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Surface markers of cloned human T cells with various cytolytic activities.

Human T cells stimulated in secondary allogeneic mixed lymphocyte culture (MLC) were cloned under limiting conditions in microculture systems using T cell growth factor and irradiated allogeneic cells. Clones with lytic activity against either phytohemagglutinin-induced blast cells bearing the stimulating alloantigen(s) (cytotoxic T lymphocyte [CTL] activity), L1210 mouse lymphoma cells coated with rabbit antibody (antibody-dependent cell-mediated cytotoxicity [ADCC]), or K562 human target cells were selected, expanded, and then analyzed for different surface markers, including rosette formation with sheep erythrocytes (E rosettes), receptors for the fc portion of IgG or IgM (Fc gamma R and Fc mu R), and a group of antigens recognized by monoclonal antibodies including Ia, 4F2, OKT8,a nd OKT4. All the cytotoxic cells were E rosette+, Ia+ and 4f2+. Expression of Fc gamma R was restricted to the clones active in ADCC. CTL clones were either OKT8+ or OKT8-. Furthermore, three of the OKT8- CTL clones were OKT4+. In addition, some cytolytic clones devoid of specific CTL activity were OKT8+. It thus appears that the claim that human CTL are OKT8+, OKT4-, and Ia- is not supported by the analysis of their phenotype at the clonal level.

Several surface membrane markers have recently been described that define T cell subpopulations in man. For example, receptors for immunoglobulin (Ig) isotypes (1) and membrane structures defined by xenoantisera (2) and, more recently, monoclonal antibodies (3), have been used to identify various subsets in peripheral T cell populations. Other markers, such as Ia antigens (4) and a group of surface antigens recognized by monoclonal antibodies (5,6), appear to be expressed on T cells primarily upon cell activation . Functional studies of T cell subsets have favored the idea that there is a link between the expression of given markers and T cell function (1)(2)(3) . However, a precise correlation between surface markers and cell function may be difficult if only a small percentage of the cells in a given subset is involved in the functional activity measured . It thus appears difficult to deduce the function of individual cells from their surface markers.
In an attempt to correlate more precisely the surface phenotype of human T cells with a given functional activity, we have analyzed 14 cloned T cells with various cytotoxic activities for a battery of surface markers, namely receptors for sheep erythrocytes (E rosettes), receptors for the Fc portion of IgG or IgM (FcyR and Fc[LR), and a group of antigens recognized by monoclonal antibodies, including la, 4F2 (6), OKT8, and OKT4 (3) .

Materials and Methods
Mixed Lymphocyte Cultures. Lymphocytes were isolated from peripheral blood lymphocytes (PBL) or spleen as previously described (1). Cells were suspended in RPMI 1640 containing 10% heat-inactivated human AB serum. Spleen cell suspensions were frozen and subsequently thawed as needed . Primary mixed lymphocyte cultures (MLC) were set up using PBL as responder cells and irradiated allogeneic spleen cells as stimulator cells, as previously described (7). For the preparation of secondary MLC, cells from day 7 (primary) MLC were centrifuged on a Ficoll-Hypaque gradient to remove dead cells, and then mixed at a 1 :10 ratio with irradiated stimulator spleen cells either in macrowells (4 X 106 total cells per well) or in tissue flasks (15 X 106 total cells per flask) . After 4-5 d of culture, secondary MLC cells were harvested, passed through Ficoll-Hypaque gradients, and washed . The majority of the recovered cells were large cells (>70%) and formed rosettes with sheep erythrocytes (SRBC) (>85%) .
Cloning of MLC Cells. Secondary MLC cells were submitted to vigorous vortex mixing (to dissociate possible cell clumps) and then seeded at 0.3 cells per well in round-bottomed microwells containing 10 5 irradiated stimulator spleen cells. The culture medium was RPMI 1640 containing 10% human AB serum and 50% supernate from phytohemagglutinin (PHA)stimulated human spleen cell cultures (as a source of T cell growth factor [TCGF]) (8) . The plates were centrifuged at 100 g for 5 min and then incubated for 4 h at 37°C . After centrifugation of the plates at 200 g for 5 min, cytolysis was assessed by counting the radioactivity of 0 .1 ml supernate for 1 min in a scintillation counter. The specific lysis was calculated according to Cerottini et al . (9) . Expansion of Clones. Cells of selected clones were added to macrowells together with irradiated spleen cells (2 X 106) of the same donor as those used as stimulator cells in RPMI 1640 containing 10% human AB serum and 50% TCGF. Under these conditions, the majority of microcultures showed strong proliferation, generating a number of cells suitable for further analysis (average 2 X 106 cells) in 7-12 d . Cells derived from the same clones were pooled, passed over Ficoll-Hypaque gradients, counted, and analyzed for cytolytic activities against the three types of target cells used in the initial selection and for surface markers .
Analysis of Surface Receptorsfor SRBC andfor the Fc Fragments of IgG or IgM (FcyR and FcUR) . The techniques have been extensively described in a previous report (1) . Neuraminidase-treated SRBC were used, and the proportion of rosetting cells was evaluated by scoring at least 400 cells . FcyR-or FcAR-bearing cells were detected by rosetting assays using ox erythrocytes coated with purified IgG or IgM fractions of rabbit anti-ox erythrocyte antisera.
Analysis of Surface Antigens by Monoclonal Antibodies . The reactivity of cloned cell suspensions or normal PBL with PTF (anti-Ia) (a generous gift of Dr . G . Damiani), 4F2 (6), OKT8, and OKT4 (3), monoclonal antibodies was determined by indirect immunofluorescent staining using fluorescein isothiocyanate-conjugated goat anti-mouse IgG . Flow cytometry of stained cells was performed on a fluorescence-activated cell sorter (B D FAGS Systems, Moutain View, Calif.) as described previously (6) .
Subclones. Subclones were obtained by seeding cells from two selected cytotoxic clones at 0 .3 cells per well in microwells under the same culture conditions used for cloning . The cytolytic activities of eight expanded subclones derived from each clone were evaluated on the three types of target cells mentioned above .  Table I .
Four expanded clones that were devoid of cytolytic activity are included for comparison . It can be seen that 8, 4, and 2 of the 14 cytolytic clones tested displayed CTL, NK, or ADCC activity, respectively.
Two clones selected for their high CTL activity were subcloned by limiting dilution .
From each parental clone, eight subclones were tested for lytic activity against the target cell types used in this study . All subclones exhibited a high lytic activity against the corresponding blast target cells without detectable NK or ADCC activity (data Cytolytic Activity and Surface Markers of Human T Cell Clones Percent of specific lysis at 30 :1 lymphocyte per target ratio. *Underlined numbers indicate clones with a given cytolytic activity . § Percent of rosette-forming cells .~P resence or absence of surface antigens was analyzed by fluorescence-activated cell sorter . Virtually 100% of the cells of each clone were either positive or negative for a single antigen. ND, not determined . not shown) . Although these results are in support of clonality of the original isolates, they also demonstrate that the cytolytic pattern of the clones tested was stable under the conditions used for their expansion . Table I, all clones were E rosette positive, with 73-94% of the cells in individual clones forming rosettes . The fact that a small percentage of these cells did not form E rosettes is probably due to limitations of the technique used . Alternatively, the possibility exists that cells at different stages of the cell cycle or with different metabolic activity vary in their expression of receptors for SRBC . In support of this contention is the finding that five individual subclones derived from the E rosettenegative fraction of a putative clonal isolate were mostly E rosette positive (unpublished observation) .

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In 12 cytolytic clones, FcyR were virtually absent, whereas 30-40% of the cells in the two clones with ADCC activity were FcyR positive . It is of interest that these two clones had no lytic activity against K562 target cells . Conversely, the four clones reactive against K562 target cells were FcyR negative and devoid of K cell activity.
It thus appears that ADCC and NK cell activities, although they are mediated by the same (FcyR-positive) subset in PBL populations, can be dissociated in clones derived from alloreactive MLC populations . Flow cytofluorometric analysis showed that all (cytolytic and noncytolytic) clones expressed relatively large amounts of la antigens . Virtually 100% of the cells in individual clones were la positive (Fig . 1) . Similar results were obtained when the clones were analyzed for expression of 41`2 antigen. In contrast, only four of the cytolytic clones were OKT8+ . Among these clones, three were cytolytic against K562 target cells, whereas one had CTL activity . Of the seven OKT8 -CTL clones, three were OKT4 + . The latter antigen was also expressed in the two clones active in ADCC and two of four noncytolytic clones . Flow cytofluorometric  analysis of individual CTL clones indicated that virtually all cells were positive or negative for either OKT8 or OKT4 antigens (Fig . 1) . Previous studies at the population level have provided conflicting results as regards la expression in human CTL. Whereas Reinherz et al. (10) reported that CTL activity displayed by in vitro alloactivated T cell populations was restricted to Ia-negative lymphocytes, earlier work from our laboratory (6) indicated that Ia+ and Ia MLC subpopulations exhibited similar levels of specific cytolytic activity . Although it cannot be ruled out that the procedure used in the present study (including the use of TCGF) for the derivation of expanded clones may favor the proliferation of lapositive cells, it is more likely that these conflicting results can be explained by differences in sensitivity of the methods used for the detection and isolation of Ia+ cells .
Previous studies have shown that CTL activity of MLC populations was restricted to an FcyR-negative subpopulation expressing 4F2 antigen (6) . The present study confirms at the clonal level that CTL generated in MLC are 4F2+ . It is evident, however, that this antigen cannot be used as a specific marker for CTL because it is also expressed in noncytolytic clones (Table I) . Moreover, these results are in agreement with our previous studies showing that CTL activity in MLC populations is confined to the FcyR-negative fraction of the 4F2 + subset (6) .
Using monoclonal antibodies of the OKT series, Reinherz and Schlossman (3) reported that CTL generated in MLC expressed the OKT8+ , OKT4-phenotype.
Such a phenotype was observed in only one of the eight CTL clones analyzed in the present study. Moreover, three of the OKT8 -CTL clones were found to express the were E rosette', la', and 4F2 + . Expression of FcyR was restricted to the clones active in ADCC . CTL clones were either OKT8 + or OKT8-. Furthermore, three of the OKT8 -CTL clones were OKT4+ . In addition, some cytolytic clones devoid of specific CTL activity were OKT8 + . It thus appears that the claim that human CTL are OKT8 + , OKT4-, and Ia is not supported by the analysis of their phenotype at the clonal level .