relationship of fc-igg and fc-igm receptors to the antigens defined by okt-antibodies and the acid...

Immunobiol., vol. 160, pp. 424-437 (1982)

Department of Clinical Immunology and Blood Transfusion, Medizinische Hochschule Hannover, Hannover, Federal Republic of Germany

Relationship of Fc-IgG and Fc-IgM Receptors to the Antigens Defined by OKT -Antibodies and the Acid a-Naphthyl-Acetate-Esterase Spot within Human T Cells

W. J. PICHLER, MARIE-LurSE LANGE, CHRISTIANE BIRKE, and H. H. PETER

Received September 1, 1981 . Accepted October 26, 1981

Abstract

The expression of receptors for Fc-IgG and Fc-IgM on human T cells was correlated to the antigens recognized by monoclonal antibodies of the OKT series (T3, T4, T8, 11 , Ml) and to the presence of the dot-like cytoplasmic a-naphthyl-acetate-esterase activity (ANAE). ANAE spots were demonstrated in cyto-centrifuged smears; Fe-receptors (Fc-R) were shown using rosetting techniques which also served to enrich the respective cell populations; OKT antigens were detected by indirect immunofluorescence and OKT4 or OKT8-enriched subsets were obtained by antibody and complement-dependent lysis. Total T and Fc-IgM-R expressing T cells had a similar distribution of OKT antigens (90-95 % T3 +, - 60 % T4 +, - 30 % T8+, :$ 9 % Ml +). The Fc-IgG-R expressing T-cell fraction differed in that it contained less OKT3 + cells (72 %), about 34 % OKMI + cells and had an elevated percentage of OKT8+ cells (53 %). Similarly, OKT8-enriched cell fractions expressed more Fc-IgG-R (44 %) than unseparated T cells (12 %) or OKT4-enriched T-cell populations (16 %). Modulation of the Fc-IgG-R by exposure to immune complexes did not alter the expression of OKT antigens. Fc-IgG-R were, however, not restricted to OKT8+ cells, as simultaneous visualization of OKT antigens and Fc-R revealed that 7-11 % of OKT4 + cells were also Fc-IgG-R-positive. Thus Fc-IgG-R appear to be preferentially expressed on OKT8+ cells, but also OKT4+ cells were able to express Fc-IgG-R.

As regards the dot-like ANAE activity, T cells expressing Fc-IgM-R exhibited 80-90 % of ANAE positivity, whereas only 20-30% of the Fc-IgG-R-bearing T cells were ANAE+ . Stimulation of T cells expressing Fc-IgM-R with ConA led to a dramatic drop in the percentage of ANAE + cells after 48 to 72 hrs (80 % to 20 %) suggesting that this cytochemical marker is lost during blast transformation. Enrichment for OKT4+ and OKT8+ subsets, on the other hand, did not result in significantly altered percentage of ANAE + cells compared to the unseparated T cells as (OKT4+ were to 88% and OKTS+ to 73%) ANAE-positive.

These data demonstrate that OKT antigens as well as Fc-IgG-R, Fc-IgM-R, and the ANAE spot are T-cell markers defining different aspects of T-cell function. This does not rule out that certain markers correlate to each other: their combined application may be useful further to dissect the T cell system.

Supported by SFB 54, Projects G12 and G13 Part of this publication has been presented in the Leukemia Marker Conference, Vienna

1981 (1). Abbreviations: ANAE = acid a-naphthyl-acetate-esterase activity; Fc-R, Fc-lgG-R, Fc

IgM-R = Fe-receptor for IgG or IgM; EA = erythrocyte-antibody complex; EAC = erythrocyte-antibody-complement complex; SRBC = sheep red blood cells.

Comparison of Different T-Cell Markers . 425

Introduction

During the last year, different attempts have been made to achieve a valuable subclassification of human T cells by using cell-surface characteristics. Of major importance was the subclassification of human T cells on the basis of expression of different Fc receptors (Fc-R) (2) or by distinct reactivity with monoclonal antibodies (3). Some of the monoclonal antibodies used to subclassify human T cells seem to identify so-called differentiation antigens and are thus comparable to the Ly-alloantigen system in mice (4). As the differentiation of T cells is also accompanied by a diversification of functional capacities, certain of the differentiation antigens can be used as markers for T cells programmed for helper/inducer or suppressor/cytotoxic functions. Other membrane structures like i.e. Fc-R or la-like antigens seem to reflect the level of activation of T cells. Expression of la-like antigens (D/DR locus) have been found to be associated with activation of T cells (5, 6). Similarly, Fc-R appear to represent markers which are expressed on relatively mature T cells only and might be associated with activation of T cells (7).

To achieve a better understanding of the meaning of the various human T-cell markers and their relationship inbetween, we analysed the antigens recognized by OKT antibodies, the expression of Fc-R and la-like antigens (D/Dr locus) and compared them also with a cytochemical marker, namely the presence of the acid a-naphthyl-esterase (ANA E) spot within T cells. The latter cytochemical marker is found in most, but not all, sheep-redblood-cell rosetting cells and has the advantage of being suitable for defining human T cells in tissue sections (8, 9).

Materials and Methods

Lymphocyte isolation

Defibrinated blood from healthy human donors was diluted with phosphate-buffered saline and with an iron filing containing salt solution (LSR, Technicon Company, Frankfurt, Germany) at ratios of 3 : 2 : 1, respectively. The mixture was placed in falcon tissue culture flasks (No. 3024), rotated at room temperature for 30 min and then layered over Ficoll Metrizoate for gradient centrifugation (all products from Seromed, Munich, Germany). The cells obtained from the interphase were depleted of adherent cells by plastic adherence for 1 hr, and T -cell-enriched fractions were prepared by rosette formation with neuraminidase-treated sheep red blood cells (SRBC) as previously described (10). The non-rosetting cell fraction, comprising both B cells and cells termed K cells, L cells, or null cells (K/ L cells), was further separated into B or KIL cell fractions by rosette formation with EAC' (bovine red blood cells coated with IgM antibody and human complement). B cells, having C' receptors, formed EAC' rosettes and sedimented in a Ficoll-Metrizoate gradient into the pellet, while KlL cells lacked C' receptors and could be harvested from the gradient interphase.

Lymphocyte surface markers

Fc-R were detected by rosetting with IgG or IgM-coated bovine red blood cells (EA-IgG or EA-IgM) as previously described (10). Rosetting with IgG was also used to separate Fc-IgG-

426 . W. J. PICHLER, M.-L. LANGE, C. BIRKE, and H. H. PETER

R-positive (TG) from Fc-IgM-R-positive T cells (TM) by gradient centrifugation. About 90 % of the Fc-IgG-R-lacking cells expressed Fc-IgM-R (10).

Reactivity with OKT antibodies was demonstrated using indirect immunofluorescence. T cells, in a concentration of 1 X 107 cells/ml, were incubated with equal amounts of 1 : 20 to 1 : 40 diluted OKT antibodies (Ortho Diagnostics, Raritan, N.J. 08869, U.S.A.) for 30 min at 4°C; normal mouse serum, diluted 1 : 100, served as control. Afer two washes with PBS, containing 2 % fetal calf Serum and O.lN NaN), the cells were stained with fluorisothiocyanate (FITC) or rhodamine-labelled goat anti-mouse antibodies (Cappel Lab, Cochranville, Pennsylvania, U .S.A.). Positive reactions were evaluated either with a Leitz orthoplane microscope using Ploem epiillumination or an FACS IV.

To test for simultaneous expression of OKT antigens and Fc-IgG-R, double-labelling experiments were performed

Monocyte/macrophage-depleted cells were first stained with the OKT antibodies and the goat anti-mouse, FITC-Iabelled antibodies. The cells were washed twice, resuspended to -1 X t07/ml and mixed with EA-IgG. After centrifugation for 5 min at - 800 rpm, the cells were incubated for 30 min at 4°C; the rosette formation and indirect immunofluorescence were evaluated using the fluorescence-microscope. At least 200 cells were evaluated.

Separation of OKT4+ or OKTa+ -T-cell subsets

OKT4 or OKT8-enriched cell fractions were prepared by the method described by THOMAS et al. (10). Briefly, T cells in a concentration of 15 X 106/200 III were incubated for 30 min at 37°C with 200 III of 1 : 10-diluted OKT4 or OKT8 antibody, respectively, and 1 : 10-diluted rabbit complement. This procedure was repeated and resulted in T-cell fractions highly enriched for the respective T cell subset (Table 2). T cells treated with complement only served as control.

Preparacion of smears and scaining for acid a-naphchyl-escerase (ANAE)

Optimal cyto-centrifuged cell smears were obtained as previously described (9). Briefly, 5-10 X 106 cells in 100 fll of RPMI/5 % FCS were spun for 10 min at 150 X g ina Shandon Elliot Cytro-centrifuge. The smears were immediately fixed by air drying at room temperature . A reaction mixture of hexazotized pararosaniline was prepared by combining two solutions : (1) 4 % pararosaniline base (E. Merck, Darmstadt, West Germany, Cat. No. 7601) dissolved in 2N HCI, and (2) freshly prepared 4 % sodium nitrite (E. Merck, Darmstadt, West Germany, Cat. No. 6549) in distilled water. Four drops of each solution were mixed together and allowed to stand for 60 s: 40 ml of PBS, pH 6.5, plus 10 mg of a-naphthyl acetate (Serva, Feinbiochernica, Heidelberg, West Germany, Cat. No. 30040) dissolved in 1 ml of acetone were then added, and the solution was filtered. The slides were incubated for 1 hr at room temperature in the reaction mixture, rinsed in distilled water, and counter-stained with hemalaun (E. Merck, Darmstadt, West Germany, Cat. No. 9249) for 10 min. After three more washes in distilled water, the slides were mounted with permount and viewed under a Zeiss microscope equipped with a high-resolution oil-immersion objective. 200-250 cells were examined for the percentage of cells expressing the typical dot-like ANAE activity. Mononuclear phagocytes and granulocytes were distinguished on the basis of their cytomorphology.

Results

Table 1 summarizes the distribution of OKT-reactive cells in total T-cell fractions and T-cell subsets enriched on the basis of Fc-IgG-R or Fc-IgM-R expression. The distribution of OKT antigens as well as the reactivity with OKI1 or OKMl was similar in total T and TM-cell fractions. The TM-cell fraction had constantly the highest percentage of OKT3-positive cells and


Table 1. Relationship between differentiation antigens and Fc receptors on human T cells

T -cell-enriched fractions'

Monoclonal Total SRBC- Fc-IgM-R-antibody rosetting cells expressing cells

OKT3 93.4b 97.6 (± 3.0) c (± 1.8)

OKT4 64.1 70.0 (± 8.2) (± 10.9)

OKT8 31.1 32.4 (± 5.1) (± 13.4)

OKIl 4.2 3.1 (± 2.5) (± 3.4)

OKMI 9.4 3.0 (± 5.2) (± 2.9)

• The respective cell fractions were enriched to > 90 %. b Mean value of 9 experiments. C Plus/minus standard deviation.

Fc-IgG-Rexpressing cells

72.6 (± 7.2)

28.3 (± 14.6)

36.9 (± 8.1)

7.8 (± 3.3)

34.4 (± 15.9)

contained only very few OKM1-positive cells. Like the unseparated T cells, Fc-IgM-R-expressing cells comprised both OKT4 and OKTS-positive cells, whereby the percentage of OKT4-positive cells was slightly increased compared to the total T-cell fraction (70 % versus 64 % ).

Quite different results were obtained by testing the T-cell fractions enriched on the basis of Fc-IgG-R expression. T G-enriched cell fractions had the lowest percentage of OKT3-positive cells (mean value 72 %), and about 34 % of the cells reacted with OKM1; these mean percentages and specifically the values of certain single experiments exceeded 100 % suggesting that some cells can express both OKMl and OKT3 antigens.

The percentage of OKT3-positive cells in TG-cell-enriched fractions was higher than in previous reports (12, 13) and allowed the analysis of the T Gcell fraction with T -subset-specific antibodies. The data were interesting as both OKT4 and OKTS-positive cells were found within the TG cells, but in contrast to the other T-cell fractions, the percentage of OKTS-positive cells was higher than the percentage of OKT4-positive cells. Taking into consideration the fact that only about 72 % of the cells were OKT3-positive and assuming that the OKT4+ and OKTS+ cells belong (mainly) to the OKT3 + -cell fraction, the mean percentage of OKTS-positive cells would be - 51 %, and of OKT4-positive cells - 39 %. In addition to this shift in the relationship between OKT4 and OKTS-positive cells, the TG-cell fraction was also distinguished by a relatively higher percentage of Iapositive cells than the other T-cell fractions (- S %).

The FACS-analysis of T, TM , and TG-cell fraction (Fig. 1) is in agreement with the data in Table 1. The graphic visualization of the fluorescence intensity provides additional information, as the different patterns of fluorescence intensity obtained suggest a wide spectrum of antigen density.

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Table 2. Expression of Fc-IgG or Fc-IgM receptors on OKT4 or OKTS-enriched T-cell fractions'

% positive cellsb

OKT4-enriched OKTS-enriched T cellsc

OKT4 91 11 72 (3.9) (9.1) (10.7)

OKTS 7 S9 27 (3.9) (5.5) (7.S)

Fc-IgG-R 16 44 12 (3.0) (6.1) (2.7)

Fc-IgM-Rd 77 51 73 (3.5) (13.7) (4.4)

OKM t< 14 21 17

• Enrichment of OKT4 + cells was achieved by treating the T cells with OKTS and C; OKTS+ cells were enriched by treating by treating the T cells with OKT 4 and C.

b mean of 4 experiments (± S.D.). C Control T cells were treated with C' only. d Fc-IgM-R were detected after culture for 24 hrs at 37°C. e Results of 1 experiment only.

The indirect immunofluorescence of OKT3, OKT4, and OKMI antigens revealed a relatively narrow spectrum of fluorescence intensity; in contrast, the reactivity with OKT8 ranged from very low to very bright fluorescence intensity, suggesting a wide quantitative distribution of OKT8 antigens. Also shown is an analysis of non-B-non-T cells, so-called K/L cells, which is a cell fraction enriched for effector cells in natural cytotoxicity. In agreement with previous reports, we found that these cells are mainly OKMI-positive and lacked most T-cell-specific antigens (14).

Table 2 depicts the Fc-IgG-R and Fc-IgM-R distribution on OKT4 or OKT8-enriched cell fractions. The complement-dependent lysis of OKT4-positive or OKT8-positive cells resulted in cell fractions enriched for OKT4 + or OKT8+ cells to over 89 %, respectively. OKT4-enriched cell fractions had a similar distribution of Fc-IgG-R or Fc-IgM -R as the total Tcell fraction. The OKT8-enriched cell fraction was enriched for Fc-IgG-Rexpressing cells, while those with Fc-IgM-R were diminished. These data substantiate the above-mentioned association between OKT8 antigen and Fc-IgG-R.

This association between expression of Fc-IgG-R and reactivity with OKT8 is further underlined by the data presented in Table 3. Simultaneous visualization of both membrane markers in freshly isolated peripheral blood lymphocytes yielded a higher percentage of cells expressing OKT8 antigens and Fc-IgG-R (19-43 %) than OKT4 antigens and Fc-IgG-R (7-11 %). However, these later results emphasize that Fc-IgG-R are not restricted to OKTS+ cells only.


Table 3. Simultaneous demonstration of OKT antigens and Fc-IgG receptors

% positive cells of freshly isolated PBL"

OKT+ OKT+ and Fc-IgG-R Fc-IgG-Rb No marker

T4 73 9(11 %)C 17 53 5 ( 8.5%) 18 26 39 3 ( 7 %) 22 36

T8 9 7 (43 %) 24 60 24 6 (.20 %) 23 41 26 6 (19 %) 22 41

" Three experiments b By EA-IgG rosetting C Percentage of Fc-IgG-R-positive cells of the total T4+ or T8+ cells.

In previous experiments, an immune-complex-induced shift in Fc-R expression of T cells could be shown as Fc-IgG-R were lost and Fc-IgM-R expressed after 24-hrs culture (10). We therefore investigated whether a change in expression of Fc-R is accompanied by a change in expression of OKT antigens. In Table 4, the effect of IgG-immune complex induced modulation of Fc-IgG-R on the expression of OKT, OKM1, and OKIl antigens is shown. In contrat to the change from Fc-IgG-R to Fc-IgM-R expression, the antigens detected by the monoclonal antibodies were not significantly affected by the exposure to immune complexes. The expression of OKTs antigen remained relatively stable: The percentage of OKT3 and OKT4-positive cells slightly increased during the 40-hrs-culture, while simultaneously the percentage of OKM1-positive cells decreased.

A third T-cell marker system, namely the dot-like, cytoplasmic ANAE staining pattern was also evaluated. Table 5 illustrates the presence of this cytochemical marker in T-cell subsets enriched on the basis of the expres-

Table 4. Fc-IgG receptor expressing T cells: reactivity with OKT antibodies before and after transition to Fc-IgM receptor expression

Isolated Fc-IgG-R-positive cells (percentage positive cells")

Before transition

Fc-IgG-R Fc-IgG R OKT3 OKT4 OKT8 OKM1 OKI1

a Mean ± S.D. of 3 experiments.

80 (± 3.1) 2 (± 1.1)

72(± 1.1) 20 (± 19.2) 37 (± 8.4) 42 (± 15.9) 12 (± 7.1)

b The cells were cultured for 40 hrs at 37°C and then retested, see ref. (10).

After transitionb

5 (± 2.2) 61 (± 2.0) 82 (± 4.0) 34 (± 5.6) 34 (± 6.2) 28 (± 4.6)

6 (± 3.1)


Table 5. Relationship of the ANAE spot within T cells to the expression of Fc-IgG or Fc-IgM receptors and to the reactivity with OKT4 or OKT8 antisera

T-cell fractions enriched' for

T Fc-IgG-R Fc-IgM-R OKT4+ OKT8+

(% positive cells)

ANAE+ 70 19.5 79 88 73 positive cells (± S.D.) (± 6.9) (± 5.6) (± 7.0) (± 7.6) (± 7.9)

'The respective cell fractions were enriched to > 89 %, using rosetting techniques or C'-dependent lysis.

sion of Fc-IgG-R, Fc-IgM-R, OKT4, or OKT8 antigens. As can be seen, the ANAE spot was clearly more expressed in TM cells than in TG cells (9). However, it was almost equally distributed in OKT4 and OKT8-enriched T-cell populations (88 % and 73 %, respectively) suggesting that the ANAE marker is present in both T helper/inducer and T suppressor/cytotoxic cell lineages.

To analyse the relationship of these three marker systems further, markers on T cells cultured up to 72 hrs in the absence and presence of ConA were investigated. As shown in Table 6, culture alone did not induce a shift in OKT markers, ANAE activity, or expression of Fc-IgM-R. The expression of the Fc-IgG~R decreased in in-vitro culture, a finding already described (10). Stimulation with ConA did not effect the expression of the OKT antigens, which appear to be stable in in-vitro culture. In contrast, both the expression of Fc-IgG-R and Fc-IgM-R as well as the expression of the ANAE spot was diminished.

Table 6. Decreased expression of ANAE spot and Fc-IgG-R and Fc-IgM-R on ConA-activated cells

Fc-IgG-R Fc-IgM-R ANAE OKT

T3 T4 T8 % positive cells

T cells' 26 21 (58)b 72 90 66 31 After 72 hrs culture 8 60 64 98 78 44 After 72 hrs culture with ConAe, d 4 31 38 96 62 48

, T cells were tested immediately after enrichment: 94% of the cells formed SRBC rosettes. b 58 % of the T cells formed rosettes with EA-IgM after culture for 18 hrs at 37°C. e The cells were washed twice with 20 mM a-methyl-mannoside-containing medium before

marker tests were performed. d ConA concentration: 6 I-Ig/ml.

(a)

(b)

(c)


Discussion

In analysing T cells by means of cell-membrane characteristics, one has to differentiate between markers reflecting the genetic commitment of aT-cell subclass, provided by the so-called differentiation antigens and cell membrane structures which reflect the level of activation of T cells. The expression of Fc-IgG-R or Fc-IgM-R by human T cells was originally considered to correlate to supressor and helper T cells (2), but various functional and morphological studies have shown that Fe-Ron T cells do not reflect the commitment to either helper/inducer or suppressor/ cytotoxic T-cell function (7). These functions, which are genetically determined, may be mirrored by the expression of antigens recognized with the recently developed monoclonal antibodies against T-cell subsets (3). A previously published comparison of the expression of Fc-R and the reactivity with monoclonal T-cell and T-subset-specific antibodies (OKT3, OKT4, OKT5) revealed that Fc-IgM-R expressing T cells comprise both OKT4 (helper/inducer) and OKTs (suppressor/cytotoxic) T-cell phenotypes (12). A surprising finding of this study was the fact that most Fc-IgGR -expressing, SRBC-rosetting cells, so-called T G cells, lacked reactivity with T-cell-specific monoclonal antibodies, but reacted with OKM1, a monoclonal antibody which was postulated to define an antigen present on cells of the granulocyte/monocyte lineage, but not on T cells (15).

The data presented here support the concept of the functional heterogeneity of Fc-IgM -R -expressing T cells. In agreement with the report by REINHERZ et al. and functional investigations of Fc-IgM -R-expressing T cells (7, 12), the Fc-IgM-R-enriched cell fractions reacted with OKT4 and OKTs antibodies. The percentage of OKT4 and OKTS-reactive cells was similar to the total T-cell fraction. Depletion of Fc-IgG-R -positive cells resulted in a small increase of OKT3 and OKT4-positive cells.

The percentage of OKT-reactive cells within the so-called TG cell fraction differed from the report by REINHERZ et al. (12) who found that 50-SO % of the cells in so-called TG + -cell fractions reacted with OKM1, but lacked reactivity with OKT3. In contrast, in our studies the majority of Fc-IgG-Rpositive, SRBC-rosetting cells reacted with the T -cell-specific OKT3-antibody; only about 32 % of the cells were positive with OKM1 in indirect immunofluorescence. The percentage of OKM1-positive cells within the so-called T G-cell fraction was thus higher than in the total T or T M-cell fractions, but lower than originally reported. This quantitative difference may have various reasons: of major importance seems to be the percentage of co-purified K/L (null) cells, contaminating the TG-cell fraction. As repeatedly discussed (7,16), these K/L cells have similar surface characteristics as so-called TG cells. They may, under certain circumstances, rosette

+- Fig. 2. ANAE-staining pattern in unseparated T cells. (a) T cells enriched forT4 + cells (b) and T cells enriched for Ts + cells (c).


with SRBC and are OKMI-positive (Fig. 1, (14». They are most likely the cells responsible for the natural cytotoxicity (NK) and accessory cell potential found in TG-cell fractions (16,17). Through extensive depletion of adherent cells as well as through a relatively vigorous resuspension of SRBC-rosetting cells after the Ficoll/Metrizoate gradient centrifugation, the percentage of OKMI-positive cells in the TG cell fraction could be kept low in our T G cell preparations.

In this context, another observation seems to be of interest. Repeatedly it could be observed that the sum of OKMI-positive and OKT3-positive cells in T -cell-enriched fractions exceeded 100 %, suggesting the presence of some cells expressing both OKMI and T-cell-specific antigens (Tables 1,2). This calculated overlap was highest in Fc-IgG-R-enriched T-cell fractions, which were also enriched for OKT8+ cells. In view of the wide spectrum of antigenic density of OKTS, it appears likely that the overlap observed is due to the simultaneous expression of OKMI and OKT8. Indeed, the existence of such a cell was supported by the analysis of a chronic lymphocytic leucemia expressing Fc-IgG-R and the phenotype 0 KM 1 +, T3 +, T 4 -, T8 + (IS). These cells had good NK-potential but no suppressive activity in pokeweed-mitogen-driven B-cell maturation. The description of such a cell with a heterogeneous cell-sudace phenotype helps to understand the longlasting dispute over the origin of the NK-effector cells, and underlines the conclusion of Fox et al. that one monoclonal antibody might not be sufficient to determine the cellular origin (13).

The TG-cell-enriched fractions demonstrated also a heterogeneity of inducer and suppressor T-cell phenotypes; in contrast to the TM-cellenriched fractions, the T G-cell-enriched fractions contained more OKTSpositive cells than OKT4-positive cells. This finding is in agreement with functional studies which attributed to TG cells mainly suppressor-cell functions (2). It was also supported by the double-marker analysis of both OKTS antigens and Fc-IgG-R, as 13-43 % of the OKT8+ cells also expressed Fc-IgG-R (Tables 2 and 3). It is, however, important to note that expression of Fc-IgG-R is not solely restricted to the OKTS suppressor! cytotoxic phenotype. Some Fc-IgG-R-expressing cells appear to belong to the OKT4 helper/inducer cell lineage: OKT4-enriched cell fractions contained Fc-IgG-R-expressing cells and TG-cell-enriched fractions contained OKT4-positive cells. Indeed, simultaneous evaluations of OKT4 reactivity and expression of Fc-IgG-R revealed that 8-12 % of freshly prepared T 4 + cells expressed also Fc-IgG-R (Table 3).

As OKT antibodies are postulated to define differentiation antigens and are thus expected to be stable membrane structures (19, 20), the relationship of OKT antigens to Fc-R was also investigated in situations where Fc-R were modulated. In previous experiments we could demonstrate that exposure to IgG-immune complexes modulates Fc-IgG-R such that they disappear and Fc-IgM-R appear (10). This transition from Fc-IgG-R to FcIgM-R expression occurs in almost all Fc-IgG-R-expressing T cells, but did


not have a significant effect on OKT antigens. Similar percentages of OKTreactive cells were found before and after transition of Fc-R. The immunecomplex-induced modulation of Fc-R does not affect the antigens recognized by OKT antibodies.

The observed decrease of OKM1-positive cells and simultaneous increase of OKT3-positive cells after 24-hrs culture is, in our opinion, not due to a change in antigenic expression. It is more likely due to a loss of OKM1-positive cells, most likely due to adherence.

As third T-cell marker system, the expression of the ANAE spot within T cells was evaluated. Previous studies have already demonstrated that ANAE is preferentially present in resting T cells, which express Fc-IgM-R (S, 21). Our studies are in agreement with these observations, as the ANAE spot was present in a higher percentage in Fc-IgM-R than Fc-IgG-Rpositive cell fractions and as activation of T cells by ConA resulted in a decrease of ANAE-positive cells (21). The percentage of ANAE-positive cells in OKT+ cell fractions was relatively high, considering that OKTS+ cells express Fc-IgG-R and that expression of Fc-IgG-R is negatively associated with the ANAE spot. This finding might be best explained by the fact that OKTS-enriched cell fractions contain more T cells (and less OKMI + cells) than TG-cell-enriched cell fractions and have therefore also more ANAE-positive cells.

In conclusion, Fc-IgG-R and Fc-IgM-R, as well as the ANAE spot, can be expressed onlin both OKT4+ and OKTS+ T cells: Fc-IgG-R are in a higher percentage expressed on OKTS+ cells. The OKTS-reactive antigens appear to be expressed in widely different densities, and some OKT3 + , OKTS+ cells may also express the antigen recognized by OKMl. The antigens recognized by OKT antibodies are stable in situations where Fc-R or the ANAE spot are modulated (mitogen activation, IgG-immunecomplex exposure). The data would fit to the concept that OKT4 and OKTS define differentiation antigens, while the Fc-R as well as the ANAE spot appear to be markers expressed only during certain stages of activation of T cells.

In case that the correlation of OKT4-positive cells = helper/inducer and OKTS-positive cells = suppressor/cytotoxic T cells can be kept up, the data presented would imply that Fc-IgG-R and Fc-IgM-R (as well as the AN AE spot) can be expressed on various immunoregulatory T-cell subsets. Therefore a conclusion regarding the functional role of Fc-R-expressing T cells should only be drawn in combination with (monoclonal) T -subsetspecific antibodies.

Acknowledgement

We would like to thank Dr. GIDEON GOLDSTEIN of Ortho Laboratories for some of the monoclonal antibodies used in this study. The secretarial assistance of E. SCHDRMANN is highly appreciated.


References

1. PICHLER, W. J., M. L. LANGE, S. SHARROW, CH. BIRKE, S. BRODER, and H. H. PETER. 1981. Relationship between differentiation antigens (OKT-series) and functional marker (Fe-receptors), in Leucemia Marker Conference, Edt. by W. Knapp, Acad. Press, p. 345.

2. MORETIA, L., M. FERRARlNI, and M. D. COOPER. 1978. Characterization of human T cell subpopulations as defined by specific receptors for immunoglobulins. Contemp. Top. Immunobio!. 8: 19.

3. KUNG, P. C., G. GOLDSTEIN, E. L. REINHERZ, and S. F. SCHLOSSMAN. 1979. Monoclonal antibodies defining distinct human T cell surface antigens. Science 206: 347.

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Dr. W. J. PICHLER, Department of Clinical Immunology, Medizinische Hochschule Hannover, Karl-Wiechert-Allee 9, 3000 Hannover 61, Federal Republic of Germany

relationship of fc-igg and fc-igm receptors to the antigens defined by okt-antibodies and the acid...

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