Immunology 1977 32 199

Relationship between E receptors and a T-specific surface antigen on human T cells

C. GATTRI N G E R & G. WICK Institute for General and Experimental Pathology, University of Innsbruck, !nnsbruck, Austria

Received 27 May 1976; acceptedfor publication 14 July 1976

cytes via specific surface receptors and thus to form E rosettes has become a standard tool for their delineation in peripheral blood and in cell suspensions of various lymphoid organs (Jondal, Holm & Wigzell, 1972). The physiological significance of these E receptors is still unknown. Because the number of E receptors seems to be increased on PHA-stimulated blast cells their possible participation in the immune reaction has been discussed (Owen & Fanger, 1975). Another, more sensitive method for the demonstration of T cells is the use of specific anti-T-cell sera for membrane immunofluorescence and cytotoxicity tests (Ablin, Baird & Morris, 1972; Aiuti & Wigzell, 1973; Kersey, Sabad, GajlReczalska, Hallgren, Yumbs & Nesbit, 1973; Smith, Terry, Buell & Sell, 1973; Bobrove, Strober, Herzenberg & DePamphilis, 1974; Brown & Greaves, 1974; Woody, Ahmed, Strong, Knudsen, Brown & Sell, 1974; Ishii, Koshiba, Ueno, Imai & Kikuchi, 1975; Wick, Ahmad, Steiner, Zeilinger, Wolner, Mittermayer & Stacher, 1976). Prior incubation of lymphoid cells with ATS inhibits subsequent rosette formation (Bach, Dormont, Dardenne & Balner, 1969). This so-called rosetteinhibition test seems to be a reliable parameter for the in vivo immunsuppressive potential of antilymphocyte sera (Bach et al., 1969). Testing by means of ATS, however, often yields higher percentages of T cells as compared to those of E rosettes. This discrepancy is especially marked with popula-

Summary. The aim of this study was the delineation of different antigenic surface determinants on the surface of adult peripheral T cells by means of a specific horse anti-human T-cell serum (ATS). It was shown that this serum reacts both with E receptors and (an) additional T antigen(s). While E receptors showed the already known susceptibility to trypsin, T antigens (as demonstrated in cytotoxicity tests) were resistant to trypsinization even at high concentration. Incubation of the trypsinized peripheral blood lymphocytes (PBL) in 5 per cent CO2 allowed the resynthesis of E-receptors. High concentrations of ATS (without complement) significantly inhibited rosette-formation. This suggests a close steric relationship between E receptors and T antigen(s). However, absorptions of ATS with trypsinized PBL left the E-rosette inhibitory capacity unaltered. Treatment of PBL with ATS in appropriate dilutions and indirect immunofluorescence tests under capping conditions followed by conventional rosette procedures showed that the E receptor and T antigens are separately mobile within the T-cell membrane. INTRODUCTION The capacity of human T cells to bind sheep erythroCorrespondence: Dr G. Wick, Institute for General and Experimental Pathology, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.

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C. Gattringer & G. Wick

tions of malignant and immature T cells (Wick G., in preparation). It was therefore deemed of interest to determine if the E-rosette test and the reaction with ATS distinguish separate surface characteristics and what would be the steric relationship of such different determinants. This issue could be of importance for the diagnosis and therapy of diseases involving immature and malignant extensions of the T-cell line. MATERIALS AND METHODS Lymphoid cells Lymphoid cells were isolated from the peripheral blood of healthy adults using a slight modification of the method described by Boyum (Bdyum, 1968). Ten millilitres of citrated blood (3 8 per cent Nacitrate; 1/10, v/v) were incubated for 20 min in a 370 water bath and then for 20 sec in a nylon-wool column (Leukopak®, Fenwal Lab., Deerfield, Illinois). This short incubation leads to the removal of phagocytes without preferential retention of B cells. After rinsing the column with 3 vol. of 370 0-15 M NaCl 30 ml of the thus diluted blood was carefully layered over 10 ml of a flotation mixture (density: 1 077) consisting of Ficoll® (Pharmacia, Uppsala, Sweden) and Ronpacon® (Cilag-Chemie, Schaffhausen, Switzerland) and then centrifuged at 250 g for 20 min at 4°. Cells harvested from the interface consisted of >95 per cent lymphocytes and were used for further testing. E-rosette tests E-rosette tests were performed according to the procedure described by Jondal et al. (1972). All cells with three and more adhering sheep red blood cells (SRBC) were counted as E-rosette forming cells

(E-RFC). Trypsinization

Suspensions of lymphocytes adjusted to a concentration of 5 x 106 cells/ml were incubated with equal vol. of trypsin solutions (Difco Lab., Detroit, Michigan) for 60 min at 370 in a shaking water bath. Ten micrograms DNAse/ml (Sigma, St. Louis, Missouri) were added to the medium to prevent clumping.

Resynthesis of E receptors After trypsinization the lymphocytes were washed five times in RPMI 1640 and then incubated for

various periods of time at 370 in a 5 per cent C02 saturated humidity atmosphere. RPMI 1640 without glutamine and with addition of 10mM HEPES (Flow Lab., Irvine, Scotland), 0 07 mg Gentamicin/ ml (Schering Corp., Port Reading, New Jersey) and 5 per cent foetal calf serum (FCS) was used as nutritive medium throughout. Antisera The serologic properties of the specific horse antiserum to human T cells used for the present study have been described in detail elsewhere (Wick, Albini & Milgrom 1976). Briefly, horses were immunized with purified human thymocytes and the sera rendered specific by exhaustive absorptions with cells from patients with chronic lymphocytic leukaemia.

Rosette-inhibition tests 2 x 106 lymphocytes/ml of phosphate-buffered saline (PBS, pH 7 2) were incubated for 60 min at 370 with an equal vol. of different dilutions of ATS, washed three times and subsequently processed for routine rosette tests.

Membrane immunofluorescence 5 x 106 lymphocytes/ml were incubated in siliconized 13 x 75 mm glass tubes with an equal volume of appropriately diluted ATS for 60 min at 370, washed three times and resuspended to the original volume in PBS. This suspension was then treated for 30 min at 370 with a fluoresceine-isothiocyanate (FITC) rabbit anti-horse Ig conjugate (lot no. 429 AA, Behringwerke, Marburg/L., FRG). The conjugate was characterized on following the guidelines of Beutner, Sepulveda & Barnett (1968); it had eight standard precipitation units, a molar fluorochrome/ protein ratio of 1-8 and was used at a final dilution of 1: 64 as determined in chessboard titrations (Beutner et al., 1968). After three further washings in PBS the cells were subjected to conventional E-rosette testing. Finally, the cells were fixed by the addition of p-formaldehyde to a final concentration of 2 per cent just before the resuspension of the E-rosettes. The total number of E-RFC and the percentage of RFC carrying fluorescent caps were then determined under a Reichert-Immunopan fluorescence microscope appropriately equipped for conventional illumination with white transmitted light (for the observation of RFC) and incident blue light for the excitation of FITC-fluorescence (for the

E receptors and T antigens observation of caps) as described elsewhere (Wick, Albini & Milgrom, 1973).

100 90 80 70 c 60 4-50 50

Cytotoxicity tests Lymphocytotoxicity tests were performed as described previously (Wick et al., 1976) using fresh guinea-pig serum as a source of complement and trypan blue exclusion as a criterion of viability.

Absorptions Absorptions of ATS with native or trypsinized lymphocytes were performed for 8 h at 40 under constant agitation on a horizontal shaking machine. The number of cells used for absorption are given in the Results section. RESULTS

Effect of trypsin treatment acteristics

on

T-cell surface char-

Up to 2 per cent of trypsin in the incubation medium did not appreciably affect the viability of human peripheral blood lymphocytes. After verifying the known susceptibility of E receptors to trypsinization (Jondal et al., 1972) a concentration of 0-02 per cent was found sufficient to decrease the number of E-RFC over 90 per cent (Fig. 1). A standard trypsin concentration of 0-05 per cent was then selected for all further experiments. As shown previously, high concentrations (up to a dilution of 1:16) of the ATS used in this investiga-

201

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I'

40 30

20 10

0*0050-01

0.030.040.05

0Q1

0-5

1-0

0-02

Trypsin concentration (/) Figure 1. Effect of trypsin concentration on the number of E-RFC. PBL

incubated with various concentrations of at 370 in a shaking water bath, washed five times and tested for rosette formation. Vertical bars represent s.d. from the mean of three to sixteen values/trypsin concentration. Dots without s.d. are arithmetic means of two values only. The medium used was PBS+ 10 pg/ml DNAse. were

trypsin for 60 min

tion proved to be cytotoxic for about 65 per cent of normal peripheral lymphocytes. This percentage corresponds to the proportion forming E rosettes and lacking surface Ig. In contrast to the findings for E-receptors the reactivity of PBL in cytotoxicity tests with ATS was not significantly reduced after trypsinization with concentrations up to 2 per cent

(Table 1). Resynthesis of E receptors: Treatment of peripheral lymphocytes with 0-05 per cent trypsin entailed a drop of the number of

Table 1. Effect of trypsin on T antigen Per cent trypsin concentration

Per cent cytotoxicity with ATS

Per cent cytotoxicity with NHoS

-

644+ 14-2* 61 47 503+6-0* 500+4-2* 60 59 2 + 20-4*

3-7+ 1 5 n.d. 6 4 8 12 11 56

005 0.1 05 10 1-5 2-0 50

i

597+19.4*

PBL were incubated with various concentrations of trypsin for 60 min at 37°. After washing five times lymphocytotoxicity tests were performed by adding ATS or NHoS in a final dilution of 1:4 to 1: 12 and fresh guinea-pig serum as a source of complement in a final dilution of 1 :3. * Mean of three experiments; no statistical significant difference (Student's t-test).

C. Gattringer & G. Wick

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E-RFC from an original mean of 68 per cent to 3 per cent. After incubation in a 5 per cent CO2 atmosphere for 13-17 h the number of E-RFC within this cell population had again risen to 56 ± 8 per cent of the original values. (Fig. 2). The use of 2 per cent trypsin completely prevented the reappearance of E receptors within the limits of the incubation time.

LA.

cr

wI 0'

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100 90 80 70 cr Iu; 60 w

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50

40 ,U 30 Of 20 10

1:128 1:64 1:32 1:16 Antiserum

a

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(A

01/21 2 3 4 5 6 7 8 9 1011 1213 Co2 Incubation time (h) I

Try psin

Figure 2. Resynthesis of E-receptors after trypsin treatment. PBL were trypsinized (0 05 per cent trypsin) for 30 min at 370, washed five times, incubated for various periods of time at 370 in a humid 5 per cent CO2 atmosphere and tested for rosette formation. Vertical bars represent s.d. from the mean of sixteen values (untrypsinized and trypsinized), five values (6 5 h) and eleven values (13-17 h) respectively.

Rosette-inhibition Fig. 3 shows that preincubation of lymphocytes with specific ATS leads to the expected inhibition of subsequent rosette formation (as known for ALS), while rabbit anti-Ig and normal horse serum (NHoS) even in very high concentrations had no such effects. An ATS dilution of 1:8 had a nearly 80 per cent inhibitory activity and the remaining E rosettes never displayed the regular 'morula' aspect, but consisted of lymphocytes with only three to six adhering SRBC. When ATS was added to the medium during the 13-17 h incubation period allowed for the resynthesis of E receptors on trypsinized cells, its rosette inhibitory capacity was significantly potentiated as compared to the action on native cells. ATS was first added to native, non-trypsinized cells and this suspension left for 13-17 h in the C02-incubator. Under these conditions of prolonged incubation a 1:90 dilution

1:8

1:4

1:2

Figure 3. Inhibition of E-rosette formation by ATS. PBL ), anti-Ig were incubated with various dilutions of ATS ( (--- -), and normal horse serum (-----) for 60 min at 370 in a shaking water bath, washed three times and tested for rosette formation. Vertical bars represent SD from the mean of 3 to 5 values. The medium used was PBS.

of the antiserum still afforded a 50 per cent inhibition of the number of E rosettes. However, if trypsinized cells were subjected to this treatment the resynthesis of E receptors was affected much more strongly. An ATS dilution of 1:90 led to over 90 per cent inhibition of E-receptor resynthesis, and with a dilution of 1:180 the number of E-RFC was still over 60 per cent lower than in trypsinized controls incubated without ATS for 13-17 h.

Absorption studies From the previously presented data it had become clear that the specific lot of ATS under investigation contained antibodies of at least two specificities: one directed against the E receptor the other against (a) T antigen(s) differing from the former e.g. by its insensitivity to trypsin. As shown schematically in Fig. 4, absorptions with trypsinized lymphocytes therefore resulted in the removal of antibodies to the T antigens leaving the antibodies to the E receptor and thus the rosette-inhibitory capacity of the antiserum intact. Absorptions with native lymphocytes removed both varieties of antibodies and thus also the potential of rosette-inhibition. Results of differential absorptions are given in Table 2.

203

E receptors and T antigens ATS

Investigations on the steric relationship of E receptors and T antigen(s) The final issue to be clarified in the present study was the separate visualization of E receptors and the T antigen(s) recognized by our ATS. For these experiments we selected an ATS dilution of 1:32 which is capable of only a partial inhibition of rosette formation (Fig. 3). It was theorized that at this concentration antibodies against the E receptor were so diluted as to give only very little rosette inhibition, while antibodies against the T antigen are still present in sufficient amount to give strong positive reactions in indirect immunofluorescence tests.

The incubation of lymphoid cells with the 1:32 dilution of ATS and the anti-horse Ig conjugate under the conditions described in the Materials and Methods section led to the capping of the T antigen. A subsequent rosette test performed with these lymphocytes gave the expected results, i.e. 30 per cent E-RFC. They again showed only three to six adhering SRBC. The analysis of these E-RFC under the fluorescence microscope revealed that all E-RFC (in addition to the rest of T cells whose rosette formation had been completely inhibited) had fluorescent caps. These caps showed no correlation to the attached SRBC and were often even located opposite to the latter (Fig. 5). NHoS and rabbit anti-human Ig serum were used for control purposes in dilutions down to 1:8 and had no effect. Finally the simultaneous capping and rosetting

Remaining anti bodies

|

NHoS ATS ATS ATS NHoS ATS ATS ATS NHoS ATS ATS ATS

1:8 1:8 1:8 1:8 1:16 1:16 1:16 1:16 1:32 1:32

1:32 1:32

ID)

Rosette inhibition

Figure 4. Principle of absorptions of ATS with native trypsinized PBL.

or

experiments were repeated with ATS concentrations which have almost no inhibitory effect. Under these conditions the surface of RFC was generally

Table 2. E-Rosette inhibition by absorbed ATS

Antiserum

Anti- E

Absorbed with -1 x 109 Native PBL/ml antiserum 1 x 109 Trypsin-PBL/ml antiserum 1 x 109 Native PBL/ml antiserum 1 x 109 Trypsin-PBL/ml antiserum

Inhibition ( 86 54 98 70 26 81 -

1 x 109 Native PBL/ml antiserum 1 x 109 Trypsin-PBL/ml antiserum

57 4 42

Absorptions of ATS were done for 8 h at 40 under constant agitation. The rosette inhibitory capacity was tested by incubating PBL with ATS for 60 min at 37°. After washing three times the conventional rosette procedure was performed.

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C. Gattringer & G. Wick

Figure 5. Differential capping of T antigens and E receptors. (a) Shows a RFC with three adherent SRBC; (b) the same area under blue illumination. The lymphocyte shows a fluorescent cap distinct from the attached SRBC. (Magnification x 280).

densely covered by SRBC, and over 90 per cent of these RFC showed fluorescent caps. Due to the dense lining of most RFC with SRBC, the latter were also found to be attached in the area of caps or in their immediate neighbourhood. DISCUSSION Besides functional tests, such as PHA stimulation, human T cells can be morphologically identified by two main approaches: the determination of E-RFC and the reactivity with specific ATS in membrane fluorescence and/or lymphocytotoxicity tests. The steric relationships between E receptors and T antigens have so far not been clarified. A prominent difference between these two surface characteristics of human T cells is the selective sensitivity of the E receptor to trypsin (which is proteolytic via its specific action on lysine and arginine). Viability and metabolism of human T cells are, however, not affected by our standard trypsin concentration of 0 05 per cent as shown by the successful stripping and resynthesis of E receptors. Rosette inhibition by ALS and also specific ATS is a standard procedure for the in vitro analysis of such sera and supposed to be a reliable parameter for their in vivo immunosuppressive potential. In principle one could explain the blocking of E receptors by ATS in spite of the chemical difference between these and the T-antigens in the following ways: (1) the T antigen constitutes a 'backbone'

structure for the E receptor; (2) rosette-inhibition is based on steric hindrance due to a close relationship of E receptors and T antigens; (3) E receptors and T antigens are completely independent membrane components and our ATS contains antibodies directed against each of these two antigenic determinants. The first possibility was excluded by the lack of a relationship between fluorescent caps and adherent SRBC. In order to reach a decision between possibilities (2) and (3) absorptions with trypsinized lymphoid cells were performed: if the ATS contained only one antibody against the T antigen, a complete removal of this specificity and the rosette-inhibitory properties of the antiserum was to be expected. However, if two antibodies were present in our ATS this procedure should leave antibodies to E receptors, and thus the capacity for rosette inhibition, unaffected which would support the third choice. In any case an absorption with untrypsinized native lymphocytes should remove all antibodies eventually present in the serum. The results presented in this paper (Table 2) prove that the third possibility, namely the presence of antibodies to the E receptors and the T antigen, holds true. The antibody against the E receptor is present in a lower concentration which explains that higher dilutions of ATS do still afford capping, but already permit rosette formation. The final point to be discussed is the markedly reduced number of SRBC adhering to RFC under the influence of higher ATS concentrations (in the

E receptors and T antigens capping experiments) which seems to suggest a certain steric relationship between the two membrane structures. One explanation would be an 'incomplete capping', leaving some antigenic determinants (in this case E receptors) behind. Such a phenomenon has recently been described by Lemonnier, Neauport-Sautes, Kourilsky & Demant (1975). A second possibility to explain this phenomenon is to suggest a non-specific co-capping of most of the E receptors caught in the dense meshwork of specific complexes of T antigen, antibody and conjugate. When higher dilutions of ATS are used this meshwork becomes considerably looser thus also making non-specific cocapping of E receptors more unlikely. This explanation would suggest a relatively low surface density of E receptors. The production of ATS without rosette-inhibitory capacity by means of heteroimmunization with trypsinized human thymocytes is now under way in this laboratory. Preliminary results confirm the expected findings, i.e. ATS which had been produced by immunizing rabbits with trypsinized human thymocytes has no rosette-inhibitory activity. This reagent should provide a valuable tool for the further characterization of the functional and morphological properties of the human T-cell membrane.

ACKNOWLEDGMENTS This work was supported by a grant from the Austrian Cancer Research Fund. Note added in proof In a recent paper by Small P., Kashiwagi N. & Kohler P.F. (Cell Immunol. 25, 302, 1976) the influence of Con A, anti-B-cell globulin (ALG) and anti-T-cell globulin (ATG) on E rosetting was investigated. ATS but not ALG or Con A showed a dose-dependent rosette inhibition. After capping with unabsorbed ATS no rosetting did occur. In contrast to the present results, these authors concluded that ATS binds to a surface structure identical with or in close proximity to the E receptors.

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species-specific antibodies in antithymocyte globulin. Transplantation, 13, 306. AIUTI F. & WIGZELL H. (1973) A study on human T-lymphocytes, their function and frequency in some diseases. Advanc. exp. Med. Biol. 29, 307. BACH J.F., DORMONT J., DARDENNE M. & BALNER H. (1969) In vitro rosette inhibition by antihuman antilymphocytes serum. Transplantation, 8, 265. BEUTNER E.H., SEPULVEDA M.R. & BARNETr E.V. (1968) Quantitative studies of immunofluorescence staining. Relationship of characteristics of unabsorbed antihuman lgG conjugates to their specific and nonspecific staining properties in an indirect test for antinuclear factors. Bull. Wid. HIth. Org. 39, 587. BOBROVE A., STROBER M., HERZENBERG L.A. & DEPAMPHILIS J.D. (1974) Identification and quantitation of thymus-derived lymphocytes in human peripheral blood. J. Immunol. 112, 520. BOYUM A. (1968) Isolation and removal of lymphocytes from bone marrow of rats and guinea-pigs. Scand. clin. Lab. Invest. 21, Supplement 97. BROWN G. & GREAVES M.F. (1974) Cell surface markers for human T and B lymphocytes. Europ. J. Immunol. 4, 302. ISHII Y., KOSHIBA H., UENO H., ImAi K. & KIKUCHI K. (1975) Surface antigenic specificities of human thymusderived lymphocytes. Clin. exp. Immunol. 19, 67. JONDAL M., HOLM G. & WIGZELL H. (1972) Surface markers on human T and B lymphocytes. J. exp. Med. 136, 207. KERSEY J.M., SABAD A., GAJL-RECZALsKA K., HALLGREN H.M., YuMas E.J. & NESBIT R.E. (1973) Acute lymphoblastic leukemic cells with T (thymus-derived) lymphocyte markers. Science, 182, 1353. LEMONNIER F., NEAUPORT-SAUTES C., KOURILSKY F. & DEMANT P. (1975) Relationship between private and public H-2 specificities on the cell surface. Immunogenetics, 2, 517. OWEN F. & FANGER M. (1975) Studies on the human T lymphocyte population III. Synthesis and release of the lymphocyte receptor for sheep red blood cells by stimulated human T lymphoblasts. J. Immunol. 115, 765. SMITH R.W., TERRY W.D., BUELL D.N. & SELL K.W. (1973) An antigenic marker for human thymic lymphocytes. J. Immunol. 110, 884. WICK G., ALBINI B. & MILGROM F. (1973) Antigenic surface determinants of chicken lymphoid cells. I. Serologic properties of anti-bursa and anti-thymus sera. Clin. exp. Immunol. 15, 237. WICK G., AHMAD R., STEINER R., ZEILINGER M., WOLNERE., MITTERMAYER K. & STACHER A. (1976) Production and diagnostic application of anti-human T-cell antisera. Postgrad., Med. J. 52, (Supplement 5), 20. WOODY J.N., AHMED A., STRONG D.M., KNUDSEN R.C., BROWN T. & SELL K.W. (1974) Delineation of human T cell functions using an antihuman T-cell antisera. J. Reticuloendothel. Soc. 15, 24a.

Relationship between E receptors and a T-specific surface antigen on human T cells.

Immunology 1977 32 199 Relationship between E receptors and a T-specific surface antigen on human T cells C. GATTRI N G E R & G. WICK Institute for...
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