V genes or non-Ig receptors in T cells. Both T and B cells derive from hematopoietic, precursor, stem cells by antigen-independent differentiation. This involves the activation of L-chain and p-type H-chain genes in both cell types. The T-cell and B-cell M-Ig differ primarily in density per cell and possibly in relative exposure on the cell surface with much of the H-chain Fc region inaccessible on T cells. Moreover, the antigen-recognition receptor of both T and B cells involves their M-Ig. On B cells, the receptor is solely the M-Ig, whereas on T cells it is suggested that the specificity of recognition controlled primarily by the M-Ig and binding of antigen to the cell can solely be by the M-Ig receptor. Finally, the activation of the T cell requires an additional interaction of the antigen with another ceIl surface structure that is coded for by a polymorphic H-2 linked gene.

Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis

The objective of this study was to find a simple method to preserve cells for subsequent flow cytometry (FACS) analysis. We determined that fixation in 1% paraformaldehyde-0.85% saline solution did not significantly alter the Coulter volume, light scatter of fluorescence properties of the cells. This method was suitable for all cell types analyzed, including mouse, human and rat lymphoid cells, erythrocytes and transformed cell lines. Furthermore, fixed cells, previously stained with fluorescein conjugated antibodies, could be stored at 4 degrees C in the dark for at least a week prior to FACS analysis. This method should prove useful to those who work with in vivo derived specimens or have limited access to flow cytometry facilities.

Correlation of Functional Properties of Human Lymphoid Cell Subsets and Surface Marker Phenotypes Using Multiparameter Analysis and Flow Cytometry

INTRODUCTION Recognition of heterogeneity in lymphocyte populations has been achieved both through the discovery of selective expression of specific cell surface antigens on lymphoid cells and by findings of different functional properties of cells bearing these antigens. The explosive development of hybridoma technology, coupled with the analytical and separation capabilities of fluorescence-activated cell sorting, has led to an extensive classification of human lymphocytes into defined subsets. Of particular importance is the fact that expression of certain cell surface antigens correlates with functional properties or the differentiation state of the cell. Although in most cases the actual physiological role of the cell surface antigen is as yet unknown, monoclonal antibodies can be used to positively or negatively select identified subpopulations of cells for further functional, biochemical or genetic studies. It is anticipated that eventually it should be possible to unequivocally identify only those cells mediating a specific and select function by analysis of their cell surface phenotype.

CD28 Costimulation Is Essential for Human T Regulatory Expansion and Function

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8–12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.

Characterization and expression of the HLA-DC antigens defined by anti-Leu 10.

The expression of HLA-DC antigens on peripheral blood mononuclear cells, in tonsil and lymph node tissue sections, on tumor cell lines, and on activated T cells was studied using monoclonal antibody, anti-Leu 10. Anti-Leu 10 reacts with HLA-DC molecules on homozygous B cell lines expressing HLA-DR 1,2,4,5,6,8, and 9. It reacts with heterozygous B lymphocytes expressing DR7 and DRw10, suggesting it also recognizes HLA-DC molecules linked to DRw10. The HLA-DC molecules detected by anti-Leu 10 are expressed on all Ig-positive and DR-positive peripheral B lymphocytes and an apparent subpopulation of DR-positive peripheral blood monocytes. Two-color immunofluorescence experiments using phycoerythrin-anti-HLA-DR (L243) and FITC-anti-Leu 10 demonstrated a correlation of the amounts of HLA-DR and DC antigens expressed on B lymphocytes. Cells expressing relatively low, or high amounts of one Class II molecule express respectively low or high amounts of the other Class II molecule. Anti-Leu 10 reacted with all B lymphocyte derived tumor cell lines not with lines of myeloid or erythroid origin, and with only one T cell derived line, HUT-78 which has an activated T cell phenotype. Consistent with this result, anti-Leu 10 binding suggested the presence of HLA-DC on activated T cells in lymphoid tissue, in addition to staining B cells. HLA-DC was also detected on mitogen and MLC activated T cells by anti-Leu 10 binding. Anti-Leu 10 is, therefore, a useful reagent for further studies of the role of HLA-DC in T cell activation and in normal B cell and monocyte functions.

Flow cytometric analysis of lymphocyte phenotypes in AIDS using monoclonal antibodies and simultaneous dual immunofluorescence

Simultaneous dual immunofluorescence and flow cytometry was used to study sixteen lymphocyte phenotypes in 209 men including: healthy homosexuals, lymphadenopathy patients (LAN), and AIDS patients. Significant differences between the distribution of lymphocytes in healthy homosexuals and healthy heterosexuals were decreased percentages of helper/inducer T cells (Leu 3), increased cytotoxic/suppressor T cells (Leu 2), and consequently a decreased Leu 3/Leu 2 ratio. The increased Leu 2 cells were identified as functionally cytotoxic subset Leu 2+ 15- phenotype rather than suppressor cells which are Leu 15+. Leu 2 and Leu 3 bearing cells exhibited an excess of membrane-bound immunoglobulins which were easily elutable at 37 degrees C. An increased percentage of an HLA-DR framework determinant bearing T cells were also detected. Within the NK cell family, Leu 7 cells were moderately increased and the functionally unidentified Leu 2+ 7+ population was strikingly elevated. LAN or AIDS patients were compared to healthy homosexual controls. Lower percentages of Leu 3 cells and higher percentages of Leu 2 cells were evident in LAN patients. These subsets were similar in LAN and AIDS patients. The increase in Leu 2+ cells was due to the Leu 2+ 15- cytotoxic subset. Fewer T cells had immunoglobulin in LAN and AIDS. A definite increase in Leu 2+ DR+ cells but not Leu 3+ DR+ cells occurred in AIDS compared to LAN or healthy controls. NK cell changes already present in healthy homosexuals persisted in LAN and AIDS patients. No differences in the distribution of B cells was detected in any intergroup comparisons. Changes in monocytes or pan-T cells were relatively insensitive measures of immunologic alterations among any of the groups. These results indicate many of the changes in lymphocyte subsets seen in AIDS and LAN subjects are already present in a carefully screened population of healthy homosexuals in San Francisco. Many of the changes in Leu 2 and NK family of cells suggest a possible adaptive response to viral or neoplastic challenge. Whether these interesting phenotypic alterations relate to functional changes in response to such challenge of the identified subsets waits further investigation.

Flow Cytometry Analysis of Murine B Cell Lymphoma Differentiation

The differentiation of lymphoid cells is a process that is fundamentally associated with the expression of all types of immune responses. Since the initial recognition of two distinct lineages of lymphoid cells in birds (Wamer et al. 1962) and mammals (Miller & Mitchell 1969, Claman & Chaperon 1969, Davies 1969), it has become increasingly evident that these two major streams of lymphocyte differentiation can become further separated into divergent pathways which lead to cells having distinctly different functional roles in the immune response. This concept has been most clearly defined in terms of T cell differentiation, in which distinct functional subpopulations of T cells mediate the different T cell activities of help, suppression, delayed sensitivity, certain alloreactivities, cytotoxicity and initiator amplifier functions (see reviews Cantor & Boyse 1976, 1977). This characterization of T cells into distinct functional subpopulations, has been shown to be associated with distinct

Incidence of cells simultaneously secreting IgM and IgG antibody to sheep erythrocytes

Abstract The open carboxymethylcellulose (CMC) hemolytic plague technique has been used to study the question of whether plaque-forming cells (PFC) exist which can secrete both IgM and IgG antibody. Using a micromanipulation transfer technique, PFC were exposed serially to three types of plaque-revealing monolayers. One of these revealed chiefly IgM plaques; the second chiefly IgG plaques, and the third achieved discrimination between the true IgM-IgG double producers and certain atypical categories of PFC. Preliminary work was done to study the efficiency of serial cell transfer. It was found that 93% of both IgM PFC and IgG (enhanced) PFC could form three plaques. In fact, most cells could be transferred successfully more frequently. IgM PFC could make up to 30 successive plaques and IgG PFC up to 10 successive plaques. Experiments involving 3 different monolayers (direct, anti-μ antibody-containing, and anti-γ antibody-containing) were performed to search for cells capable of (a) forming IgG antibody of sufficient hemolytic activity to produce a plaque without enhancing serum; (b) forming IgM antibody of such potency as to break through the inhibitory capacity of anti-μ reagents; (c) forming non-complement-fixing IgM. Cells of categories (a) and (b) were easily found and distinguished from each other. Cells of category (c) were not found by us. The serial transfer approach outlined above was next used with the modification that the anti-γ antibody-containing monolayer was replaced by one containing a serum mixture capable of inhibiting most IgM PFC and of enhancing IgG PFC. Cells capable of making plaques in both this “enhancing-inhibitory” monolayer and in a direct monolayer, but not in the category of “breakthrough IgM PFC” or “lytic IgG PFC” were considered as true double-producers of IgM and IgG. Of 900 selected antibody-forming cells examined in either the primary or the secondary response of mice to SRBC, 14 (1.5%) were double producers. The present status of the hypothesis of an IgM to IgG transition in expanding immunocyte clones is briefly considered.

Regulatory T Cells and Human Myeloid Dendritic Cells Promote Tolerance via Programmed Death Ligand-1

Human regulatory T cells inhibit graft-versus-host disease that can occur after tissue transplantation, in part through expression of programmed death ligand 1 and modulation of antigen-presenting cells.