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A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chapter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts into the field. Other chapters in this book will provide more details, more references, and even an intriguing view of the future of cytometry.

A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8(+) cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8(+) lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods. We have monitored tumor specific CD8(+) as well as CD4(+) lymphocyte precursor frequencies in the peripheral blood using a Dye Dilution Proliferation Assay (DDPA). We summarize our experience applying DDPA in a multi-parameter, antigen-specific assay, detailing some of its complexities and advantages. We provide examples of our clinical trial results showing tumor-specific CD8(+) and CD4(+) precursor frequency (PF) data in patients being treated on novel immunotherapy trials.

Cell-tracking dyes stain cells with bright fluorescence which is partitioned between daughter cells after each cell division, so that the daughter cells have closely half the intensity of the parent cells from which they were derived. Therefore, the intensity of a cell, relative to its intensity at the time of staining, provides information about how many divisions have occurred. Knowing the number of division cycles that have occurred, one can calculate the number of cells in the original population (before culture) that were going to go on to proliferate. In this way, cell-tracking dyes provide a flow cytometric method for determining the proportion of cells in a population that will go on to proliferate in response to stimulation. This dye-dilution methodology can, therefore, detect proliferation of rare antigen-specific cells that increase in number during division and, importantly, can be used to back-calculate the precursor frequency of these rare cells. Simultaneously, the phenotype of these cells can be determined, as well as their ability to synthesize cytokines, and to express or not relevant antigen-binding receptors and activation markers.

The T-cell receptor provides T cells with specificity for antigens of particular molecular structure (the "epitope"); the T-cell pool in an individual responds to the presence of many different antigenic epitopes, but any particular T cell will respond preferentially to one defined epitope. After stimulation of a T cell by the binding of its receptor to its cognate antigen in the context of a major histocompatibility complex (MHC) molecule on an antigen-presenting cell, the T cell will begin to proliferate and synthesize cytokines. Tetramer binding and the enzyme-linked immunospot (ELISPOT) method have been used to determine what proportion of cells in the T-cell pool can bind to a defined antigenic peptide or will secrete cytokines in response to a particular antigenic stimulation. The method described here uses tracking dyes to determine what proportion of T cells will proliferate in response to stimulation. As a flow cytometric "single-cell" method, it can be combined with tetramer and cytokine staining to determine the precursor frequencies of cells in the T-cell pool able to bind tetramer, to synthesize cytokines, and to proliferate in response to antigen.

A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chapter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts into the field. Other chapters in this book will provide more details, more references, and even some controversy about specific topics.

Triggering of the T-cell receptor by cognate antigen induces a variety of cellular events leading to cell proliferation and differentiation. While the plasticity and diversity of T-cell responses have been recognized for a long time, few quantitative studies have been conducted to measure what proportion of specific T cells will enter a given differentiation program after antigen stimulation. In the present study, we analyzed human T cells cultured with influenza-peptide-loaded dendritic cells. We compared three individual methods for assaying the frequency of antigen-specific T cells: ELISPOT, tetramer-binding, and proliferation. The three methods yielded similar but not identical results. In order to study these differences at the single cell level, we developed a multiparameter flow cytometric method, which allows simultaneous analysis of antigen-specific tetramer binding, T-cell proliferation, and cytokine production. Based on these data, we used flow precursor frequency analysis to calculate the proportion of eight different precursor subsets in the original, resting population. We conclude that approximately half of the cells that bound specific tetramers actually proliferated and synthesized IFNgamma in response to antigen. In addition, similar numbers of cells that did not bind tetramer proliferated (but did not synthesize IFNgamma). The method allows for an estimate of the precursor frequency of each functional subset within the initial population. It could be applied to additional markers of function and differentiation, combining all parameters into a description of the complex response potential of a T-cell pool.

CD163, a monocyte and macrophage-specific surface glycoprotein, which is increased by interleukin-10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia-associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI-0 indicate that a metalloproteinase is responsible for LPS-mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.

Unlabelled: We previously reported that a continuous insulin infusion improves neutrophil phagocytic function after cardiac surgery in diabetic patients. These data suggested that hyperglycemia impairs neutrophil function, and because nondiabetic patients also experience hyperglycemia during cardiac surgery, we hypothesized that a continuous insulin infusion would improve glucose control and neutrophil function in nondiabetic cardiac surgical patients. Patients were randomized to receive either no insulin (Control group) or a continuous insulin infusion (Insulin group), with glucose measurements every 10 min during cardiopulmonary bypass (CPB). Blood glucose was significantly lower in the Insulin group immediately after surgery but not during surgery. When assessed as the percentage of phagocytic cells, neutrophil function was similar in the Control and Insulin groups at baseline (55% and 57%, respectively) and after CPB (38% and 43%, respectively). However, a quantitative determination of neutrophil phagocytic activity showed that whole blood neutrophil phagocytic capacity increased significantly in both groups at 60 min after CPB when compared with their respective baseline values and that the increase in total neutrophil phagocytic capacity was significantly more in the Insulin group compared with the Control group (P = 0.036). This observation was primarily due to a larger increase in the peripheral blood neutrophil count and not to increased activation of neutrophils., Implications: IV insulin, as used in this study, had effects on blood glucose only after cardiac surgery, when it was associated with an increased neutrophil count and a greater total capacity of peripheral blood neutrophils to ingest foreign particles.

Studies from our laboratory and others have established that both mononuclear phagocytes and neutrophils mediate very efficient cytotoxicity when targeted through Fc receptors using a suitable monoclonal or bispecific antibody (BsAb). Cross-linking an Fc receptor for IgG (FcgammaR) triggers multiple anti-tumor activities including superoxide generation, cytokine and enzyme release, phagocytosis and antibody-dependent cellular cytotoxicity (ADCC). In this report, using unfractionated leukocytes and two color flow cytometric analysis, we describe the phagocytic capacity of peripheral blood polymorphonuclear cells (PMN) and monocytes isolated from patients enrolled in a phase I clinical trial of MDX-H210 given in combination with IFNgamma. MDX-H210 is a BsAb targeting the myeloid trigger molecule FcgammaRI and the HER-2/neu proto-oncogene product overexpressed on a variety of adenocarcinomas. In this trial, cohorts of patients received escalating doses of MDX-H210 3 times per week for 3 weeks. Interferon-gamma (IFNgamma) was given 24 h prior to each BsAb infusion. Our results demonstrate that monocytes from these patients were inherently capable of phagocytosing the HER-2/neu positive SK-BR-3 cell line and that addition of MDX-H210 into the assay significantly enhanced the number of targets phagocytosed. Two days after administration of an immunologically active dose of MDX-H210 (10 mg/m2), monocytes from these patients were able to phagocytose greater amounts of target cell material, indicating that these cells remained armed with functionally sufficient BsAb for at least 48 h. PMN from these patients very efficiently mediated phagocytosis through FcgammaRI after being treated with IFNgamma, but not before. We conclude that phagocytosis is not only an efficient mechanism of myeloid cell-mediated cytotoxicity, but may also be a mechanism by which antigens from phagocytosed cells can enter a professional antigen presenting cell for processing and presentation.

Background: Most assays used to measure invasion are based on manual counting of the number of cells that have migrated completely through commercial coated filters. We describe here a confocal fluorescence-imaging method that can assess the relative rates of invasion of cells into a matrix., Methods: After being seeded on the matrix and a period of incubation, the cells are fixed and treated with RNase. Propidium iodide is then added to stain the double-stranded DNA. A confocal microscope system is used to obtain high-resolution images of the red propidium iodide fluorescence and laser reflectance from optical sections at increasing depths in the matrix. The section with high laser reflectance marks the top of the matrix., Results: Data were calculated as the total area of red fluorescence above background in each section and were plotted as a percentage of the summed fluorescent areas in all sections., Conclusions: Because the distance into the matrix of the nuclei can be calculated by measuring from the reflective upper surface of the matrix, the method is useful for assessing the rate of cell migration and for comparing the ability of different cells to invade through different matrices under varying conditions., (Copyright 2000 Wiley-Liss, Inc.)

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.

Matrix metalloproteinases (MMPs) play an important role in tumor cell invasion and metastasis. These processes require the dissolution of the basement membrane and invasion of the stromal matrix (ECM), and are mediated by MMPs. Consequently, MMP inhibitors may be attractive as new anticancer agents. To examine the potential contribution of collagenase-1 (MMP-1) in invasion of stromal matrix, we used the highly invasive and metastatic breast cancer cell line MDA-MB-231 as a model system. These cells express procollagenase-1 constitutively and this expression can be repressed by all-trans retinoic acid. Invasion of these cells into a collagen type I matrix was assessed by scanning electron microscopy (SEM), and was quantitated with a computer program and confocal laser scanning microscopy (CLSM). We found that MDA-MB-231 cells freely invaded the collagen type I matrix, suggesting that these cells possess a mechanism for activating the latent collagenase-1. In contrast, down-regulation of collagenase-1 expression by all-trans retinoic acid caused these cells to become less invasive. To confirm a role for collagenase-1 in mediating collagen type I invasion, assays were carried out in the presence of FN-439, an inhibitor of collagenase-1 enzyme activity. We found that in the presence of the proteinase inhibitor, invasion of type I collagen by MDA-MB-231 cells was also reduced. These results indicate that collagenase-1 produced by the breast tumor cells may enhance stromal matrix degradation by enabling the tumor cells to modulate their own invasive behavior, and suggest that decreasing collagenase-1 levels may be effective in breast cancer therapy.

A nondestructive fluorescence-imaging assay is described for quantitating the number and size of plaques formed over time by cytotoxic effector cells in a monolayer of target cells. It can also be used to assay the growth of adherent cells toward confluence. The method involves the use of fluorescein conjugated to high molecular weight dextran. The dextran is excluded by adherent cells, thereby making the medium around cells more fluorescent than the cells themselves. The area of the plate that is fluorescent can be determined by confocal fluorescence imaging microscopy. With this new method, changes in the confluency of adherent cells or in the number and area of cytotoxic plaques can be assayed repeatedly over an extended period of time, without manipulation of the cells or of the medium.

Objective: To quantify the numbers of leukocytes in the normal cervix and relate these numbers to the diagnosis of cervicitis., Methods: Isolated cell suspensions were prepared from cervical tissue recovered at hysterectomy from 37 women who had no obvious cervical disease. The percentages of CD45+ cells (leukocytes) in these preparations were determined using immunofluorescence-based flow cytometric analysis. These percentages were compared with the pathologist's assessment of cervicitis., Results: Leukocytes were present in all cervical samples tested. For endocervical samples, the mean (+/- standard error of the mean [SEM]) percentage of CD45+ cells was 12.4 +/- 1.9% of cells in patients with a diagnosis of cervicitis (n = 16) and 9.1 +/- 1.1% in patients without cervicitis (n = 17). For ectocervical samples, the mean (+/- SEM) percentage was 14.8 +/- 3.0% in those with cervicitis (n = 16) and 9.5 +/- 1.6% in those without cervicitis (n = 19). The differences between samples from patients with cervicitis and those without cervicitis were not statistically significant at the .05 level. Intra- and interassay variabilities were 5.7 +/- 1.2% and 7.3 +/- 1.6%, respectively., Conclusion: Our study demonstrates there is a resident population of leukocytes in the cervix. Leukocyte number did not relate clearly and consistently to the diagnosis of cervicitis made by the pathologist. We suggest that the resident population of leukocytes, in the absence of other indicators of infection, may confuse determinations of cervicitis.

Problem: The tissues of the human female reproductive tract (Fallopian tube, uterus, cervix, and vagina) may play different roles in the provision of mucosal immunity. The purpose of this study was to develop a uniform method suitable for quantitative comparison of the leukocytes from all these tissues., Method of Study: Tissues, typically 0.5-1.0 g, were dispersed by enzyme treatment. A flow cytometric gating procedure based on CD45-positivity and low far-red autofluorescence permitted unfractionated, freshly dispersed cells to be phenotyped with respect to T lymphocytes, B lymphocytes, macrophages, and granulocytes., Results: Reproductive tract tissues contain leukocytes that represent approximately 6-20% of the total number of cells, with the Fallopian tubes and uterus containing a higher proportion of leukocytes than the cervix and vagina. The uterine endometrium from post-menopausal women has fewer leukocytes than does uterine endometrium from pre-menopausal women. T lymphocytes are a major constituent (30-60%) of leukocytes from all tissues. The Fallopian tube contains granulocytes as another major constituent; granulocytes are significantly less numerous in the other tissues. All tissues contain B lymphocytes and macrophages as clearly detectable but minor components., Conclusions: Three-color flow cytometry is an appropriate method for quantitative comparison of leukocytes from the different tissues of the female reproductive tract, during all phases of the menstrual cycle and within post-menopausal samples. Results indicate that the tissues differ from each other, particularly with respect to the large number of granulocytes in the Fallopian tubes.

The human female reproductive tract (RT) has been analyzed by others with respect to NK cell cytolytic activity, but not CD3+ T cell (CTL) cytolytic activity. Here, we describe the cytolytic capacity of mucosal CD3+ T cells both longitudinally within the RT (Fallopian tube, uterine endometrium, endocervix, ectocervix, and vaginal mucosa) and temporally throughout the menstrual cycle, using a redirected lysis assay system. Cytolysis by CD3+ CD8+ T cells is found throughout the RT and appears to be hormonally regulated, since in the uterine endometrium, the capacity for CD3+ T cell cytolytic activity is present during the proliferative phase of the menstrual cycle and absent during the subsequent secretory (postovulatory) phase. In contrast, in postmenopausal women the entire RT, including the uterus, retains the capacity for strong CD3+ T cell cytolytic activity. These findings suggest that the high levels of estradiol and progesterone present during days 14 to 28 of the menstrual cycle down-regulate CTL activity in the uterus. As a consequence, the absence of this activity may allow implantation of a semiallogeneic embryo that would otherwise be rejected. Further, these studies indicate that CTL activity is regulated differentially in different regions of the RT, persisting in the cervix and vagina throughout the menstrual cycle.

Problem: To investigate the mucosal immune system in the cervix and vagina of premenopausal women in terms of immune cells present and cytolytic capacity of mucosal CD3+ T cells in the lower reproductive tract., Methods: Fresh tissue fragments prepared by vibratome sectioning were analyzed for the presence of cells by confocal scanning laser microscopy (CSLM). Isolated reproductive tract cells prepared by enzymatic were analyzed for CD3+ T cell phenotype by FACS analysis and for cytolytic function by an anti-CD3 mAb mediated redirected lysis assay., Results: As determined by CSLM, CD3+ cells as well as macrophages and dendritic cells are distributed throughout the lower female reproductive tract in both the epithelium and subepithelial mucosa. It was found that cervical and vaginal tissues from pre- and post-menopausal women contain CD3+ T cells (CTL) that have cytolytic activity, when measured in an antigen non-specific anti-CD3 mAb mediated redirected lysis assay., Conclusions: These results indicate that the lower reproductive tract of women is immuno-competent as judged by the presence of CD3, CD4, CD8, macrophage, and dendritic cells in the endocervix, ectocervix, and vagina of premenopausal and postmenopausal women. Further, these studies demonstrate that CD3+ T cells with cytolytic activity are present in the cervix and vagina during the proliferative and secretory phases of the menstrual cycle and following menopause.

Several previous studies, including our own, have indicated that flow cytometric assays can identify an at-risk population of kidney transplant recipients. We used the assay for recipient selection for a period of twelve months. Recipients with donor T cell-directed IgG were excluded from transplantation and those with B cell-directed IgG were treated with increased immunosuppression. The transplants performed over this period (n = 126) were compared with an earlier series (n = 118) where, although the flow cytometric crossmatches were performed, the results did not influence patient management. In the series where the flow cytometric crossmatch was used in management, a lower failure rate was found at three months (P = 0.037 chi square), primary non-function was reduced (P less than 0.0001, Mann-Whitney), rejection episodes were reduced (P less than 0.0001, Mann-Whitney) and the hospital stay was shorter (P less than 0.0001, Student's t). The risk factors of ischemic times, panel reactivity, exposure to previous grafts and A/B locus matching were identical between the two groups. However DR matching was found to be higher in the series with the improved results (P less than 0.0001, Mann-Whitney). In view of the significant improvement in graft success and low complication rate, we intend to continue with the policy of recipient selection by flow cytometric crossmatching and DR matching.

Previous studies have shown that flow cytometric crossmatch assays can identify an at risk population in renal transplantation. We used the assay for recipient selection for 1 year. Recipients with donor T cell directed IgG were excluded from transplantation and those with B cell directed IgG were treated with increased immunosuppression. The transplants performed over this period (n = 126) were compared with an earlier series (n = 118) in which flow cytometric crossmatch results did not influence patient management. The results were evaluated for mortality and graft outcome at 3 months and 1 year. In addition, postoperative complications and duration of hospital stay were also assessed.

A novel in vitro assay of renal epithelium tight junction function was used to assess the efficacy with which rabbit anti-thymocyte globulin (ATG) blocks epithelium damage mediated by lymphokine-activated killer (LAK) cells. It was found that LAK cells lysed renal epithelial cells poorly in standard chromium-release assays but that they caused a rapid, and almost total, reduction in trans-epithelium monolayer resistance, indicating tight junction failure and, hence, loss of tissue function. LAK cell-mediated cytolysis of the sensitive K562 cell line was completely blocked in the presence of ATG at a concentration of 200 micrograms/ml. Addition of ATG at this concentration to damaged renal cell monolayers in the presence of LAK cells allowed the trans-monolayer resistance to recover rapidly to levels approaching the values recorded before initial addition of LAK cells. On this basis it seems likely that the rapid restoration of renal function frequently observed after appropriate "rescue" therapy during episodes of acute rejection may reflect subtle changes in tissue function rather than recovery from widespread graft cell cytolysis.

The DNA content of breast tumours from 170 patients who presented between 1978 and 1980 was measured by flow cytometry. The relationship between tumour ploidy and disease outcome was assessed and its association with other prognostic factors evaluated. Compared with those with diploid tumours, patients with aneuploid tumours had significantly earlier relapse and shorter survival (P less than 0.0001). Tumour ploidy was strongly related to grade (P less than 0.001), but there was no significant association between DNA ploidy and c-erb-B-2 expression, lymph node status or tumour size. In lymph node negative and c-erb-B-2 negative patients, aneuploid tumours were associated with a poorer prognosis (P less than 0.001) than diploid tumours. Multivariate analysis showed that tumour ploidy gave independent information on disease free and overall survival. Tumour ploidy may be used as an independent prognostic variable in patients with breast cancer and it may be helpful in defining patients within the node negative or c-erb-B-2 negative groups likely to have a poor outcome who might benefit from adjuvant treatment.

Human umbilical vein endothelial cells (HUVEC) were cultured and treated for varying periods with a range of concentrations of tumour necrosis factor-alpha (TNF-alpha). After this treatment the proportion of peripheral blood mononuclear cells (PBMC), previously depleted of plastic adherent cells, capable of binding to the endothelial cells was assessed. Few PBMC bound to HUVEC which had not been pretreated with TNF-alpha but up to 36% bound after pretreatment of the endothelial cells with TNF-alpha for 10 hr at a concentration of 10 U/ml. Phenotypic characterization of the adherent and non-adherent PBMC subpopulations revealed that natural killer (NK) cells (CD16+) and a proportion of memory helper T cells (CD4+ CD45RA-) bound to TNF-alpha pretreated HUVEC but that few naive helper T cells (CD4+ CD45RA+) showed similar binding. Cytotoxicity assays for NK activity were used to analyse functionally the adherent and non-adherent PBMC subpopulations. It was found that the cell subpopulation which did not adhere to TNF-alpha pretreated HUVEC mediated little lysis of K562 target cells. Conversely, the endothelial cell-adherent PBMC subpopulation produced active lysis supporting the phenotypic evidence that NK cells were concentrated within this subpopulation. These results suggest that TNF-alpha has a rapid and profound up-regulatory effect on the expression of adhesion molecules on the surface of HUVEC. Furthermore, it is apparent that these up-regulated adhesion molecules preferentially bind NK cells and a subset of memory helper T cells from the PBMC population.

To test the hypothesis that major histocompatibility complex (MHC) molecules protect target cells from lysis by natural killer cells (NKC), we transfected the MHC- B16 melanoma line F10 with the class I genes encoding Dd, Kb, and Kk. Only low levels of Dd expression could be obtained and there was no protection against NKC. By contrast, Kb and Kk transfectants were obtained which displayed significant resistance to NKC, and with the latter transfectants resistance was clearly related to the level of transgene expression. Various mutants of the F10 line with altered patterns of MHC expression were also obtained. These mutant lines provided evidence that (i) the Db molecule is also capable of inducing resistance to NKC and (ii) high MHC class I expression does not by itself guarantee lowered susceptibility to NKC.

When antigen density on the surface of a cell population is low and variable, the percentage of that population determined to express the antigen (i.e., to be positively stained) depends directly on the sensitivity of the flow cytometer for resolving particles which are dimly fluorescent from those which are unstained. In this study, the sensitivity of a commercial flow cytometer has been improved by changes in the photomultiplier tube, the fluorescence filter, and the amount of stray light entering the fluorescence channel. In a model system with human lymphocytes, modifications to these factors increased the percent of the B-lymphocyte population found to express the CD5 antigen.

The ability of lymphokines to affect the development and differentiation of mouse thymocytes in vitro was evaluated in a carefully controlled 3-day organ culture system. Concanavalin A (Con A)-induced supernatant (SN) from the T-cell clone D10.G4, which contains high concentrations of interleukin-3 (IL-3), IL-4 and IL-5, but lacks IL-1, IL-2 and interferon (IFN), markedly increased the proportion of CD4+CD8- cells, and decreased the proportion of CD4+CD8+ cells. These effects were unaffected by dialysing the SN, showing them to be caused by macromolecular factors. Highly purified recombinant IL-3 and IL-4 could exert similar effects, rIL-3 and rIL-4 both increasing the proportion of CD4+CD8- cells, and rIL4 in addition reducing the proportion of CD4+CD8+ cells. In conjunction with the findings of other investigators, these results indicate that at least four lymphokines (IL-1, IL-2, IL-3 and IL-4) can control T-cell development in the thymus.

We have demonstrated that serum from appropriately sensitized patients can contain IgG antibodies that bind to cultured renal epithelial cells. The presence of such antibodies on the surface of renal cells enables otherwise nonlytic PBMC to lyse these renal cells by an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. Experiments involving cell-sorting and specific complement-mediated lysis showed that the ADCC effector cells were of the CD3 -ve, C16 +ve phenotype characteristic of NK cells. In this report it is argued that an ADCC mechanism may be of importance in mediating chronic renal cell damage in the absence of acute allograft rejection.

Lymphocyte function and cell surface phenotype were examined in fifteen patients with late onset hypogammaglobulinaemia. The percentage of surface immunoglobulin-positive B cells in fourteen of the fifteen patients was in the normal range. Patients' B cells expressed MHC class II antigens at normal levels. For one patient, there was relatively high sIgD and low sIgM expression on B cells; the rest of the patients did not differ from controls in surface immunoglobulin density. The proportion of B cells positive for CD5 in patients was comparable to normal controls, and considerably less than in cord blood. However, the pattern of immunoglobulin isotype secretion in vitro by patients' B cells closely paralleled responses of cord blood B cells. Spontaneous secretion of IgM and IgG by patients' B cells was very low. Following polyclonal activation in the presence of autologous T cells, cells from thirteen patients secreted IgM within the normal range in response to at least one activator. The response of patients' purified B cells to IL-2 and gamma-IFN was variable. For four of six tested, B cells cultured with IL-2 and gamma-IFN together with polyclonal activators secreted normal levels of IgM. B cells from the other two patients secreted little or no IgM in response to these cytokines. For fourteen patients, IgG secretion following polyclonal activation remained low both when B cells were cultured with T cells or with a combination of IL-2 and gamma-IFN. IgG subclass imbalance was seen in one patient, whose cells secreted an unusually high proportion of IgG3, and undetectable IgG2 and IgG4; this pattern was consistent whether T cell help was provided by autologous or allogeneic T cells. Similarly purified B cells from this patient showed deficient IgG2 and IgG4 production in response to IL-2 and gamma-IFN.

In many transplant centers, the T lymphocytes of transplant patients are routinely monitored, both to predict and diagnose the cellular events that result from transplantation and to evaluate the effectiveness of immunosuppressive therapy. During the course of this monitoring, we have observed anomalous results: the number of T cells said to be present varies greatly depending on the surface marker used for the assay. It is shown, in this study, that these "anomalous" results can be predicted from a knowledge of the spectrum of specificity of the anti-lymphocyte antibody therapy being administered. Because polyclonal antibodies have varied and unstated specificities, it is difficult to interpret results obtained from monitoring T-cell numbers or subset ratios without some knowledge of the specificities of the drugs being used.

The ac-20 mutant strain of the unicellular green alga, Chlamydomonas reinhardii, lacks both chloroplast ribosomes and ribulose bisphosphate carboxylase activity when grown on organic medium. Under these conditions, the cells do not posses pools of either the large or small subunit of this enzyme. When transferred to inorganic medium, the carboxylase activity recovers. During this recovery, de novo synthesis of both subunits occurs. Synthesis of both subunits is inhibited by chloramphenicol even when possible free subunit pools rather than just the subunits incorporated into whole enzyme are examined.

Lymphocytotoxic immunoglobulin is routinely assayed before human renal transplantation. If IgG directed against donor T cells is detected in the serum of the potential recipient, transplantation is not performed as it is associated with a poor graft outcome. Poor sensitivity of the conventional assay has been postulated as being the cause of some graft failures. Two new flow cytometric assays are described which are more sensitive than the conventional test. The first assay requires manual separation of T and B lymphocytes and therefore takes a similar time to perform as the conventional assay. The second assay utilises a two-colour system and lymphocyte's separation is by fluorescence. This assay takes half the time to perform, thereby decreasing graft ischaemic time before transplantation.

Efficient adsorption of bacteriophages SP 50 and phi 25 occurred only to bacilli that contained wall teichoic acid and neither phage bound to phosphate limited bacilli that contained teichuronic acid instead of teichoic acid. Though both phages require the presence of teichoic acid, their receptors are not identical. Efficient binding of phage phi 25 required the presence of greater proportions of teichoic acid in the wall and the receptor for this phage was destroyed when bacteria or isolated walls were heated at pH 4 whereas the ability of these samples to bind phage SP 50 was unaffected by such treatment. Efficient binding of phage SP 50 was not highly dependent on the presence of glucosyl substituents on the teichoic acid. Such substituents were required for phage phi 25 binding though their anomeric configuration appeared to be unimportant since the phages bound well to both strains W23 and 168, the wall teichoic acids of which carry glucosyl substituents of opposite anomeric configuration. The differences in the nature of the receptors may be of value in the use of the phages as probes for the location and distribution of teichoic acid in the wall.

Chloramphenicol at 50 and 100 μg/ml inhibited synthesis of ribulose diphosphate carboxylase in the ac-20 mutant strain of Chlamydomonas reinhardii. Cycloheximide at 0.5 and 1.0 μg/ml significantly stimulated synthesis of this enzyme. By comparison, chloramphenicol had no effect on induction of isocitrate lyase by acetate, and cycloheximide completely inhibited this induction. The data suggest either that the carboxylase is made completely on 70S ribosomes or, less likely, that pools of subunit made on 80S ribosomes exist with long life times in the C. reinhardii cell.

While the importance of the standard preoperative crossmatch in predicting renal graft success is accepted, a more rapid and sensitive assay may be of additional clinical benefit. We have developed a flow cytometric assay to detect the presence of antibodies (IgG) in the recipient sera directed against donor lymphocytes, prior to transplantation. This assay is more rapid and sensitive than the conventional cytotoxic test. In a clinical study the sera of 75 renal graft recipients were tested, all of which were negative in their conventional crossmatch; 12 of these were identified as having T cell-directed IgG, and 4 had B cell antibody. Graft failure was not significantly different in the positive and negative antibody groups, as defined by flow cytometry (P = 0.147, chi square test). The incidence of postoperative complications was studied in the 60 grafts functioning at three months. Recipients with donor B or T cell directed antibodies had a longer primary nonfunction (P = 0.0098, Mann-Whitney U test), and showed a higher number of rejection episodes (P = 0.014, Mann-Whitney U test); accordingly they were more likely to require strong immunosuppressive agents such as OKT3 or ATG (P less than 0.05, chi square test). Patients with donor-directed antibodies were also hospitalised for a longer period (P = 0.015, Mann-Whitney U test) and had a higher creatinine level 3 months after transplantation (P = 0.021 Mann-Whitney U test). This study shows that the described preoperative flow cytometric crossmatch is capable of defining a population of renal transplants who form an at-risk group. Thus this assay has considerable potential in pretransplant matching of recipients with a particular graft donor.

A method for dual staining of mononuclear cells for lymphocyte phenotypic markers and DNA is described. The cells were stained with fluorescein-conjugated monoclonal antibodies and then rendered permeable to propidium iodide using saponin. Propidium iodide stains DNA and, using flow cytometry, cell cycle analysis of individual lymphocyte subpopulations can be determined. Saponin acts within 1 min, preserves expression of surface antigens and is effective at all concentrations from 0.001% to 1%. This technique is simple, rapid and gives reproducible results.

Flow cytometric measurements of nuclear DNA content often involve the calculation of a DNA index, which compares the DNA fluorescence from two different populations. Such DNA indices have been used to classify aneuploid peaks from tumour tissue into different categories relative to normal diploid cells. This report describes a correction based on the channel for unstained particles that is required if DNA index values are to give a true and reproducible indication of relative DNA content.

It is apparent that B cells are heterogeneous with respect to, for example, the antigens they express on their surface, and the stimuli to which they can respond. It is still unclear to what extent these differences relate to the stage of differentiation (eg. virgin B cells differing from activated B cells or memory cells), or whether distinct developmental lineages might exist. It has been proposed by some authors that, in the mouse, B cells expressing the ly-1 antigen constitute a separate lineage. In man also, a minor population of B cells expresses detectable levels of the CD5 antigen, but far less information is available about these cells. Interest in the CD5+ and ly-1+ B cell subpopulations has been further stimulated by the suggestion that these cells might play a special role in autoimmune disease. Although, in mouse, ly-1+ B cells differ in several respects from ly-1- B cells, the main evidence that they form a separate lineage derives from experiments in which ly-1+ B cells could not be reconstituted with adult bone marrow. It should be borne in mind that the situation is quite different in humans where, following bone marrow transplantation, CD5+ B cells are rapidly restored. Moreover, in the irradiated mice, at least in some of the experiments ly-1+ B cells were in fact reconstituted by adult bone marrow. Furthermore, at least in humans, expression of CD5 can sometimes be induced. There is, as yet, no good evidence that human CD5+ B cells form a distinct lineage, and it is possible that CD5 expression depends upon microenvironmental influences acting on the B cell during its differentiation. Several interesting properties have been attributed to ly-1+ B cells, including the ability to provide help to other B cells, and the secretion of autocrine factors. However there is also evidence that these features are not exclusive to B cells expressing ly-1. It has also been suggested that ly-1+ B cells might be long-lived. It is not yet known whether some of the properties of ly-1+ B cells might be a direct result of their expressing this antigen; this may become more clear when the function of CD5 is elucidated. The suggestion that the repertoire of ly-1+ B cells might be biased towards the expression of certain V genes is very interesting. Many of the hybridomas from neonatal mice produce antibodies which are multi-specific, and therefore well suited to form a first line of defence against potential pathogens.(ABSTRACT TRUNCATED AT 400 WORDS)

Whilst several centres have reported lymphocytotoxic antibody detection using single and dual fluorescent stains with analysis of the fluorescence emitted from the cell population present in a well of a multiwell plate, problems are encountered with cell concentration and light emission overlap. A method we have developed using flow cytometry produces similar values of percentage cell death using single or double staining techniques (correlation coefficient = 0.9896). This method is not influenced by slight variation in cell number or light emission overlap. The effect of introducing red cell impurities into the normal lymphocyte preparation is described.

Electron transport activity and absorbance changes associated with P700 were investigated in a mutant strain of Chlamydomonas reinhardi with impaired photosynthesis. This mutant strain, ac-8oa, cannot reduce NADP with electrons from either water or dye and ascorbate, but it has considerable Hill activity. The mutant strain shows none of the absorbance changes characteristic of P700. Although unable to carry out cyclic photosynthetic phosphorylation, ac-8oa is able to synthesize ATP when ferricyanide is provided as an electron acceptor. These observations lead to the conclusion that a site for the coupling of photosynthetic phosphorylation with electron transport must exist between the 2 photochemical systems.

Intact chloroplasts isolated from leaves of Vicia faba L. var. the Sutton show a decline in the endogenous level of alpha-ketoglutarate upon illumination. alpha-Ketoglutarate supplied to the chloroplasts is similarly utilized in this light-dependent reaction, and its consumption is paralleled by a concomitant increase in the level of glutamate. There is no photostimulation of glutamate synthesis in chloroplasts broken by osmotic shock, but it can be somewhat restored by addition of ferredoxin and NADP. These results suggest that in the isolated chloroplast the synthesis of glutamate from alpha-ketoglutarate is regulated by the availability of reduced pyridine nucleotide generated by photosynthetic electron transport. This conclusion is supported by the finding of an apparent competition between the photoreduction of phosphoglycerate to triose phosphate and the photoutilization of alpha-ketoglutarate.