Assessing the potential role of photopheresis in hematopoietic stem cell transplant.

The First International Symposium on Photopheresis in Hematopoietic Stem Cell Transplantation was held in Vienna, Austria with an educational grant from Therakos Inc. from 25 May to 27 May 2005. Three general issues were addressed: (1) pathophysiology of graft-versus-host disease (GvHD), (2) induction of immune tolerance and the immunology of phototherapy and (3) current standard treatment and prevention strategies of acute and chronic GvHD and the use of extracorporeal photopheresis (ECP). The objectives of the meeting were to open a dialogue among leading researchers in photobiology, immunology, and hematopoietic stem cell transplantation; foster discussions and suggestions for future studies of the mechanism of action of ECP in acute and chronic GvHD; and promote collaboration between basic scientists and clinicians. As can be seen from the summaries of the individual presentations, important advances have been made in our understanding of GvHD, including the use of photoimmunology interventions and the development of robust model systems. It is our expectation that data from photoimmunology studies can be used to generate hypotheses in animal models that can further define the mechanism of action of ECP and help translate the findings to clinical trials of ECP for the prophylaxis and treatment of both chronic and acute GvHD.

T-cells infiltrating renal cell carcinoma display a poor proliferative response even though they can produce interleukin 2 and express interleukin 2 receptors.

The fact that progressing tumors contain a significant infiltrate of T-cells brings into question the competency of the infiltrating T-lymphocytes (T-TIL). We have examined the role of the T-cell receptor/CD3 complex and/or the interleukin 2 receptor (IL2R) in responsiveness of T-cells that infiltrate human renal cell carcinoma. T-TIL display a poor proliferative response to interleukin 2 (IL2) alone, IL2 in combination with antibody to CD3, or mitogen stimulation. The proliferative unresponsiveness was not related to low expression of CD3 or IL2R beta as the percentage of T-cells expressing CD3 and IL2R beta were comparable in both T-TIL and peripheral blood T-cells obtained from the same patient. In contrast to the lack of proliferative activity, stimulation of T-TIL or peripheral blood lymphocytes with phytohemagglutinin or anti-CD3 resulted in comparable levels of both IL2 and gamma-interferon mRNA and protein expression. While levels of IL2R alpha were low in unstimulated T-TIL and peripheral blood lymphocytes, anti-CD3 antibody or IL2 were capable of inducing surface expression of this protein in both cell populations. IL2R alpha mRNA levels were comparable in T-cells from the tumor and peripheral blood although in some experiments both the percentage of IL2R alpha-positive cells and the density of surface expression per cell were reduced in T-TIL. This reduced IL2R alpha expression on T-TIL was not responsible for the proliferative unresponsiveness since T-TIL that expressed both IL2R alpha and/or IL2R beta still failed to respond to high doses of IL2. Thus T-TIL display a selective loss of response to at least two well defined extracellular stimuli. While T-TIL exhibit a poor proliferative response regardless of the form of stimulation these cells remain sensitive to both anti-CD3 and IL2 in terms of IL2 and gamma-interferon or IL2R alpha expression, respectively. The fact that proliferative unresponsiveness exists even though T-TIL can produce IL2 and express IL2R alpha/beta suggests that T-TIL have a selective loss of a common intracellular signaling pathway which is requisite to proliferation but not other aspects of response to antigenic stimulation.

A simplified method of CD34+ cell determination for peripheral blood progenitor cell transplantation and correlation with clinical engraftment.

Enumeration of CD34+ cells by flow cytometry is the recognized standard for quantitating progenitor cells for peripheral blood progenitor cell (PBPC) transplantation. Although many clinical studies have confirmed that the time to neutrophil and platelet engraftment is inversely proportional to the number of CD34+ cells infused, the minimum number of CD34+ cells necessary to acheive rapid engraftment has not been satisfactorily determined. The lack of a standardized method for quantitation of CD34+ cells by flow cytometry (FCM) is often cited as the reason for this ambiguity. This report describes an FCM method for CD34+ cell determination that is simple, highly reproducible, comparatively inexpensive, and validated by excellent correlation with clinical engraftment. Pheresis samples are stained and fixed within 4 hours of collection. Two hundred fifty thousand events are acquired as list mode data using a forward scatter threshold. The discrete CD34+ population is enumerated using a CD34-phycoerythrin FL2 vs. side scatter plot and Paint-A-Gate Pro software. The method was validated by excellent statistical correlation with clinical engraftment. Using this method, we determined the number of CD34+ progenitor cells necessary to achieve rapid engraftment to be 2 x 10(6)/kg.

Improved sensitivity and resolution in the flow cytometric DNA analysis of human solid tumor specimens. Use of in vitro fine-needle aspiration and uniform staining reagents.

Twenty-five unselected fresh colon or breast tumors were studied to identify specific components of sample preparation, sample staining, and flow cytometer operation affecting the sensitivity of DNA stemline analysis. Solid tumors were disaggregated using both a published method for mechanical/enzymatic whole cell (M/EWC) isolation and a fine-needle aspiration (FNA) technique. Staining FNA samples with CycleTEST propidium iodide reagents demonstrated improved sensitivity in the recognition of near diploid and near tetraploid aneuploid populations: 9 of 20 resolvable aneuploid DNA stemlines identified in FNA suspensions were not detected or clearly resolved in M/EWC preparations. These results suggest that previously reported discordances between flow and static image cytometry in the recognition of near tetraploid DNA stemlines may be related to inherent limitations of M/EWC. The in vitro FNA technique, when compared to M/EWC, may yield increased sensitivity and precision in clinical DNA stemline analysis when using fresh, unfixed, solid tumor specimens.

A comparative study of DNA quantitation in breast carcinoma with image cytometric analysis and in vitro fine-needle aspiration with flow cytometric analysis.

The DNA ploidy status of 53 fresh primary breast carcinomas was analyzed in a comparative study of flow cytometric (FCM) and image cytometric (ICM) analyses. Samples for FCM analysis were obtained with an in vitro fine-needle aspiration technique. Touch imprints from the same tumors were analyzed by three independent observers by ICM analysis. An ICM comparative study of "sequential" and "visually selected" nuclei also was performed. There was an overall concordance of 0.85 in the classification of diploid and nondiploid tumors by FCM and ICM analyses. In most discordant cases, a nondiploid population was identified by FCM analysis alone. This is attributed to the superior resolution of and sampling/preparatory method used for FCM analysis. There was an overall concordance of 0.91 in the classification of diploid and nondiploid tumors by ICM analysis. Selective analysis of atypical nuclei resulted in increased sensitivity in the detection of DNA aneuploid populations by ICM analysis.

A prospective comparison of DNA quantitation by image and flow cytometry.

Advances in computer and video technology suggest that image analysis may be practical method of measuring DNA that also allows visual confirmation of cell type. The purpose of this study was to prospectively compare DNA quantitation from 92 solid tumors in which DNA indices had been measured by image analysis of touch preparations (CAS 100) and flow cytometry of cell suspensions (FACScan). For 81 cases, there was excellent correlation between the two methods. For nine cases, however, an aneuploid population, usually near tetraploid, was identified by image but not by flow cytometry. Three cases had aneuploid peaks by flow cytometry that were not identified by image. Although these methods show good correlation, rare populations may be missed by CAS, presumably because of sampling errors in the touch preparation. Aneuploid populations may also be missed by flow cytometry, either because of cell loss during processing or because visual identification by image can increase sensitivity.

Host tumor infiltrating lymphocytes in B cell non-Hodgkin's lymphomas.

Tumor-infiltrating T lymphocytes (TIL-T) were quantitated by three color flow cytometry in cell suspensions from excisional biopsy specimens of 43 B cell non-Hodgkin's lymphomas (NHL) and 8 benign lymphoid hyperplasias (BLH) to identify potential differences in host T cell responses. We quantitated three TIL-T subsets: CD3+CD4+CD8- (helper-inducer), CD3+CD4-CD8+ (suppressor-cytotoxic) and CD3+CD25-HLADr+ (long term activated TIL-T) and compared them in three diagnostic groups: BLH, low grade B cell NHL (LG NHL) and intermediate-high grade B cell NHL (IG-HG NHL). The following results were obtained: Mean percentage +/- s.e. of activated TIL-T for BLH, LG-NHL and IG-HG NHL: 10.3 +/- 1.9, 23.2 +/- 4.6 and 38.8 +/- 9.5, respectively. Mean percentage +/- s.e. of suppressor-cytotoxic TIL-T for same groups: 13.9 +/- 1.5, 14.9 +/- 1.9 and 34.4 +/- 4.5, respectively. Mean percentage +/- s.e. of helper-inducer TIL-T cells for the same groups was 38.2 +/- 12.7, 32.1 +/- 7.2 and 22.5 +/- 4.6, respectively. Helper/suppressor ratio +/- s.e. for same groups was 3.0 +/- 1.1, 2.4 +/- 0.6 and 1.3 +/- 0.4, respectively. Activated and suppressor-cytotoxic TIL-T percentage progressively increased from BLH toward IG-HG NHL. The percentage of these two TIL-T subsets were significantly higher in IG-HG NHL than in BLH and LG-NHL (P < 0.0007, 0.0002, 0.0001 and 0.0260 for the comparisons TIL-T in BLH vs IG-HG NHL and LG-NHL vs IG-HG respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Memory T cell subsets in B cell non-Hodgkin's lymphomas.

Memory T cells were quantitated by three color flow cytometry in cell suspensions from biopsy specimens of 34 B cell non-Hodgkin's lymphomas (NHL) and 10 benign lymphoid hyperplasias (BLH). CD3+CD45R0-CD45RA+ (naive), CD3+CD45R0+CDRA- (memory) and total CD3+ T cells were compared in BLH, low grade (LG), intermediate (IG) and high grade (HG) B cell NHL. Mean percentage +/- s.e. of CD45R0 + T cells for BLH, LG, IG and HG NHL were: 14.7 +/- 3.8, 11.2 +/- 3.5, 28.9 +/- 7.5 and 45 +/- 15, respectively. Mean percentage +/- s.e. of CD45RA+ T cells were: 10.2 +/- 2.6, 7.1 +/- 2.3, 5.4 +/- 1.4 and 3.5 +/- 1.2, respectively. Mean percentage +/- s.e. of total CD3+ T cells were: 42.5 +/- 11, 22.4 +/- 7.5, 39.3 +/- 10.1 and 62.7 +/- 22.2, respectively. Memory and total T cells progressively increased from LG toward IG and HG NHL while naive T cells simultaneously decreased. Although the differences in naive T cells were non-significant, the differences in memory T cells were significant between LG and IG and LG and HG NHL (both p < .0100). We previously reported an increase in activated-cytotoxic T cells in IG -HG B cell NHL. We now report that this subset of lymphocytes has also acquired memory function. Future studies should determine if in vitro expansion and adoptive transfer of this subset of T cells results in tumor cytolysis.

The use of isotypic control antibodies in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets by flow cytometry. Are they really necessary?

OBJECTIVE: Isotypic control reagents are defined as irrelevant antibodies of the same immunoglobulin class as the relevant reagent antibody in a flow cytometry panel. The use of the isotypic control antibody has been advocated as a necessary quality control measure in analysis of flow cytometry. The purpose of this study was to determine the necessity of an isotypic control antibody in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets. MATERIALS AND METHODS: We performed a prospective study of 46 consecutive patient samples received for lymphocyte subset analysis to determine the need for the isotypic control. For each sample, a sham buffer (autocontrol) and isotypic control reagent were stained for three-color immunofluorescence, processed, and identically analyzed with Attractors software. The Attractors software allowed independent, multiparametric, simultaneous gating; was able to identically and reproducibly process each list mode file; and yielded population data in spreadsheet form. RESULTS: Statistical analysis (Fisher's z test) revealed no difference between the CD3+ autocontrol and CD3+ isotypic control (correlation = 1, P < .0001) or between the CD3+, CD4+ autocontrol and the CD3+, CD4+ isotypic control (correlation = 1, P < .0001). The elimination of the isotypic control reagent resulted in a total cost savings of $3.36 per test. Additionally, the subtraction of isotypic background can artifactually depress population enumeration. CONCLUSIONS: The use of an isotypic control antibody is not necessary to analyze flow cytometric data that result in discrete cell populations, such as CD3+ and CD3+, CD4+ lymphocyte subsets. The elimination of this unnecessary quality control measure results in substantial cost savings.

Activated T-cell subsets in benign lymphoid hyperplasias and B-cell non-Hodgkin's lymphomas.

Activated tumor-infiltrating T lymphocytes (TIL-T) were quantitated prospectively in excisional biopsy specimens of 49 B-cell non-Hodgkin's lymphomas (NHL) of various grades and compared with eight benign lymphoid hyperplasias (BLH) to identify any potential difference in host T-cell response. Immunotyping of tissue-cell suspensions was done by three-color flow cytometry, which was complemented by immunocytology by using cytocentrifuged preparations. Two activated T-cell subsets were studied: acutely activated TIL-T (CD3+ CD25+ HLADr-) and chronically activated TIL-T (CD3+ CD25- HLADr+). Results showed an association of the more aggressive intermediate/high-grade B-cell NHL with a higher percentage of late-phase activated TIL-T and a progressive increase with the grade of malignancy: 10.29%, 23.25%, 33.87%, and 47.78% (means) for BLH and for low-, intermediate- and high-grade B-cell NHL, respectively. Differences for this subset were significant (P less than 0.050) for the following comparisons: hyperplasia versus intermediate-grade NHL (P less than 0.0012), hyperplasia versus high-grade NHL (P less than 0.0002), and low versus high-grade NHL (P less than 0.0080). The percentage of acutely activated TIL-T cells did not show a statistically significant difference between the groups. The results suggest a host T-cell response to proliferating neoplastic cells in B-cell NHL. Paradoxically, the response does not appear to be protective for the host since the intensity is directly proportional to the grade of malignancy. However, the recognition of this response may have clinical applications since its amplification with biological response modifiers may result in effective adoptive immunotherapy of B-cell NHL. Further clarification of the specificity and biologic significance of host T-cell activation in B-cell NHL will require functional studies of isolated lymphocytic subpopulations from neoplastic tissue.

Monocyte interactions with solid substrates monitored by chemiluminescence.

The kinetics of interaction of solid substrates with mononuclear cells is a little explored by important feature in the evaluation of potential prosthetic materials. Monocyte production of chemiluminescence in a luminol-enhanced system was used to explore such kinetics. A theoretical model of cell/surface interaction is developed and alternatives in the evaluation of data generated by chemiluminescent curves are presented. The interactions of monocytes with a polytetrafluoroethylene (PTFE) surface were similar to those seen in studies on phagocytosis and were dependent on an absorbed serum protein layer. This study illustrates the use of a simple laboratory test for monocyte oxidative function in assessing the potential inflammatory effects of various prosthetic materials.

T lymphocytes infiltrating renal cell carcinoma have a reduced expression of transferrin receptor.

T-cell responses have been reported to be impaired in cancer patients, and lymphocytes infiltrating human tumors (T-TIL) appear to be more affected than those in the peripheral blood. T-TIL display a poor proliferative response when compared to peripheral blood T (T-PBL) cells that show a strong response to all stimuli. Here we report that T-TIL from patients with renal cell carcinoma (RCC) also have a defect in transferrin receptor (TfR) expression that is not present in T-PBL cells. Immunocytometry studies (dual staining for CD3 epsilon and TfR) demonstrated that autologous T cells from the peripheral blood but not from the tumor expressed TfR following stimulation with IL2, anti-CD3 or PHA. Expression of TfR correlated with the capacity of T cells from the blood and tumor to proliferate. Gene expression studies using reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that TfR mRNA levels in T-TIL were undetectable or low relative to T-PBL following stimulation. The failure to detect TfR mRNA in T-TIL after stimulation was not due to a shift in kinetics of mRNA accumulation since TfR mRNA was not detectable at any of the times tested (4, 12, 24 and 36 hr). The defect in TfR gene expression is selective since IL2R alpha gene expression was induced in T-TIL. Because IL2 binding to its receptor results in TfR expression, the defect in TfR induction in T-TIL appears to be distal to IL2R alpha expression. Our studies illustrate another alteration in T-TIL that is not observed in T cells from the peripheral blood. The absence of TfR gene expression may contribute to the poor proliferative response of T cells from the tumor.

L3T4 (CD4+) cells that mediate contact sensitivity to trinitrochlorobenzene express I-A determinants.

The present study has further characterized the T cell-mediated inflammatory response of contact sensitivity (CS) to the hapten trinitrochlorobenzene (TNCB) in mice. A discernible CS response was found to be induced as early as 2 days after epicutaneous application of TNCB. The response peaked on Days 4 to 5 and it then declined to a nearly undetectable level by Days 10 to 11. Examination of the draining lymph nodes demonstrated that development of CS coincided with an increase in cellular proliferation and in the total number of cells present. Despite a severalfold increase in the cellular contents of the draining lymph nodes of sensitized mice, the relative percentages of most subsets of T cells remained unchanged. Flow cytometric studies revealed that the subpopulation of T cells characterized as Thy 1.2+ L3T4+ I-A+ increased substantially in comparison to its presence in unsensitized mice. Whether the Thy 1.2+ L3T4+ I-A+ cells that increased following sensitization represented the effector population that mediates CS was then examined. Four-day immune lymph node T cells or L3T4 cells positively selected from them were capable of adoptively transferring CS to normal mice. However, these cells, after treatment with anti-Ia antibody or anti-I-A monoclonal antibody and complement, were unable to transfer CS. These findings imply that expression of I-A determinants may indicate antigen-induced T cell activation in vivo and that L3T4 cells that mediate CS are I-A positive.

Characterization of tumor-infiltrating lymphocyte subsets from human renal cell carcinoma: specific reactivity defined by cytotoxicity, interferon-gamma secretion, and proliferation.

The detection of T cells with specificity for human renal cell carcinoma (RCC) has been difficult to document. In an attempt to improve our identification of RCC-reactive T cells, tumor-infiltrating lymphocytes (TIL) were expanded in interleukin-2/interleukin-4 (IL-2/IL-4) and then separated into CD4+ and CD8+ subsets using antibody-coated biomagnetic beads. TIL grown in IL-2/IL-4 expanded to greater numbers than TIL grown in IL-2 alone. From 16 patients in whom subset separation was performed, three CD4+ and three CD8+ TIL consistently had specificity for RCC that was detected by cytotoxicity, proliferation, or interferon-gamma (IFN-gamma) production. Four of the six lines were derived from the IL-2/IL-4 cultures. Two CD8+ TIL lines displayed specific lytic activity, lysing the autologous tumor but not allogeneic RCC or nonrenal tumors. Moreover, the lytic activity of these lines was blocked by anti-CD3 antibody, suggesting that tumor recognition was through the TCR/CD3 complex. Two additional TIL lines showed preferential lysis of RCC because they were cytotoxic for autologous tumor and one or more allogeneic RCC but not other tumor types. Two nonlytic CD4+ lines as well as the two CD8+ lines that were specifically lytic also produced IFN-gamma in response to the autologous tumor but not allogeneic RCC. Although these TIL lines produce IFN-gamma when stimulated with tumor alone, the addition of 5 U/ml of IL-2 significantly enhanced IFN-gamma secretion. The four TIL lines that showed specificity for RCC in terms of IFN-gamma production also had enhanced proliferation to the autologous RCC plus IL-2 but not to multiple allogeneic RCC plus IL-2. These studies demonstrate that TIL from RCC patients contain both CD4+ and CD8+ T cells that have specificity for RCC. In addition to cytotoxicity, specificity to RCC can be defined by IFN-gamma production and proliferation.

Responses to T cell receptor/CD3 and interleukin-2 receptor stimulation are altered in T cells from B cell non-Hodgkin's lymphomas.

T cells infiltrating (T-TIL) B cell non-Hodgkin's lymphomas (NHL) are thought to represent a local host response to the tumor. However, tumor progression in the presence of this T cell infiltrate suggests that the T-TIL may be functionally impaired. To address this issue we determined whether response to stimulation of T-TIL from 25 patients with NHL through the T cell receptor (TCR/CD3) and the interleukin-2 (IL-2) receptor (IL-2R) was intact, since activation of these receptors is important for proliferation and cytokine production. Our results demonstrate defects in response to stimulation via TCR/CD3 and the IL-2R in T-TIL cells from patients with NHL that were not observed with T cells from the peripheral blood. T-TIL showed minimal proliferation to anti-CD3 and only modest proliferation to IL-2 alone or when combined with anti-CD3. Moreover, cytokine production in T-TIL was impaired since stimulation through the TCR/CD3 complex did not induce mRNA for interferon gamma (IFN gamma), IL-2, IL-4 or IL-10. The functional unresponsiveness of these cells may be linked to altered signalling through the TCR/CD3 since an abnormal tyrosine phosphorylation pattern was detected in T-TIL after stimulation with anti-CD3.

Molecular regulation of intercellular adhesion molecule 1 (ICAM-1) expression in renal cell carcinoma.

Intercellular adhesion molecule-1 (ICAM-1) mediates two important functional aspects of tumor biology, namely enhancement of tumor metastasis and mediation of host defense mechanisms such as lymphocyte-mediated tumor cytotoxicity. Since ICAM-1 is expressed by most renal cell carcinomas (RCC), the regulation of ICAM-1 expression is important in understanding the biological behavior of RCC. We report an investigation on ICAM-1 expression and molecular regulation by cytokines and protein kinase C activator on RCC cell lines. Of the various cytokines, tumor necrosis factor alpha (TNF alpha), interferon-gamma (IFN gamma), and phorbol myristate acetate (PMA) strongly upregulated ICAM-1 protein expression on RCC. The kinetics of ICAM-1 message induction was studied by Northern analysis of total RNA extracted from RCC and normal kidney proximal tubular (NKPT) cells. Time course studies showed that ICAM-1 mRNA was upregulated by INF gamma, TNF alpha, and PMA, plateaued after 2 h, and remained increased for up to 24 h. Although ICAM-1 mRNA in NKPT cells was upregulated by these cytokines, their messages returned to basal levels after 24 h. ICAM-1 mRNA stability assays showed that both unstimulated and stimulated RCC cells had very stable ICAM-1 mRNA up to 24 h. In order to investigate whether increased gene transcription contributes to ICAM-1 upregulation, RCC cells were treated with TNF alpha, IFN gamma, or PMA with or without simultaneous addition of actinomycin D. ICAM-1 message induction-blocking studies suggested that primary upregulation of ICAM-1 mRNA may be caused by transcriptional upregulation. These results suggest that long-lasting ICAM-1 message upregulation in response to cytokines or PMA may be due to transcriptional upregulation in the early phase and stabilization of ICAM-1 message in the later phase (after 4 h). These observations suggest that RCC may lack the normal downregulatory mechanisms which control ICAM-1 expression and may explain the high frequency of ICAM-1 expression observed on primary human RCC.

Phosphorus metabolites and the distribution of cell cycle phase of RIF-1 tumors in response to 14 Gy irradiation.

Simultaneous measurements of DNA cell phase cycle distributions and in vivo 31P NMR spectroscopy were performed on 40 RIF-1 murine tumors irradiated with 14 Gy of X-radiation. Diploid and tetraploid tumor populations were observed. The cells blocked in G2/M phase were measured as a function of the ratios of tetraploid cell number in G2/M phase versus total cell population measured. The G2/M population reached a maximum at 32 h post irradiation, dropping to control values by 72 h, while the ratio of inorganic phosphate to beta-nucleotide triphosphate dropped significantly at 32 h and remained significantly lower than control up to 72 h post irradiation. Measurements of PME, PDE, PCr, and pH showed no significant variations at any time point. No significant change in host cell population could be observed. Since the measured G2/M population never increased to more than 3% of the total cell population, the change observed in the 31P NMR spectra were not simply the result of possible differences in NMR profiles of the different cell phase populations but were more likely due to a change in the metabolic characteristics or environment of a majority of the cells.

Expression of cell adhesion molecules in an established and characterized new human renal cell cancer line, CCF-RC7.

In order to investigate the importance of cell adhesion molecules (CAMs) in renal cell carcinoma (RCC), a cell line, designated as CCF-RC7, was established from a human RCC of the clear cell type. CCF-RC7 was passaged over 50 times in vitro for 3 1/2 years. The cell line has an epithelial morphology and a doubling time of 30 h, forming colonies in soft agar with an average efficiency of 10.4% and producing clear cell tumors in athymic nude mice. CCF-RC7 cells have an aneuploid-hypotetraploid karyotype with a modal chromosome number of 82 and rearrangements in chromosomes 9, 12 and 14. Immunohistochemical and flow immunocytometric analyses revealed high expression of ICAM-1 (CD54), and Hermes antigen (CD44), which was significantly upregulated by cytokine and PMA treatment. VLA-4 was expressed on approximately 20% of tumor cells and could not be altered by cytokine or PMA stimulation. High expression of sialyl Lewis X was also demonstrated by immunohistological examination. This newly characterized cell line will serve as a useful model for the study of CAMs during hematogenous metastasis and host defense mechanisms in human RCC.

Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using three-color immunocytometry.

Accurate and consistent enumeration of B-cell subpopulations in lymphoid tissue was achieved through multiparameter three-color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)-anti-CD19 (Leu12) and biotinylated anti-HLADr/streptavidin-Duochrome (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti-surface immunoglobulin (SIg) antibodies, effectively separated non-specific binding and background fluorescence from true B-cell surface FITC immunofluorescence, while concomitantly analyzing for HLADr and CD19 phenotypic expression/deletion. Autofluorescence was measured to establish a fluorescence threshold. A second control measured non-specific binding of isotypic control mouse Ig and non-immune rabbit IgG. Cell suspensions from 128 samples of various lymphoid proliferations were studied. In 116 of the 128 samples, kappa/lambda ratios determined by flow cytometry correlated well with immunocytology results obtained using cytospins from the same cell suspension and with histopathologically established diagnosis. Clonality and lineage as defined immunotypically by flow cytometry was concordant with genotypic results in 64 of the 67 cases evaluated. SIg, HLADr, and CD19 deletions were demonstrated by flow cytometry in 8, 4, and 1 case(s), respectively. Discordance was usually attributable to selective loss of large neoplastic cells in flow cytometry specimens or absent expression of SIg by some cytoplasmic Ig (CIg+) lymphomas.