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H E Prince

Publications and source records attributed to H E Prince.

At least 19 recordsLinked to original sources
PubMed1994

Spontaneous lymphocyte proliferation in human T-cell lymphotropic virus type I (HTLV-I) and HTLV-II infection: T-cell subset responses and their relationships to the presence of provirus and viral antigen production.

Spontaneous lymphocyte proliferation (SLP) during in vitro culture of mononuclear cells (MCs) characterizes over half of asymptomatic individuals infected with human T-cell lymphotropic virus type I (HTLV-I) or HTLV-II. Both CD4 and CD8 T-cell subsets within MC cultures are activated during SLP, as judged by high-density CD25 (CD25bright) expression; it is unclear, however, whether both cell subsets can directly undergo SLP. In the present investigation, the SLP capacities of purified CD8 and CD4 cells were examined in subjects infected with HTLV-I (n = 19) or HTLV-II (n = 54) in relation to the SLP status of MCs from each subject. No increase in SLP was observed for CD8 or CD4 cells from SLP-negative (SLP-) HTLV-infected subjects, whereas robust SLP characterized CD8 cells from all SLP-positive (SLP+) individuals, regardless of HTLV type. In contrast, SLP+ CD4 cells characterized only 23% (7 of 31) of HTLV-II+ SLP+ individuals, whereas SLP+ CD4 cells characterized 100% of HTLV-I+ SLP+ individuals. In cocultures of HTLV-II+ SLP+ CD8 cells and autologous SLP- CD4 cells, sizable proportions of both CD8 cells and CD4 cells coexpressed CD25bright, suggesting that SLP- CD4 cells were activated in the presence of SLP+ CD8 cells. PCR analysis for tax sequences detected provirus in most CD4- and CD8-cell preparations from HTLV-seropositive individuals, regardless of type and the SLP status of cell subsets. To determine whether SLP was associated with activation of viral genes, levels of HTLV-I and HTLV-II core antigen (Ag) in supernatants were measured. Viral Ag production and SLP responses were significantly correlated for both CD4 and CD8 cells in both HTLV-I and HTLV-II infections. However, inhibition of CD8- or CD4-cell SLP by cyclosporin A or anti-Tac (anti-CD25) did not reduce Ag production, indicating that Ag production is not coupled to SLP. These findings show that CD4 cells from SLP+ HTLV-I+ and SLP+ HTLV-II+ individuals differ in SLP capacity, that the absence of SLP does not indicate a lack of infection, and that production of viral Ag is associated with, but not dependent on, SLP.

Base Sequence↗
PubMed1992

Phenotypic comparison of the three populations of human lymphocytes defined by CD45RO and CD45RA expression.

Published reports indicate that CD45RO-CD45RAbright T cells are native T cells, CD45RObrightCD45RA- T cells are memory T cells, and that concomitant loss of CD45RA expression and gain of CD45RO expression occurs during transition from naive to memory status. Thus, following in vitro activation of CD45RO- CD45RAbright T cells, a subset of transitional CD45ROdimCD45RAdim T cells is observed before conversion to a CD45RObrightCD45RA- phenotype is completed. Interestingly, all three of these phenotypic subsets are represented in the circulating human lymphocyte pool. We thus used dual-color flow cytometry to phenotypically characterize CD45RObrightCD45RA-, CD45ROdimCD45RAdim, and CD45RO- CD45RAbright lymphocytes. Both the CD45RObrightCD45RA- and CD45ROdimCD45RAdim subsets consisted almost entirely of T cells, whereas the CD45RO-CD45RAbright subset contained T cells plus essentially all of the B and natural killer cells. Additional studies used three-color flow cytometry to assess activation markers on T cells within the three subsets defined by CD45RO/CD45RA expression. CD25 expression increased with conversion from naive to memory status (5% of CD45RO-CD45RAbright, 24% of CD45ROdimCD45RAdim, and 42% of CD45RObrightCD45RA- T cells), whereas CD38 expression decreased during conversion (76, 53, and 27%, respectively). We also assessed the fluorescent intensities of CD11a, CD2, and CD44, shown by others to be increased on memory, compared to naive T cells. Visual inspection of fluorescence cytograms confirmed these findings, and further showed that transitional T cells express these markers at levels indistinguishable from those for naive T cells. These findings suggest that acquisition of CD25 and loss of CD38 occur relatively early in the naive-to-memory transition process, being evident in the transitional cell subset. In contrast, increased expression of CD11a, CD2, and CD44 appear to represent late events, occurring after loss of CD45RA and gain of CD45RO has been completed.

Humans↗
PubMed1992

Lymphocyte subsets in HTLV-II-infected former blood donors: relationship to spontaneous lymphocyte proliferation.

Previous studies showed that over 70% of HTLV-seropositive blood donors from the Los Angeles area are infected with HTLV-II; further, mononuclear cells from about half of these HTLV-II+ donors exhibit spontaneous lymphocyte proliferation (SLP) during in vitro culture. To determine if HTLV-II+SLP+ donors exhibit more marked immune system changes than HTLV-II+SLP- donors, lymphocyte subsets for these two HTLV-II+ groups were compared to an uninfected control group. The percentage of lymphocytes expressing CD3 was significantly increased and the percentage expressing a CD16/56+CD3- phenotype (natural killer cells) was significantly decreased in the HTLV-II+SLP+ group (N = 34) versus the control group (N = 49). On the basis of absolute numbers, the lymphocyte number was significantly higher in the HTLV-II+SLP+ group than in the control group and reflected significant increases in the numbers of both CD4 and CD8 subsets of T cells. Analysis of proportional changes in CD4 and CD8 cell subsets revealed significant increases in the proportions of CD4 cells expressing HLA-DR, CD8 cells expressing HLA-DR, and CD8 cells expressing CD45RO for the HTLV-II+SLP+ group versus the control group. For all phenotypic parameters measured, no significant differences were noted when comparing the HTLV-II+SLP- group (N = 21) and the control group. Cell culture experiments utilizing purified CD4 cells and CD8 cells from a subset of each study group revealed that in vitro spontaneous proliferative capacity resides within both the CD4 cell and CD8 cell populations from SLP+ individuals. These findings show that changes in circulating lymphocyte subsets in HTLV-II infection are found only in association with SLP, and that the capacity to exhibit SLP characterizes both CD4 and CD8 lymphocyte subsets.

Blood Donors↗
PubMed1992

Unaltered lymphocyte subsets in hepatitis C virus-seropositive blood donors.

Lymphocyte subsets were evaluated by dual-color flow cytometry in whole blood specimens from 35 blood donors who were seropositive on enzyme-linked immunosorbent assay (ELISA) for hepatitis C virus (HCV) and whose sera reacted in a four-antigen recombinant immunoblot assay (RIBA) (referred to as the HCV+R group), 15 donors who were seropositive on ELISA for HCV with indeterminate or negative RIBA results (the HCV+I/N group), and 25 HCV-seronegative controls (HCV-group). The cell subsets assessed included natural killer cells, B cells, T cells, CD4 and CD8 subsets of T cells, and T-cell subsets defined by the coexpression of markers that appear (HLA-DR, CD25, CD38) or disappear (CD45RA) after activation. A one-way analysis of variance revealed no significant differences among the three study groups. These findings show that, unlike cytomegalovirus- and human immunodeficiency virus-positive individuals, HCV-positive individuals do not exhibit lymphocyte alterations indicative of the immune activation caused by chronic viral infection.

Antigens, CD↗
PubMed1992

Culture-associated enhancement of LECAM-1 expression by lymphocytes and partial inhibition of enhancement by IL-4.

Recent studies have shown that the human leukocyte endothelial cell adhesion molecule-1 (LECAM-1) functions as a homing receptor, mediating leukocyte binding to high endothelial venules in peripheral lymph nodes. Increasing evidence has demonstrated that cytokines, such as IL-4, can modulate the expression of surface proteins such as homing receptors on a variety of cells. We thus investigated the modulatory effects of cytokines on LECAM-1 expression by lymphocytes using single- and dual-color flow cytometry. We found that the density of LECAM-1 expression increased markedly during 3 days of culture and that this culture-associated enhancement (CAE) of LECAM-1 expression was significantly inhibited by IL-4. B cells and both major T cell subsets (CD4, CD8) exhibited CAE of LECAM-1 expression, but the inhibitory effect of IL-4 on this response occurred only in the T cell populations. The inhibitory effect of IL-4 on enhanced LECAM-1 expression was reversible, and characterized all 3 LECAM-1 epitopes assessed. Natural killer cells, in contrast, did not exhibit CAE of LECAM-1 expression, and IL-4 had no modulatory effect on LECAM-1 expression by these cells. Another adhesion molecule, CD44, showed enhanced expression during culture, but this enhancement was not inhibited by IL-4. The results show that LECAM-1 expression by T and B lymphocytes is significantly increased during culture and that the inhibitory effect of IL-4 on this increase is restricted to T cells. These findings suggest that IL-4, generated during an immune response, may play a role in regulating the migration and localization of T lymphocytes to lymphoid tissues.

Antibodies, Monoclonal↗
PubMed1991

Immunologic correlates of spontaneous lymphocyte proliferation in human T-lymphotropic virus infection.

Previously we showed that mononuclear cells from about half of human T-lymphotropic virus (HTLV)-seropositive persons exhibit spontaneous proliferation in vitro. We sought to determine if proliferation was associated with other immunologic changes characteristic of HTLV infection. The parameters assessed were (1) percentages of lymphocytes expressing CD4 and/or CD25 (interleukin-2 receptor), (2) serum levels of soluble CD25, (3) serostatus for other viruses, (4) anti-HTLV antibody levels, and (5) HTLV type determined by polymerase chain reaction or serologic reactivity with type-specific peptides. The proliferation+ HTLV (PROL+) group, proliferation HTLV (PROL-) group, and control group showed similar percentages of CD4+, CD25+, and CD4+CD25+ lymphocytes; serum levels of soluble CD25 were also similar. Antibodies to cytomegalovirus, hepatitis B core, and hepatitis C were present in similar proportions of PROL+ and PROL+ groups. However, a significant association was found between spontaneous proliferation and anti-HTLV antibody levels; sera from 67% of PROL+ persons, but only 18% of PROL- persons, required dilution to yield absorbance values within the linear range of the anti-HTLV antibody assay. In the PROL+ group, persons whose sera required the most dilution had proliferative responses significantly higher than those whose sera required no dilution. The PROL+ and PROL groups were similar with regard to the relative distribution of HTLV-I and HTLV-II infection. These findings indicate that HTLV-related spontaneous lymphocyte proliferation is related to levels of circulating anti-HTLV antibodies, and characterizes both HTLV-I and HTLV-II infection.

Antibodies, Viral↗
PubMed1991

HIV-related alterations in CD8 cell subsets defined by in vitro survival characteristics.

Previously we showed that over 50% of CD8 cells from HIV-infected persons do not survive in 3-day cultures of mononuclear cells; this loss occurred preferentially in subsets with phenotypes indicative of in vivo activation. In the studies reported here, we asked if cytokines enhanced CD8 cell survival. Of IL1, IL2, IL4, IL6, tumor necrosis factor, and interferon-gamma only IL2 specifically enhanced CD8 survival in the HIV group, compared to the control group. Further studies thus focused on characterizing CD8 cell survival in the presence of IL2. In both study groups, three subsets of CD8 cells were identified based on in vitro survival: (a) those surviving in culture medium alone (survivors), (b) those surviving only when IL2 was included in the culture medium (IL2-dependent survivors), and (c) those failing to survive even in the presence of IL2 (nonsurvivors). By dual-color cytofluorometry, the CD8 survivor subset was similar in the two study groups, and expressed nonactivated phenotypes (Leu8+, CD45RA+, HLA-DR-). The IL2-dependent survivor subset was also similar in the two study groups and expressed the phenotypes Leu8-, CD45RA+, CD57+, HLA-DR+, and CD38+, suggesting prior activation. The CD8 nonsurvivor subset, in contrast, was markedly different in the study groups: compared to the control group, the HIV group contained significantly higher proportions of CD8 cells expressing the phenotypes Leu8-, CD57+, and HLA-DR+, also suggesting activation. These findings indicate that, in HIV infection, the activated CD8 cell subsets that do not survive in medium alone consist of a "normal" component that requires IL2 for survival and an "abnormal" component that does not survive even in IL2.

Antigens, CD↗
PubMed1991

Spontaneous lymphocyte proliferation in HTLV-I/II infection reflects preferential activation of CD8 and CD16/56 cell subsets.

Previous studies have shown that lymphocytes from HTLV-infected persons spontaneously proliferate when cultured in vitro. We investigated which cell subsets become activated in this response. Mononuclear cells from 16 HTLV-seropositive former blood donors and 9 seronegative controls were cultured for 7 days; activation was then assessed by measuring DNA synthesis in cultured cells and by monitoring CD25 expression by CD3, CD4, CD8, CD19, and CD16/56 lymphocyte subsets. Of the 16 cultures of HTLV + donor cells, 10 showed spontaneous proliferation (Prol + group) and 6 did not (Prol - group). Cytofluorometric analysis revealed a significant increase in the fractions of CD8 cells and CD16/56 cells expressing CD25 for the Prol + group, compared to the Prol- and control groups. Similarly, the fractions of CD25 cells expressing CD8 or CD16/56 were significantly increased in the Prol + group. Although neither the fraction of CD4 cells expressing CD25 nor the fraction of CD25 cells expressing CD4 were increased for the Prol + group, the modal fluorescence intensity value for CD25 expression by CD4 cells was increased, suggesting some CD4 cell activation occurred as well. Blastoid cells were, on average, 79% CD25 +, whereas the sum of CD4 + CD25 + (27%), CD8 + CD25 + (30%), CD19 + CD25 + (3%), and CD16/56 + CD25 + (35%) subsets was 95%; the presence of 17% CD8 + CD16/56 + cells accounted for most of this discrepancy. These findings indicate that spontaneous lymphocyte proliferation in HTLV infection reflects preferential activation of CD8 and CD16/56 cell subsets, apparently including the minor CD8 + CD16/56 + subset.

Antigens, CD↗
PubMed1991

Three-color cytofluorometric analysis of CD8 cell subsets in HIV-1 infection.

Previous studies have shown that CD8 cell subsets, some expressing activation markers, are elevated in human immunodeficiency virus (HIV) infection. To assess the overlap of these subsets, we used three-color flow cytometry to phenotype CD8 cells in cryopreserved mononuclear cells from uninfected controls and from people infected with HIV, in CDC classes II, III, and IV (n = 12 per group). There were several CD8 subset changes observed in association with HIV infection. A shift from a naive (CD45RA+CD45RO-) to a memory (CD45RA-CD45RO+) phenotype occurred in the CD8 subset, but the intermediate phenotype (CD45RA+CD45RO+) was unchanged. Increases in DR+CD8 and CD38+CD8 cells were noted in both naive and memory CD8 subsets, defined by CD45RA or CD45RO expression. Both the CD57+ and CD57- subsets of DR+CD8 cells were increased, whereas only the CD57+ subset of CD38+CD8 cells was elevated. The increase in CD57+CD8 cells reflected a selective rise in CD57+CD8 cells coexpressing CD38, DR, and CD45RO. The CD38+ DR+ CD8 subset was markedly increased and was apparently derived from both the CD38-DR-CD8 and CD38+DR-CD8 subsets. Compared with classes II and III, the CDC class IV group showed an increased proportion of CD8 cells expressing CD38; higher percentages of CD38+DR+CD8, CD38+CD45RA-CD8, and DR+CD45RO+CD8 subsets; and decreased percentages of CD38-CD45RA+CD8 and CD38-CD57-CD8 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosyl Cyclase↗
PubMed1990

Normal expression of p55 interleukin 2 receptor (CD25) by lymphocytes from former blood donors seropositive for human T lymphotropic virus.

Dysregulated expression of the p55 interleukin 2 receptor (CD25) is characteristic of adult T cell leukemia (ATL) associated with human T lymphotropic virus (HTLV) infection. In order to determine if similar changes characterize HTLV infection in the apparent absence of ATL, CD25 expression by peripheral blood lymphocytes from HTLV-seropositive former blood donors was measured using a sensitive dual-color cytofluorometric assay. When comparing the HTLV-seropositive group (N = 19) and a seronegative control group (N = 20), no significant differences were observed in either the proportions of the major lymphocyte subsets (CD3, CD4, CD8, CD19, CD16/56) coexpressing CD25 or the phenotypic distribution of CD25+ cells among these lymphocyte subsets. Similarly, the total percentages of CD3, CD4, CD8, and CD19 cell subsets were unchanged; however, the percentage of CD16/56+ cells was significantly decreased in the HTLV group and reflected a decrease in the percentage of CD16/56 cells lacking CD25. These findings indicate that HTLV infection without ATL is characterized by normal CD25 expression by lymphocytes and a decreased percentage of lymphocytes with a phenotype characteristic of natural killer cells.

Antigens, CD19↗
PubMed1990

Interrelationships between serologic markers of immune activation and T lymphocyte subsets in HIV infection.

Relationships among four serologic activation markers and T cell subsets were measured in HIV-seropositive former blood donors (N = 64) and seronegative controls (N = 61). Significant correlations were observed for the HIV group in pairwise comparisons of soluble IL-2 receptor (sIL-2R), beta 2-microglobulin (beta 2M), neopterin (NEOP), and soluble CD8 (sCD8). CD4 cell levels (number/microliter) in the HIV group showed significant negative correlation with all four serologic markers; CD8 cell levels, in contrast, showed no significant correlation with any serologic activation marker measured. Significant correlations were observed, however, among various cell surface activation markers and serologic activation markers. Specifically, the proportion of CD8 cells expressing CD45RA showed significant negative correlations with NEOP and B2M levels, whereas the proportion of CD8 cells expressing HLA-DR showed significant positive correlations with B2M and sIL-2R levels. Further, the proportion of CD8 cells expressing CD38 showed significant positive correlations with all four serologic activation markers. These findings indicate that sIL-2R, B2M, NEOP, and sCD8 show similar quantitative changes and correlational relationships to CD4 cell destruction in HIV infection; they differ, however, in their relationships to proportional changes in activated CD8 cell subsets.

Antigens, CD↗
PubMed1989

Preferential loss of Leu 8-, CD45R, HLA-DR+ CD8 cell subsets during in vitro culture of mononuclear cells from human immunodeficiency virus type I (HIV)-seropositive former blood donors.

Recent data from our laboratory showed that the CD4:CD8 cell ratio increased significantly during in vitro culture of unstimulated mononuclear cells (MC) from HIV-infected persons. To test the hypothesis that this increase reflected a decline in CD8 cell levels, changes in CD4 and CD8 cell levels during culture of MC were assessed quantitatively. These analyses were accomplished using a Spectrum III flow cytometer, which analyzes a constant volume (0.02 ml) of cell suspension. The number of cells counted within this volume (termed the sip count) thus reflects the cell number in the suspension. To establish day 0 sip counts, aliquots consisting of 200,000 lymphocytes were treated with monoclonal antibodies, resuspended in 1 ml of buffer, and analyzed. Also on day 0, identical cell aliquots were placed in microtiter wells and cultured for 3 days. Cells retrieved from individual wells were then analyzed as on day 0. The mean relative recoveries (RR) of lymphocytes, CD4 cells, and CD8 cells were significantly lower in the HIV group (N = 28) than in the control group (N = 26). For the HIV group, CD8 cell RR was significantly lower than CD4 cell RR. Dual-color analyses showed that CD4 cell loss in the HIV group did not occur preferentially within CD4 subsets defined by Leu 8 or CD45R expression. Similarly, CD8 cell loss did not occur preferentially within CD8 subsets defined by Leu 7 expression. In contrast, CD8 cell loss did preferentially affect Leu 8-, CD45R-, and HLA-DR+ CD8 subsets, compared to the reciprocal CD8 subsets.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1989

Simultaneous determination of absolute total lymphocyte and CD4+ lymphocyte levels in peripheral blood by flow cytometry.

A distinguishing feature of the Spectrum III flow cytometer is its capacity to analyze a constant volume of cell suspension. The authors have capitalized on this feature to directly and simultaneously measure the absolute numbers of all lymphocytes and CD4+ lymphocytes per microliter of peripheral blood. The study group consisted of 42 hospital patients, 12 former blood donors seropositive for human immunodeficiency virus (HIV), and 12 HIV-seronegative donors. Regression analysis revealed a highly significant correlation (r = 0.97) between Spectrum lymphocyte counts and lymphocyte counts determined by a Coulter Counter S-Plus IV. Similarly, Spectrum CD4 cell counts were significantly correlated (r = 0.98) with CD4 cell counts calculated from the Coulter lymphocyte counts and % CD4+ lymphocytes determined by flow cytometry. These findings indicate that the absolute numbers of lymphocytes and subsets of lymphocytes in peripheral blood can be rapidly and simultaneously measured by flow cytometry. Such an assay should prove useful in studies of HIV infection, where total lymphocyte and CD4 cell levels are important parameters for clinical staging and assessing responses to treatment.

CD4-Positive T-Lymphocytes↗
PubMed1988

Soluble IL-2 receptor levels in serum from blood donors seropositive for HIV.

Serum levels of soluble IL-2R (sIL-2R) were measured in blood donors seropositive for HIV. Approximately one-half (53/101) of these seropositive donors exhibited increased serum sIL-2R levels compared with seronegative controls (n = 65). Further, a significant inverse correlation was observed between serum sIL-2R levels and CD4 cell levels in seropositive study participants. These findings suggest that increased serum sIL-2R levels in HIV infection may be linked to CD4 cell loss, and thus reflective of the stage of HIV-induced disease.

Blood Donors↗
PubMed1988

In vitro activation of T lymphocytes from human immunodeficiency virus (HIV)-seropositive blood donors. I. Soluble interleukin 2 receptor (IL2R) production parallels cellular IL2R expression and DNA synthesis.

We investigated the relationship of soluble interleukin 2 receptor (sIL2R) production to cellular IL2R expression and DNA synthesis by mitogen-stimulated mononuclear cells from blood donors seropositive for human immunodeficiency virus (HIV). SIL2R was measured using an enzyme-linked immunosorbent assay which employed 2 anti-IL2R monoclonal antibodies recognizing distinct IL2R epitopes. Decreased phytohemagglutinin-induced DNA synthesis and cellular IL2R expression were accompanied by decreased levels of sIL2R in cell culture supernatants. Similar findings were observed for pokeweed mitogen-induced responses. There was no detectable spontaneous secretion of sIL2R into culture supernatants by unstimulated mononuclear cells from either HIV-seropositive or control seronegative donors. These findings indicate that the in vitro T-cell activation defects which characterize HIV infection include decreased sIL2R production, as well as decreased cellular IL2R expression and DNA synthesis. Further, they show that assessment of supernatant sIL2R levels can be used as a valid, reliable assay for T-cell activation.

Acquired Immunodeficiency Syndrome↗
PubMed1988

In vitro activation of T lymphocytes from HIV-seropositive blood donors. II. Decreased mitogen-induced expression of interleukin 2 receptor by both CD4 and CD8 cell subsets.

Mononuclear cells (MC) from many individuals exposed to human immunodeficiency virus (HIV) exhibit a reduced proliferative response to a suboptimal concentration of phytohemagglutinin (PHA). However, the relative contributions of the 2 major T-cell subsets, namely CD4 and CD8 lymphocytes, to this reduced response remain unclear. Based on reports that interleukin 2 receptor (IL2R) expression correlates well with proliferative responses in HIV infection, we used dual-color cytofluorometry to measure IL2R expression by CD4 and CD8 cells following PHA activation of MC from HIV-seropositive blood donors. For data analysis, this study group was divided into two subgroups on the basis of DNA synthesis responses (seropositive with normal DNA synthesis, designated sero + NML, or seropositive with decreased DNA synthesis, designated sero + LOW). When compared to the seronegative control and sero + NML groups, the sero + LOW group exhibited significant reductions in the percentage of MC expressing IL2R, the proportion of CD4 cells expressing IL2R, and the proportion of CD8 cells expressing IL2R. In contrast, these parameters were unchanged in the sero + NML group compared to the control group. These findings show that reduced PHA-induced proliferative responses by MC from HIV-infected persons are associated with decreased IL2R expression by both CD4 and CD8 lymphocyte subsets.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Flow cytometric analysis of lymphocyte activation in the mixed lymphocyte response.

Interleukin 2 receptor (IL2R) expression may be a useful parameter for assessing lymphocyte activation in the mixed lymphocyte response (MLR). However, the contribution of irradiated stimulator (S*) cells to the levels of IL2R+ cells recovered must first be defined. We have used a flow cytometric parameter termed the sip count to assess this potential contribution of S* cells. This parameter, which is the number of cells within a defined cell gate, is a reflection of the viable cell number per culture well, since (a) a constant number of cells were plated per well on day 0, (b) cells recovered from a well were resuspended in a constant volume, (c) the flow cytometer aspirated (sipped) a constant volume of cell suspension, and (d) nonviable cells were not included in the gate. Sip count assessment showed that only 5% of S* cells were recoverable by day 4 of culture; in contrast, 70% or more of unstimulated responder cells were recoverable. Sip counts of MLR cultures identified an increase in cell number beginning on day 5, reflecting DNA synthesis and cell division. We then used the sip count to assess changes in the levels of IL2R+ cells in MLR cultures. The number of IL2R+ cells continued to increase up to day 7, even though maximal DNA synthesis occurred on day 5. Further, dual color analysis revealed that the proportion of CD4 cells expressing IL2R was maximal on day 5, whereas the proportion of CD8 cells expressing IL2R continued to increase until day 9. These findings show that flow cytometry can be used to study lymphocyte activation by alloantigens.

Antigens, Differentiation, T-Lymphocyte↗