Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes.

Two long-term cultured cell lines were established from BALB/c mouse axillary and cervical lymph nodes that exhibited a combination of functional, morphological, and phenotypic characteristics consistent only with high endothelial venule cells. Spleen lymphocytes selectively bound and migrated across the cell lines. On Matrigel, these cell lines formed tubules with lumens, a characteristic unique to endothelial cells.

Effect of anti-alkaline phosphatase monoclonal antibody on B lymphocyte function.

Alkaline phosphatase (APase) is a glycosylphosphatidyl-inositol (GPI)-anchored protein appearing on the membranes of mitogen-stimulated B cells after progression into S phase of the cell cycle. Maximal APase expression occurs after peak proliferation and precedes maximal immunoglobulin (Ig) secretion. While APase is clearly an activation marker for mitogen-stimulated B cells, the physiologic role of APase in B cells has not been defined.

Expression of CD45 isoforms by Epstein-Barr virus-transformed human B lymphocytes.

CD45 is the most common protein tyrosine phosphatase (PTPase) in the membrane of white blood cells, serving as a potent regulator of lymphocyte activation and signal transduction. While the amino acid sequence of the intracellular domain of the molecule is conserved, that of the extracellular domain occurs in multiple isoforms, each of the result of alternative mRNA splicing. In T lymphocytes, the lowest relative molecular mass (Mr) form, CD45RO, is associated with acquisition of memory function, whereas the highest Mr isoform, CD45RA, occurs in "naive" T cells.

Alkaline phosphatase on activated B cells characterization of the expression of alkaline phosphatase on activated B cells. Kinetics and membrane anchor.

Recently we reported that the expression of the enzyme alkaline phosphatase (APase) is a marker for B cell activation. Enzymatic activity was found only in activated B cells and not T cells. Using flow cytometry we showed that some of the APase was found on the cell membranes (mAPase) and by functional assays, some was spontaneously released into the tissue culture medium.

Expression of the low Mr isoform of CD45 (CD45RO) in B-cell non-Hodgkin's lymphomas.

The CD45 antigen family consists of multiple molecular isoforms ranging from 180 to 220 relative molecular mass (Mr). The highest Mr isoforms are recognized by monoclonal antibodies (MoAbs) designated CD45RA, while those recognizing the low Mr isoform are designated CD45RO. About half of the T-cells in peripheral blood express CD45RA while the remainder express CD45RO.

Polyclonal activation of rat B cells. II. Dextran sulfate as a cofactor in mitogen-induced and antigen-induced differentiation of rat B lymphocytes.

Salmonella typhimurium mitogen (STM) is a polyclonal activator of rat B lymphocytes, triggering them to proliferate, but not differentiate, to antibody-secreting cells. When lymphokines in the form of a supernatant from Con A-stimulated splenocytes (CAS) are added to B cell cultures activated by STM, only a small number of cells are driven to differentiate. Only with the addition of a third signal provided by the polyanionic polysaccharide dextran sulfate (DXS) is significant rat B cell differentiation observed.

Late events in B cell activation. Expression of membrane alkaline phosphatase activity.

Alkaline phosphatase (APase) has been previously described as a membrane marker correlating with B cell proliferation after stimulation by selected B cell mitogens. We have found, however, that the appearance of B cell membrane APase correlates more closely with differentiation than with proliferation. This conclusion has been drawn from the following observations: 1) APase activity appears well after peak B cell thymidine uptake, 2) mitogens which stimulate only B cell proliferation (Salmonella typhimurium mitogen) fail to induce expression of the enzyme, and 3) when proliferation of mitogen-activated B cells is inhibited, APase activity is not suppressed and may even be augmented.

Lymphokine-induction of memory B-cell differentiation: differential stimulation of large virgin and memory B-cell differentiation.

In order to compare and contrast the requirements of virgin and memory B cells for B-cell differentiation factors, a model system was developed in which low-density rat B cells isolated from 4-week primed antigen-draining lymph nodes were cultured in vitro. This large low-density cell population contained B cells which were 90% surface IgM positive and 60% IgD positive and showed moderately elevated Ia staining. When the cell population was stimulated with antigen plus lymphokines or lymphokines alone, antigen-specific IgG antibody was secreted; this was used as a measure of memory cell differentiation.

Lymphokine regulation of surface Ia expression on rat B cells.

Class II major histocompatibility antigens (Ia) play a major role in regulating T-B cell interactions; therefore, regulation of the amount of Ia on B cells may be an important point of control in the immune response. Mitogens in human and murine systems have been reported to increase Ia expression on B cells, and in the mouse the lymphokine BSF-1 (IL-4) markedly enhances Ia expression. This report describes studies of lymphokine and mitogen regulation of class II expression on rat B cells.

An alternative method of panning for rat B lymphocytes.

A new method of panning for B lymphocytes is described in which the ability of the sIg+ cells to adhere depends on the nature and concentration of nonspecific protein used rather than on the use of anti-immunoglobulin. Rat lymph node cells were suspended in 3% bovine serum albumin in Tris-buffered Hanks' and incubated in tissue culture flasks to allow adherence to the plastic. The recovered bed of adherent cells was shown by flow cytometry to be greater than 90% surface immunoglobulin positive and MHC class II positive while containing very few T cells.

Polyclonal activation of rat B cells. I. A single mitogenic signal can stimulate proliferation, but three signals are required for differentiation.

A water-soluble, proteinaceous preparation derived from the cell walls of Salmonella typhimurium Re mutants has recently been tested in our laboratory for its ability to act as a mitogen for rat lymphocytes. We have found this preparation (STM) to be a potent stimulator of B lymphocyte proliferation, as measured both by 3H-TdR incorporation and by cell cycle analysis performed with flow cytofluorometry. STM stimulates approximately 50% of rat B cells to enter cycle.

Polyclonal activation of primed rat B cells.

In recent years, murine and human virgin B lymphocytes have been used to examine the steps necessary for polyclonal activation. In these models mitogens are used in conjunction with lymphokines to determine which signals are responsible for regulating B-cell triggering, proliferation, and differentiation. While progress has been made in understanding these events as they occur in virgin B cells, very little evidence exists to suggest whether these models of activation also apply to the memory B-cell population.

Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation.

Lymphocyte proliferation and differentiation result from ordered cellular interactions governed by soluble products (lymphokines). Dissecting the individual steps in these processes has been difficult, due to a paucity of pure lymphokines. Recently, it was reported that the derivatized ribonucleoside 8-mercaptoguanosine (8MGuo) has both mitogenic and differentiative effects on murine B cells.

Role of complement in the immune response.

Evidence has accumulated that shows that fragments of C3 are potent inhibitors of immune responses. A low-molecular-weight fragment of C3 and fragments possessing leukocyte-mobilizing activity have been shown to block both antigen- and mitogen-induced human T cell proliferation, and to block mixed lymphocyte culture responses and the generation of cytotoxic lymphocytes. The same fragments inhibit the development of secondary in vitro antibody responses of rat lymphocytes.

Complement inhibits immune responses: C3 preparations inhibit the generation of human cytotoxic T lymphocytes.

C3 fragments have been shown to inhibit mitogen- and antigen-induced human lymphocyte blastogenesis. In this study, C3 preparations and a small fragment of C3 (contained in a preparation called Fraction 2) were examined for their capacity to regulate cytotoxic T lymphocytes (CTL) and proliferative mixed lymphocyte responses (MLR). We found that both preparations inhibited the generation of allogeneic human CTL as well as MLR in a dose-related manner.

The correlation between the activation state of B cells and their capacity for in vitro propagation of immunologic memory.

The B-cell population responsible for in vitro antigen-mediated proliferation and expansion of the memory B-cell population is a large activated blast. Such cells predominate early after antigen priming and can be regenerated by adjuvant (Bordetella pertussis) stimulation in vivo. Although these cells are proliferating in vivo, additional stimuli are needed for expansion of the memory population in vitro.

Complement fragments suppress lymphocyte immune responses.

There has been controversy as to whether complement augments immune responses or inhibits them. In this article John Weiler and his colleagues discuss recent evidence that complement fragments can inhibit immune responses that depend upon cellular proliferation.

Inhibition of human lymphocyte blastogenesis by C3: the role of serum in the tissue culture medium.

Preparations of the third component of human complement (C3) inhibit human lymphocyte blastogenic response to mitogens and antigens when cultured in serum-free medium or in medium supplemented with 5% autologous serum (AS). In contrast, when the culture medium was supplemented with 5% foetal calf serum (FCS), C3 failed to inhibit responses to mitogen (concanavalin A) or to antigen (streptolysin O); some FCS lots allowed stimulation rather than inhibition of the lymphocyte responses. Moreover, when lymphocytes were cultured in serum containing equal amounts of FCS and AS, no inhibition was seen.

Inhibition of secondary in vitro antibody responses by the third component of complement.

Purified human C3 was found to inhibit rat in vitro secondary antibody responses. Fifty percent inhibition of antibody-forming cell development occurred with C3 concentrations of 26 micrograms/ml. This decrease was not the result of a general toxicity or a shift in the antibody response kinetics.

In vitro antigen-mediated clonal expansion of memory B lymphocytes.

An in vitro model for the propagation and expansion of the memory B lymphocyte population is described. DNP-BGG immune cells were mixed with OVA immune cells and challenged immediately with DNP-OVA. After the 1st response had begun to wane, the cells were rechallenged with DNP-OVA (day 11 of culture).

Role of T cells in the development of memory B cells. Quantitative and qualitative analysis.

The purpose of this investigation was to address the current controversy regarding the T-cell requirement for the generation of B-memory cells. We have circumvented the possible objection to previous experiments regarding residual T cells in T-deprived animals by examining memory cell generation in relation to the numbers of T cells participating in the immune response. Thymectomized and lethally-irradiated rats were reconstituted with foetal liver or a more mature stem cell source, neonatal liver.

The third component of complement inhibits human lymphocyte blastogenesis.

Purified human C3 was studied for its ability to regulate human peripheral blood lymphocyte activation in a serum-free tissue culture system. C3 and its fragments formed by trypsin digestion were not mitogenic. However, C3 inhibited both mitogen- and antigen-induced lymphocyte proliferation in a dose-related manner.

The effect of growth hormone and insulin upon MLC responses and the generation of cytotoxic lymphocytes.

The ability of growth hormone (GH) and insulin to influence positively T lymphocytes responding to an alloantigen stimulus in vitro was analyzed through the use of a serum substitute system. The presence of insulin but not GH, enables the generation of a successful mixed lymphocyte culture (MLC) blastogenic response. However, the presence of GH during a 5-day MLC allowed for the generation of cytotoxic T lymphocytes (CTL).

Immunologic aspects of the prostate.

Our experiments involving the use of the Dunning R3327 adenocarcinoma as an animal model of prostatic cancer as well as clinical studies on the immunocompetence of prostatic cancer patients are described. Utilizing the Dunning tumor, we have demonstrated that this transplantable adenocarcinoma of the rat prostate was similar to human prostatic cancer with respect to its macroscopic and microscopic appearances, growth rate, growth differential in male and female recipients, and some of its metastatic potential. Cryosurgery was capable of destroying the primary tumor as it can in man.