CD45 isoforms associated with distinct functions of CD4 cells derived from unusual healthy donors lacking CD45RA- T lymphocytes.

We now report two healthy individuals whose T lymphocytes were over 95% positive for CD45RA antigen expression. However, these donors normally expressed both the CD29 high (CD29+) and CD45RO high (CD45RO+) antigens on approximately 40 and 50% of their CD4 cells, respectively. Despite the strong CD45RA expression on the surface of almost all CD4 cells, the CD29 marker allowed T cells from these donors to be divided phenotypically into subsets having distinct in vitro function. CD4+CD29+ cells from these donors responded maximally to recall antigens such as TT and provided strong helper function for B cell Ig synthesis. In contrast, CD4+CD29- cells responded poorly to recall antigens and had poor helper function for B cell Ig synthesis, but had strong suppressor activity. Thus, CD29 antigen expression was still predictive of the in vitro functional activity as previously described for normal donors. Furthermore, biochemical analysis of the distribution of individual CD45 isoforms on the surface of these subsets of CD4 cells revealed distinct differences. The CD4+CD29 high (CD4+CD29+) subset of cells primarily expressed the 180-, 190-, and 205-kDa CD45 isoforms, while the CD4+CD29 low (CD4+CD29-) cells primarily expressed the 190-, 205-, and 220-kDa CD45 isoforms. These results suggest that despite the superficial phenotypic similarity of CD4 cells in these donors, distinctions in the distribution of both CD29 and the 180- and 220-kDa CD45 isoforms exist and might play a role in the different functions of freshly isolated CD4 lymphocytes.     

Reduced ecto-5'-nucleotidase activity and enhanced OKT10 and HLA-DR expression on CD8 (T suppressor/cytotoxic) lymphocytes in the acquired immune deficiency syndrome: evidence of CD8 cell immaturity

Markedly reduced ecto-5'-nucleotidase activity was found in peripheral blood lymphocytes from 27 out of 30 homosexual men with the acquired immune deficiency syndrome (AIDS) in association with Kaposi's sarcoma (AIDS-KS; 2.67 +/- 1.70 U/10(6) cells; n = 13), opportunistic infections (AIDS-OI; 9.29 +/- 7.32; n = 7), or the AIDS-related complex (ARC; 9.82 +/- 6.12; n = 10). These values were significantly different from healthy controls (22.70 +/- 4.58; p less than 0.001). In AIDS-KS patients, both T cells and non-T cells exhibited significantly reduced ecto-5'-NT activity (p less than 0.001). AIDS-KS CD8 cells contained 20% of the mean ecto-5'-NT activity (7.04 +/- 3.53) displayed by control CD8 cells (34.07 +/- 4.86; p less than 0.001). No significant difference in enzyme level was observed between control and AIDS-KS CD4 cells (11.93 +/- 4.98 vs 7.98 +/- 3.28, respectively). In AIDS patients, lymphocyte ecto-5'-NT activity was inversely related (r = -0.518; p less than 0.01) to the absolute number of OKT10+ cells, but no correlation was found with the number of HLA-DR+ cells (r =-0.224). Two-color analysis of lymphocytes from AIDS-KS patients revealed that 75 +/- 12% of circulating CD8 cells expressed the OKT10 antigen, whereas only 10 +/- 6% of control CD8 cells did. HLA-DR antigens, which are not normally found on circulating resting T cells, were expressed in AIDS-KS CD8 cells, although to a lesser extent than OKT10. These data demonstrate that most AIDS CD8 cells differ from control CD8 cells. Although it has been suggested that these cells are activated cytotoxic or suppressor cells, the data presented here support the hypothesis they are immature. Reduced T cell ecto-5'-NT activity and enhanced expression of OKT10 and HLA-DR antigens on circulating CD8 cells, in conjunction with lack of transferrin receptor-(OKT9) and IL 2 receptor-(Tac) bearing lymphocytes, sustain this latter hypothesis. The correlation of the numerical reduction of CD4 cells with the reduced levels of ecto-5'-NT (r = 0.606; p less than 0.01) suggests that the abnormal maturation of CD8 cells seen in AIDS might be a consequence of the CD4 deficiency characteristic of this syndrome.     

The functional heterogeneity of CD8+ cells defined by anti-CD45RA (2H4) and anti-CD29 (4B4) antibodies.

The monoclonal antibodies, anti-2H4(CD45RA), and anti-4B4(CD29), along with UCHL1-(CD45RO), identify reciprocal populations of CD4 cells with distinct suppressor inducer (CD45RA+CD29-CD45RO-) and helper inducer (CD45RA-CD29+CD45RO+) functions. Although the CD8+ population is known to contain precytotoxic, cytotoxic, suppressor, and some natural killer cells, the exact phenotypic identities of these functional CD8 subsets has not been established. In this study, we tried to determine whether these monoclonal antibodies could distinguish functionally distinct subsets of cells within the CD8+ population. For this purpose, whole T cells or fractionated T cells were sensitized with irradiated allogeneic non-T cells for 6 days, following which, CD8+ or CD8+CD11b- cells were isolated and cellular functions such as suppressor, killer precursor, and killer effector activity were assessed. The results showed that both class I-restricted alloantigen-specific killer effector and killer precursor cells belonged to the CD8+CD11b-CD45RA-CD29+ population. Moreover, these killer effector cells expressed the CTL-associated S6F1 molecule, an epitope of the LFA-1 antigen. In contrast, suppressor effector cells belonged to the CD8+CD11b-CD45RA+CD29- cell population. Although the UCHL1 antigen has been reported to define the CD4+CD29+ helper inducer cell, over 90% of allo-activated CD8+ cells expressed this antigen, whereas only 40-60% of these cells expressed either CD45RA or CD29 antigens. These results suggest that anti-CD45RA and anti-CD29 antibodies may provide useful tools for distinguishing between suppressor effector versus killer effector and killer precursor cells within the CD8+CD11b- population.     

Two-color flow cytometry and functional analysis of lymphocytes cultured from human renal allografts: identification of a Leu-2+3+ subpopulation

The phenotype of T lymphocyte subsets present in renal biopsies showing acute cellular allograft rejection in six patients on cyclosporine have been characterized in situ by immunoperoxidase staining, and after expansion in vitro in interleukin 2 (IL-2) by two-color flow cytometry, sorting, and functional analysis. After 8 to 42 days in organ culture, both Leu-3+ (CD4) and Leu-2+ (CD8) subsets were found in each culture, in a ratio that varied from 0.2 to 5.0, which was not significantly different than the results of in situ immunoperoxidase staining of the uncultured biopsy. The cultured cells were almost all Leu-4+ (CD3) T cells (89% +/- 4), which expressed the activation markers DR (82% +/- 6) and the IL 2 (CD25) receptor (15% +/- 4). The Leu-3+ cells were largely Leu-8- (90% +/- 6), whereas a minority of the Leu-2+ cells were Leu-15+ (CD11) (26% +/- 4). Only a small fraction of the Leu-2+ cells stained for Leu-7 (8% +/- 6). Functional analysis of FACS-purified Leu-2-3+ and Leu-2+3- populations indicated that both subsets proliferated in response to graft donor antigens in a mixed lymphocyte reaction (MLR) and produced IL 2. Only the Leu-2+3- population demonstrated donor-specific cytotoxic activity. A minor subpopulation in each culture were both Leu-3+ and Leu-2+ (2.0%). Leu-2+3+ cells from one biopsy were purified to homogeneity (99.8%), and were found to express the T cell antigen receptor complex Ti/CD3 (WT-31+, Leu-4+), but not the common thymocyte antigen CD1 (OKT6). The Leu-2+3+ cells neither responded in the MLR, nor showed any cytotoxic capacity. The Leu-2+3+ cells were capable of IL 2 but not interferon-gamma production. None of the purified cultures demonstrated NK activity. A subset of the purified Leu-2+3+ cells lost Leu-2+ during 1 to 3 wk in culture, and became Leu-2-3+. These studies provide evidence that the cells that infiltrate renal allografts during rejection include alloproliferative, lymphokine-producing cells of both Leu-2+ and Leu-3+ subsets. The Leu-2+3- cells are also highly cytotoxic against donor lymphocytes, indicating the presence of helper independent cytotoxic T cells. A minor population of Leu-2+3+ T cells that do not express donor specific function was also identified.     

Human lymphokine-activated killer (LAK) cells: identification of two types of effector cells

We analyzed the antigenic phenotype of lymphokine-activated killer (LAK) effector cells. Human blood lymphocytes were cultured for 3 days with 100 U/ml recombinant interleukin 2 (rIL 2), subpopulations isolated with monoclonal antibodies and a fluorescence-activated cell sorter (FACS) and assayed for cytotoxic activity against 51chromium labeled noncultured melanoma tumor cells. Initial experiments compared the LAK effector function of CD5+ T lymphocytes vs CD5- cells (predominantly CD16+ NK cells). The mean percent specific release at a 10:1 effector:target (E:T) ratio was 25% +/- 16 for CD5- cells, 10% +/- 6 for CD5+ cells, and 22% +/- 9 for unsorted cells. In contrast, when lymphocyte subpopulations were isolated before rIL 2 culture (LAK precursors), CD5- cells but not CD5+ cells developed LAK activity (28% +/- 12 vs 1% +/- 1, mean percent specific release, 10:1 E:T ratio), confirming our previous results showing that only CD16+ cells were LAK precursors. The discrepancy between LAK effector and precursor phenotypes suggested that LAK precursors acquired CD5 determinants during rIL 2 culture; however, double label immunofluorescence of rIL 2 cultured CD16+ cells showed that this was not the case. The data suggested that in the presence of other cell types, some T lymphocytes may develop LAK activity, but purified blood T lymphocytes do not develop LAK function when cultured with rIL 2 alone. We also analyzed LAK effector function in lymphocyte subpopulations defined by CD4 and CD8 antigens. The data showed that lymphocytes with a low density expression of CD8 and no expression of CD4 were enriched for LAK effector cells, whereas CD4+ and CD8- had less activity than unsorted cells. Lymphocytes with a high density expression of CD8 had activity similar to unsorted cells. We also assessed the contribution of Leu-7 (HNK-1) granular lymphocytes to LAK effector function. After culture with IL 2, lymphocytes were depleted of Leu-7+ cells by antibody and complement treatment and then were sorted into CD5+ and CD5- fractions. The cytotoxic activity of Leu-7-CD5+ cells was a mean 5% +/- 5 vs a mean 14% +/- 8 for the total CD5+ population (20:1 E:T ratio). The activity of Leu-7- CD5- was slightly less than the total CD5- fraction (21% +/- 9 vs 28% +/- 14, 10:1 E:T ratio). In conclusion, LAK effector function was highest in non-T cell (CD5- CD16+) populations and some activity was also present in T cell populations (CD5+ and predominantly Leu-7+).     

Activated and memory T lymphocytes in human milk.

Since activated macrophages and cytokines are found in human milk (HM), a flow cytometry study was conducted to determine whether T cells in HM display phenotypic markers of recent or previous activation. HM was collected during the first 3 d of lactation. The Paint-a-Gate program was used to optimize gating on the lymphocyte population. A mean +/- 1 SD of 4 +/- 3% of total HM leukocytes were lymphocytes and 96 +/- 3% were macrophages and granulocytes (N = 33 subjects). HM lymphocyte populations were further analyzed in five subjects. T cells (CD3+) represented 83 +/- 11% and B cells (CD19+) were 6 +/- 4% of HM lymphocytes. The mean CD4/CD8 ratio of T cells in HM was 0.88 (range 0.40-1.25). This ratio was significantly decreased compared to the peripheral blood (PB) of control adults (P less than 0.02) and postpartum women (P less than 0.02), due mostly to a significant increase in CD8+ CD3+ cells in HM compared to the PB of control adults (P less than 0.002) and postpartum women (P less than 0.05). T cells bearing markers of recent activation were significantly increased in HM compared to the PB of control adults: 85 +/- 7% of CD3+ cells in HM were HLA-DR+ (controls, 10 +/- 4%; P less than 0.001), and 15 +/- 6% of CD3+ cells in HM were IL-2R+ (controls, 6 +/- 2%; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.     

In situ localization of CD45 isoforms in the human thymus indicates a medullary location for the thymic generative lineage.

Previous work has suggested that the generative lineage within the human thymus can be defined by the selective expression of CD45 isoforms and is CD45RO- and predominantly CD45RA+. In order to physically localize these cells we have stained frozen sections of human thymus with antibodies to CD45RO (p180), and CD45RA (p205/P220), as well as with CD1 and HLA class I to define cortical and medullary areas, respectively. In the cortex, 70 to 90% of thymocytes were CD45RO+, whereas only 0.5% expressed CD45RA. Medullary cells were 30% CD45RO+, 29% CD45RA+; approximately 40% did not express detectable levels of either isoform but did express CD45 common determinants. To assess the degree of proliferation of cells expressing CD45 isoforms, we stained adjacent sections, or used double staining, with Ki67, an antibody that detects a nuclear Ag on proliferating cells. We found that CD45RA+ thymocytes are predominantly a resting medullary population with a small component in cell cycle, consistent with our analysis of human thymocytes by immunofluorescence, and with data in murine systems defining the generative lineage. To confirm that the CD1- or low, CD45RO-CD45RA+ thymocytes defined by immunofluorescence analysis were likely to have a medullary location, we analyzed the CD4/CD8 subset distribution of CD1-cells. From 80 to 90% of CD1-thymocytes are CD4+ or CD8+ single positives or CD-8- double negatives. CD1-thymocytes also include 12 to 14% CD4+8+ cells with a probable medullary location. A similar analysis of lymphocytes expressing a high density of HLA class I, which have a medullary location, confirmed the existence of CD4+8+ thymocytes in the medulla. Purified CD3-4-8- cells, previously shown to be CD1-CD45RA+, were also shown to bear a high density of HLA class I, indicating a medullary location. Correlative localization of a panel of Ag thus supports the argument for a medullary location of the thymic generative lineage.     

Immunoregulatory T lymphocytes in man. Soluble antigen-specific suppressor-inducer T lymphocytes are derived from the CD4+CD45R-p80+ subpopulation

Regulation of the immune response in man is largely dependent on interactions between cells of the cluster designation 4+ (CD4+) helper/inducer sublineage and the CD8+ suppressor/cytotoxic sublineage. When cultured with autologous antigen-primed CD4+ lymphocytes, CD8+ cells differentiate into suppressor T cells (Ts) that specifically inhibit the response of fresh autologous CD4+ cells to the priming antigen only. The current study was undertaken to analyze the roles in this suppressor circuit of subpopulations of the CD4+ sublineage distinguished from one another on the basis of their binding (or lack of binding) to monoclonal antibodies against molecules p80 (Leu8) and CD45R (p220/Leu18/2H4). When examined for the proliferative responses to alloantigenic stimuli, each of the four: CD4+p80+, CD4+p80-, CD4+CD45R+, and CD4+CD45R- populations proliferated vigorously, synthesized interleukin 2 (IL-2) and interferon and released soluble IL-2 receptors. However, the responses to soluble antigens such as Candida and diphtheria toxoid were exhibited by CD4+CD45R-, CD4+p80+, and CD4+p80- cells, but not by CD4+CD45R+ cells. When examined for their ability to induced CD8+ Ts in the Candida-driven suppressor-induction culture system, only CD4+p80+ and CD4+CD45R- cells induced strong suppression. Further, when CD4+CD45R- cells were separated into CD4+CD45R-p80+ and CD4+CD45R-p80- subpopulations, despite the ability of both subpopulations to respond to Candida, only CD4+CD45R-p80+ cells induced autologous CD8+ Ts. Activated CD8+ Ts suppressed not only proliferation but also the release of soluble IL-2 receptors by autologous antigen-activated CD4+ cells. Thus, the antigen-specific suppressor-inducer T cells appear to be derived from the CD4+CD45R-p80+ (Leu3+, Leu8+, 2H4-) subpopulation of the CD4+ sublineage.     

Distribution of ecto-5'-nucleotidase on subsets of human T and B lymphocytes as detected by indirect immunofluorescence using goat antibodies

Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin. Lymphocyte cell surface antigens were detected with phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD16, and anti-CD19. HB-4, an antigen present on a major subset of human peripheral blood B cells, was detected with murine monoclonal anti-HB-4 and PE-anti-mouse-kappa. Analysis showed that ecto-5'-NT was expressed on 32 +/- 7% of CD3+, 19 +/- 6% of CD4+, and 50 +/- 21% of CD8+ T cells, but not on CD16+ lymphocytes. Ecto-5'-NT was also expressed on 81 +/- 8% of adult peripheral blood B cells as defined by PE-anti-CD19; HB-4 was expressed on 84 +/- 7% of CD19+ cells. The two populations of B cells were not identical, however, because HB-4 was co-expressed on only 79 +/- 18% of ecto-5'-NT+ B cells. Two-color immunofluorescent staining of T cells from a patient with congenital agammaglobulinemia and low T cell ecto-5'-NT activity revealed reduced percentages of ecto-5'-NT+ cells in his CD3+, CD4+, and CD8+ populations. Thus, reduced ecto-5'-NT activity by enzyme assay was paralleled by reduced numbers of 5'-NT molecules on the cell surface. Two-color immunofluorescent staining of B cells from a patient with hypogammaglobulinemia and low B cell ecto-5'-NT activity also revealed markedly reduced expression of 5'-NT. HB-4 expression was normal, however, suggesting that the patient's B cells were blocked in maturation subsequent to the acquisition of HB-4 but prior to that of ecto-5'-NT. These results demonstrate that anti-5'-NT antibodies will be valuable tools for analyzing ecto-5'-NT expression and lymphocyte maturation in patients with immuno-deficiency diseases.     

Reactivity of lymphocytes to a progesterone receptor-specific monoclonal antibody

In this study we present evidence for reactivity of pregnancy lymphocytes, but not nonpregnancy lymphocytes, with the progesterone receptor-specific monoclonal antibody mPRI. Using an avidin-biotin peroxidase detection system, we found a nuclear staining in 14.6 +/- 3.7% (mean +/- SEM, N = 27) of pregnancy lymphocytes, while only 0.47 +/- 0.33% (mean +/- SEM, N = 15) of nonpregnancy lymphocytes reacted with the antibody. To characterize the receptor-bearing subset, CD8+ and CD4+ cells were depleted by complement-dependent lysis. Depletion of CD8+ cells was accompanied by 62 +/- 18% loss of progesterone receptor-bearing cells, while depletion of CD4+ cells resulted in a twofold increase in the number of positively staining lymphocytes. In nonpregnancy lymphocytes a 3-day PHA treatment, as well as allogeneic stimulation, resulted in a significant increase in the number of receptor-containing cells. These results suggest that pregnancy, but not nonpregnancy, lymphocytes contain progesterone binding structures, and that these are inducible by mitogenic or alloantigenic stimuli.     

Alloantigen-specific T suppressor-inducer and T suppressor-effector cells can be activated despite blocking the IL-2 receptor

To determine IL-2 requirement for activation of suppressor cells, PBMC were primed in one-way MLR in the presence of 10 micrograms/ml anti-IL-2R beta-chain antibody 2A3 (CD25) or control antibody, then irradiated and added as regulators in a fresh MLR. Cells primed in the presence of antibody 2A3 suppressed the proliferative response to fresh autologous lymphocytes to specific alloantigen but had no effect on the response to cells from third party donors. Priming in the presence of an antibody of irrelevant specificity induced only limited suppressor activity. Activated suppressor cells did not show cytolytic activity specific for the stimulators when tested at the time of the suppressor cell assay. To identify the subset(s) responsible for suppression, cells primed in the presence of antibody 2A3 were separated into CD4+/CD45RA+, CD4+/CD45RA-, and CD8+ subsets, which were irradiated and then tested. The suppressive activity was found predominantly in the CD4+/CD45RA+ subset, whereas CD8+ cells had some activity and CD4+/CD45RA- cells had none. No subset suppressed the response of autologous cells to third-party cells. When primed CD4+/CD45RA+ cells were cocultured with fresh autologous lymphocytes depleted of CD8+ cells, no suppression was observed, indicating that, although the CD4+/CD45RA+ cells can function as inducers of suppressors, they cannot function as suppressor-effectors. Conversely, CD8+ cells activated in MLR in the presence of 2A3 caused suppression, regardless of whether the fresh autologous responder population contained CD8+ cells. CD4+/CD45RA+ and CD8+ subsets isolated after priming in the presence of 2A3 also demonstrated Ag-specific suppression in the generation of cytotoxic T lymphocytes whereas CD4+/CD45RA- cells had no activity. Our data are consistent with the model that suppression of alloreactivity requires the cooperation of two types of cells, a CD4+/CD45RA+ suppressor-inducer and a CD8+ suppressor-effector population. Activated Tsi and fresh Tse or activated Tse alone can suppress lymphocyte proliferation and generation of CTL in response to specific Ag. Activation of Ag-specific T suppressor-inducer and T suppressor-effector cells appears to be relatively IL-2 independent and presumably require one or more other growth factors.     

Effector activity of decidual CD8+ T lymphocytes in early human pregnancy

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7-14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR(32)] of 32.7 +/- 5.8) and against K562 target cells (mean SCR(32) of 20.3 +/- 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8(+) T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.     

Lymphocyte subset analyses in healthy adults vaccinated with yellow fever 17DD virus

In this study the kinetics of humoral and cellular immune responses in first-time vaccinees and re-vaccinees with the yellow fever 17DD vaccine virus was analyzed. Flow cytometric analyses were used to determine percentual values of T and B cells in parallel to the yellow fever neutralizing antibody production. All lymphocyte subsets analyzed were augmented around the 30th post vaccination day, both for first-time vaccinees and re-vaccinees. CD3+ T cells increased from 30.8% (SE +/- 4%) to 61.15% (SE +/- 4.2%), CD4+ T cells from 22.4% (SE +/- 3.6%) to 39.17% (SE +/- 2%) with 43% of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2% (SE +/- 2.9%) to 27% (SE +/- 3%) with 70% corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7% (SE +/- 3%) to 80% (SE +/- 2.3%), CD4+ T cells from 24.9% (SE +/- 1.4%) to 40% (SE +/- 3%) presenting a percentage of 95% CD4+CD45RO+ T cells, CD8+ T cells from 19.7% (SE +/- 1.8%) to 25% (SE +/- 2%). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6% in first-time vaccinees and 20.7 to 62.6% in re-vaccinees. Regarding neutralizing antibodies, the re-vaccinees presented high titers even before re-vaccination. The levels of neutralizing antibodies of first-time vaccinees were similar to those presented by re-vaccinees at day 30 after vaccination, indicating the success of primary vaccination. Our data provide a basis for further studies on immunological behavior of the YF 17DD vaccine.     

Functional heterogeneity of vaccine-induced CD8(+) T cells

The functional status of circulating vaccine-induced, tumor-specific T cells has been questioned to explain their paradoxical inability to inhibit tumor growth. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited CD8(+) T cell precursor frequency among PBMC in 13 patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209-217(210 M) epitope. T cell precursor frequency increased from undetectable to 12,400 +/- 3,600 x 10(6) CD8(+) T cells after vaccination and appeared heterogeneous according to previously described functional subtypes: CD45RA(+)CD27(+) (14 +/- 2.6% of tHLA-staining T cells), naive; CD45RA(-)CD27(+) (14 +/- 3.2%), memory; CD45RA(+)CD27(-) (43 +/- 6%), effector; and CD45RA(-)CD27(-) (30 +/- 4.1%), memory/effector. The majority of tHLA(+)CD8(+) T cells displayed an effector, CD27(-) phenotype (73%). However, few expressed perforin (17%). Epitope-specific in vitro stimulation (IVS) followed by 10-day expansion in IL-2 reversed this phenotype by increasing the number of perforin(+) (84 +/- 3.6%; by paired t test, p < 0.001) and CD27(+) (from 28 to 67%; by paired t test, p = 0.01) tHLA(+) T cells. This conversion probably represented a change in the functional status of tHLA(+) T cells rather than a preferential expansion of a CD27(+) (naive and/or memory) PBMC, because it was reproduced after IVS of a T cell clone bearing a classic effector phenotype (CD45RA(+)CD27(-)). These findings suggest that circulating vaccine-elicited T cells are not as functionally active as inferred by characterization of IVS-induced CTL. In addition, CD45RA/CD27 expression may be more informative about the status of activation of circulating T cells than their status of differentiation.     

P-selectin (CD62) binds to subpopulations of human memory T lymphocytes and natural killer cells.

P-selectin (CD62) is a Ca(2+)-dependent lectin expressed on activated platelets and endothelium. Although P-selectin is known to function as a receptor for myeloid cells, previous studies indicated that P-selectin also bound to a subset of lymphocytes. Using a multi-color immunofluorescence assay we found that purified P-selectin bound to 12.2 +/- 4.1% of peripheral blood lymphocytes and that P-selectin could mediate adhesion of activated platelets to lymphocytes. A subpopulation of CD4+, CD8+, and CD16+ lymphocytes bound P-selectin. There was a marked preference for P-selectin binding to memory cells (CD45RO+) in both the CD4+ and CD8+ populations. Binding to all cell types was Ca(2+)-dependent and blocked by pretreatment of the cells with sialidase. These data suggest that P-selectin may play a role in the recruitment of specific lymphocyte populations to sites of inflammation.     

Adhesion molecules on peripheral blood lymphocyte subpopulations in the elderly

Adhesion molecules, such as CD49d, CD50 and CD62L, have important roles in many adhesive interactions involving cells of the immune system. Since it has been shown that many immunological alterations are present in aged subjects, we studied, by means of triple colour whole blood immunostaining and multiparametric flow cytometry, the expression and intensity level (MFI) of these molecules on peripheral blood lymphocyte subpopulations from 23 healthy elderly subjects and 13 young controls. In the elderly a decrease in total peripheral blood lymphocytes bearing CD62L antigen was observed (39 +/- 13% vs 63 +/- 6% and 745 +/- 312/mm3 vs 1,393 +/- 407/mm3; p<0.001), whereas the numbers of lymphocytes expressing CD49d and CD50 antigens were comparable in aged and young subjects. In addition, CD50 and CD62L MFI values on total peripheral blood lymphocytes were higher in elderly than in young subjects (5.23 +/- 1.03 vs 4.18 +/- 0.44, p = 0.001 and 2.60 +/- 0.35 vs 2.21 +/- 0.40, p = 0.005 respectively) while the intensity expression of CD49d was unchanged. The percentages and absolute numbers of T and B lymphocytes expressing CD62L were decreased in elderly compared to young subjects (CD62L+CD3+: 43 +/- 15% vs 66 +/- 9% and 581 +/- 257/mm3 vs 1,028 +/- 418/mm3, p<0.001; CD62L+CD19+: 78 +/- 12% vs 90 +/- 4%, p < 0.005 and 103 +/- 64/mm3 vs 207 +/- 98, p < 0.001). A decrease in the proportion of CD62L bearing NK cells was also observed in the elderly (25 +/- 14% vs 46 +/- 24%, p<0.005), although their absolute number was unchanged. No significant differences were detected in the proportion of T, B and NK lymphocytes expressing CD49d and CD50 antigens and only the absolute numbers of B cells expressing these adhesion molecules were lower in elderly (CD49d+CD19+: 121 +/- 71/mm3 and CD50+CD19+: 107 +/- 73/mm3) compared to young donors (CD49d+CD19+: 248 +/- 112/mm3 and CD50+CD19+: 235 +/- 120/mm3, p < 0.001). Moreover, the intensity of adhesion molecule expression was differentially modulated in the elderly depending on the specific lymphocyte cell population considered. The densities of CD49d, CD50 and CD62L antigens on B and NK lymphocytes from the two age groups were not different; on the contrary, T lymphocytes from elderly donors exhibited increased CD49d (1.69 +/- 0.09 vs 1.62 +/- 0.07, p < 0.05), CD50 (4.98 +/- 1.16 vs 3.77 +/- 0.46, p < 0.001) and CD62L (2.26 +/- 0.38 vs 1.99 +/- 0.37, p < 0.05) MFI values compared to young donors.     

Expression of variable exon A-, B-, and C-specific CD45 determinants on peripheral and thymic T cell populations.

A mAb (I/24) has been generated that is specific for a determinant on mouse CD45 molecules. Reactivity of this mAb with a panel of CD45 transfected cell lines demonstrated that the determinant recognized is dependent upon expression of one or more CD45 variable exons and that exon C is sufficient for its expression. The exon C-specific epitope detected by I/24 is expressed at high density on essentially all B lymphocytes and at an intermediate density on the vast majority of CD8+ splenic T cells. Two distinct subpopulations of CD4+ splenic T cells were detected, a minor subpopulation that expresses this exon determinant at high density and a major subpopulation that expresses it at a much lower density. This first identification of a CD45RC-specific reagent allowed a comparison of the expression of exon A-, exon B-, and exon C-specific determinants on peripheral and thymic lymphoid populations. When splenic lymphocytes were analyzed for expression of CD45RA (reactive with mAb 14.8), CD45RB (reactive with mAb 23G2 or mAb 16.A), and CD45RC (reactive with mAb I/24) determinants, it was found that each of these CD45 determinants had a distinct pattern of expression on CD4+ and CD8+ T cells and B cells. CD45RB and RC epitopes were also detected at high density on a small proportion (0.7 to 4.1%) of thymocytes. Both CD45RB and RC epitopes were found predominantly on CD4-CD8- and CD4-CD8+ thymocytes but were also found on small numbers of CD4+CD8+ and CD4+CD8- cells. The population of thymocytes that expressed CD45RB and CD45RC determinants displayed a novel TCR CD3 phenotype characterized by a level of expression that was intermediate between that seen in the larger CD3 bright and CD3 dull populations of thymocytes.     

Phenotypic analysis of a large number of normal human bone marrow sample by flow cytometry

Bone marrow aspirates from 48 healthy donors (34 adults, 14 children) were analyzed by flow cytometry (FACS Analyzer) after purification of low-density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labelling with 23 anti-hematopoietic cell monoclonal antibodies. Based on physical properties, these Ld BMC could be divided into four different populations called E, My, Mo and L, which comprised 14% +/- 9%, 31% +/- 16%, 10% +/- 5% and 45% +/- 14% of these cells, respectively. The phenotypic analysis of these different populations enabled the identification in E, of erythrocytes (Glycophorin A+, Rhesus D+, but negative for early erythroid differentiation markers such as the transferrin receptor (Tf. R) and the FA6-152 antigen); in My of cells of the myeloid lineage (VIM2+, HLA DR-); in Mo of cells of the monocytic lineage (VIM2+, CD14+) plus some myeloblasts (VIM2+, CD14-, HLADR+) and finally in L of a heterogeneous population including: 1. T lymphocytes labelled to the same extent by CD2, CD3, CD5 and CD6 (28% +/- 10%), B lymphocytes assessed by CD19 and CD20 (12% +/- 8%), Pre-B cells (CD10+ = 8% +/- 7%), less than 5% of "natural killer" cells (CD16+ or Leu7+) and finally, less than 6% of myelomonocytes (CD14+ and/or VIM2+). 2. The erythroid lineage (rhesus D+ = 42% +/- 20%, Tf.R+ and FA6-152+ = 32% +/- 12%). 3. Undifferentiated cells or progenitor cells (CD34+ = 7% +/- 5%). 4. Cells unlabelled by any antibodies (approximately 6%). We observed no difference between bone marrow samples from adults or children, with respect to physical properties, and with all but four immunological markers. A significantly higher proportion of B cells (CD19+ and CD10+) (P less than 0.001) and undifferentiated cells (CD34+ and HLADR+) (P less than 0.02) was observed in children. These data, obtained from a large number of bone marrow samples, could be used to quantify the imbalance of some bone marrow disorders.     

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.