Flow cytometric studies of the binding of monoclonal antibodies OKT3, OKT4 and OKT8

Journal of Immunological Methods, 115 (1988) 159-167

159

Elsevier JIM04973

Flow cytometric studies of the binding of monoclonal antibodies OKT3, OKT4 and OKT8 Tadahiro Oonishi 1, Kanako Sakashita 2 and Nobuhiro Uyesaka 3 Department of Pathology, and 2 The Medical Information Institute, The Kanto Teishin Hospital, 5-9-22, Higashi-Gotanda, Shinagawa-ku, Tokyo 141, Japan, and 3 Department of Physiology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113, Japan

(Received 7 September 1987, revised received 21 March 1988, accepted 8 July 1988)

The binding of monoclonal antibodies (OKT3, O K T 4 and OKT8) to h u m a n T cells was investigated by flow cytometry. A flow cytometer was calibrated with standard fluorescence microspheres, which permitted quantitation of the number of bound antibody molecules. Considerable care was taken to perform the flow cytometric assay at a constant temperature and the effect of temperature on the binding reaction was examined. The binding of OKT3, O K T 4 and O K T 8 exhibited saturation kinetics. The m a x i m u m binding varied with temperature. Kinetic analysis according to the Hill equation revealed that the value of the Hill coefficient for OKT3 changed from 1.8 to 1.0 when the temperature was raised from 1 2 ° C to 36 o C, whereas the corresponding values for O K T 4 and O K T 8 did not vary with temperature. Thermodynarnic functions obtained from the Van't H o f f plot showed that the binding of OKT3 was exothermic whereas the binding of O K T 4 and OKT8 were endothermic. Key words: Flow cytometry; Binding reaction; OKT3; OKT4; OKT8; T cell, human

Introduction In combination with the use of monoclonal antibody (mAb), flow cytometry is widely used to identify and characterize particular types of cell. However, as Bohn (1976) has pointed out, the most valuable application of flow cytometry is in

Correspondence to: T. Oonishi, Department of Pathology, The Kanto Teishin Hospital, 5-9-22, Higashi-Gotanda, Shinagawa-ku, Tokyo 141, Japan. Abbreviations: mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; KLH, keyholelimpet hemocyanin; HBS, Hepes-buffered saline (15 mM Hepes buffer, 145 mM NaCI, pH 7.3); PBMC, peripheral blood mononuclear cell; FCMS, FITC-conjugated microsphere; FITC-m_Ab, FITC-conjugated monoclonal antibody; AIDS, acquired immune deficiency syndrome.

the quantitative investigation of ligand-receptor interactions. In order to perform such quantitative experiments, we have developed a new standard fluorescence microsphere to calibrate the flow cytometer (Oonishi et al., 1985), and have analyzed the interactions of OKT3, O K T 4 and OKT8 with h u m a n T cells. These m A b have been extensively used to identify the subsets of T cells, and the structures of the corresponding surface molecules (T3, T4 and T8) have now been determined (Littman et al., 1985; M a d d o n et al., 1985; Van den Elsen et al., 1985). It has also become evident that these molecules play a crucial role in the interactions between T cells and various target celIs ( G o v e r m a n et al., 1986; Meuer e t al., 1983b), and that the binding of O K T 3 induces an activation of T cells which mimics the binding of antigen (Van Wauwe et al., 1980; Meuer et al., 1983a; Weiss et

0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

160 al., 1984). Here, we present a standard method for investigating ligand-receptor interactions by flow cytometry, as exemplified by the binding characteristics of the T3, T4 and T8 molecules with their respective mAb.

Materials and methods

Monoclonal antibodies Mouse mAb directed against the T3 molecule (OKT3, IgG2a), the T4 molecule (OKT4, IgG2b), the T8 molecule (OKT8, IgG2a) and these mAb conjugated with fluorescein isothiocyanate (FITC) were purchased from Ortho Diagnostic Systems (Raritan, N J). FITC-conjugated mouse IgG2a and IgG2b mAb recognizing keyhole limpet hemocyanin (KLH) were purchased from Beckton Dickinson Immunocytometry Systems (Mountain View, CA). Preparation of human peripheral blood mononuclear cells Venous blood was collected from the antecubital veins of healthy individuals into 1 / 1 0 vol. of 150 U / m l heparin. After removal of plateletrich plasma (2 ml) from 10 ml of the blood sample by centrifugation at 220 × g for 10 min at 20 o C, 8 ml of blood cells were diluted with 10 ml of Hepes-buffered saline (HBS, 15 mM Hepes buffer, 145 mM NaC1, p H 7.3). An aliquot of 6 ml of diluted blood was layered over 4 ml of lymphocyte separation medium (FicoU-Conray, 1.077 g / m l ) and centrifuged at 570 × g for 30 min at 20 ° C. The material at the interface was used as the peripheral blood mononuclear cell (PBMC) fraction after twice washing with resuspension and centrifugation at 220 x g for 10 rain at 20 ° C in HBS. These PBMC were resuspended in cold HBS and the cell suspension was adjusted to 1 × 10 7 cells/ml. Calibration of flow cytometer and estimation of the number of FITC-conjugated mAb molecules bound to lymphocytes A flow cytometer (Epics V, Coulter Electronics, Hialand, FL) was calibrated with standard fluorescence microspheres as described previously (Oonishi et al., 1985). The standard fluorescence microspheres were prepared by covalently con-

jugating FITC to plastic microspheres (Micropearl-FT, Sekisui Fine Chemical Co., Osaka, Japan). The amount of bound FITC molecules was determined after lysis of FITC-conjugated microspheres (FCMS) in strong alkaline solutions (1 N N a O H solution). In flow cytometry, fluorescence intensity was related in a linear fashion to the amount of bound FITC molecules. The fluorescence intensity of the FCMS suspension (1 x 10 6 particles/ml) was measured in a fluorescence spectrophotometer, and the fluorescence intensity per microsphere was determined from the FITC concentration in 0.1 N N a O H solution. The fluorescence intensity of one FITC molecule bound to the microsphere was equivalent to that of 2.86 x 10 -is nM FITC in 0.1 N N a O H solution. The fluorescence intensities of the different FCMS were calculated by multiplying the fluorescence intensity of 2.86 x 10 -15 nM by the numbers of bound FITC molecules. The fluorescence intensities of FITC-conjugated OKT3, OKT4 and OKT8 solutions (100 ng I g G / m l ) were equivalent to the fluorescence intensity of 13 nM FITC in 0.1 N N a O H solution, which was the same in these three FITC-conjugated monoclonal antibodies (FITC-mAb). Assuming a molecular weight of 150000 for mouse IgG, the fluorescence intensity of each FITC-mAb was 3.25 x 10 -14 nM in 0.1 N N a O H solution. Therefore, the fluorescence intensity of FCMS bound with N molecules of FITC was equivalent to that of N × 2.86 × 10-1~/3.25 × 10 -14 ( = 0.088 × N ) molecules of FITC-conjugated OKT3, OKT4 or OKT8. Just prior to the binding study of lymphocytes with FITC-mAb, a flow cytometer was calibrated with FCMS. The channel numbers of the peak positions in the fluorescence histograms were plotted against the numbers of FITC molecules bound to FCMS. The light-scatter-gated fluorescence intensity of the lymphocytes was measured and the peak channel number in the fluorescence histogram was determined. This was then converted to the number of FITC molecules bound to FCMS from the calibration curve. The number of FITC-conjugated OKT3, OKT4 or OKT8 molecules bound to lymphocytes was estimated by multiplying the number of bound FITC molecules by a factor of 0.088 as described above.

161

Binding of FITC-mAb to lymphocytes Lyophilized FITC-conjugated OKT3, OKT4 and OKT8 were reconstituted with distilled water and diluted to an appropriate concentration with HBS. For studies of the binding of FITC-mAb to lymphocytes, 50 /tl of PBMC suspension were added to 200 /xl of FITC-mAb of known concentrations (the final cell concentration was 2 X 10 6 cells/ml), and incubated in an isothermobath. As a control sample, PBMC were added to FITCconjugated anti-KLH mouse IgG2a and IgG2b mAb under the same conditions as above. By circulating cold or warm water through a jacket surrounding the sample cup and line, the sample was kept at a constant temperature throughout the experiment. The temperature of the sheath stream was also adjusted to the test temperature by cooling down or heating the sheath tank. All experiments were carried out within 3 h of cell separation and 5 h from drawing the blood.

Results

Binding study of FITC-mAb to lymphocytes by flow cytometry Fluorescence signals gated on the single cell fraction in the forward light-scatter histogram were collected, and the relative numbers of cells were plotted against the logarithm of the integral fluorescence intensities. The shapes of the frequency distributions for fluorescence did not vary with antibody concentration or temperature. The channel number of the main peak in the fluorescence histogram was used as a representative value of fluorescence intensity of the lymphocytes. The channel number was converted to the number of FITC molecules bound to FCMS, from which the number of bound FITC-mAb molecules was estimated as described in the materials and methods section. The fluorescence intensity of lymphocytes interacting with FITC-conjugated OKT3 showed saturation kinetics. After washing twice with resuspension and centrifugation at 220 x g for 10 rain at 20°C, the fluorescence intensity of lymphocytes decreased to a considerable extent. The washed lymphocytes reacted with the original concentrations of FITC-conjugated OKT3 and re-

FITC molecules bound to FCMS (xlO s) 15

10

~

°~

0

5

/

0

-

~

50

t

I

,iV

1000 1500 2000 Concentration of FITC-OKT 3[ ngJml]

Fig. 1. Effects of the washing procedure on the binding of FITC-conjugated OKT3. The PBMC were reacted with FITCconjugated OKT3 at 20 o C and were subjected to flow cytometry. Fluorescence intensities were measured without washing (I) and after washing (II). The washed lymphocytes were reacted with the original concentrations of FITC-conjugated OKT3 (III) or with the same concentrations of FITC-conjugated anti-KLH mouse IgG2a mAb (IV). Control samples were reacted with FITC-conjugated anti-KLH mouse mAb (V). Fluorescence intensity is expressed as the number of FITC molecules bound to FCMS (see materials and methods section). Points are the means of six determinations from two independent experiments.

covered the initial intensity of fluorescence, but the substitution of anti-KLH mouse mAb for OKT3 resulted in no change in the intensity (Fig. 1). This experiment demonstrates clearly that the washing procedure exerts a critical influence on the interaction between FITC-mAb and lymphocytes, and that the fluorescence intensity measured by the flow cytometer is due solely to the cell-bound fluorescence without interference from co-existing unbound fluorescence.

Effect of temperature on the binding of FITC-conjugated OKT3, OKT4 and OKT8 As it was difficult to maintain stable conditions in which to measure fluorescence intensity at temperatures below 10 ° C, this experiment was carried out at temperatures between 12°C and 36 ° C.

162 Bound antibody molecules per lymphocyte (xlO 4)

io, x

:~i

1.4--

15

o 36°C

1.2--

26,,

24',

1.0--

= 16', 12",

0.8--

10

:

o/

o 0.6--

I

0.4--

,

0.2

5

__//I

0

2.0

/2.5

3.0

log

-0.2 I 0

I 500

I 1000

I 1500

I 2000

I 2500

-0.4

Concentration of FITC-OKT 4 [ ng/ml ]

(B) (A) Fig. 2. Effects of temperature on the binding of FITC-conjugated O K T 4 to P B M C as illustrated by binding curves (A) and Hill plots (B). The points are the m e a n s of nine determinations from three independent experiments. Bound antibody molecules per lymphocyte xlO=) 15

X t-X

~

1.2--

t2°c 16 *

. 12°C

1.0

2O 24

0.8

1C

24 28

28

0.6 0.4--

o

0.2--

--4/ i

2.0

Y2.5

3.0

log x

-0.2 j'-

0

t 500

I 1000

I 1500

I 2000

-0.4

Concentration of FITC-OKT 3 [ ng/ml ] (A)

(B)

Fig. 3. Effect of temperature on the binding of FITC-conjugated O K T 3 to P B M C as illustrated by binding curves (A) and Hill plots (B). The points are the m e a n s of nine determinations from three independent experiments.

163 X

k~ 1-X 1.2--

Bound antibody molecules per lymphocyte (x104)

o 36°C 1.0-

/

/ * 08-....----~ 36oc 28 ",

3°r I

j j . j ~. ..~_. o_~ . o-"JJ.J

/ /

24,,6

28 "

//-24 ///.

16

06-

t2

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I

log x

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0

I 250

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I 750

I 1000

I 1250

Concentration of F I T C - O K T 8 [ n g / m I]

/ - 0 . 6 --

/

,/

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-/ (A)

-to"

(BI

Fig. 4. Effects of temperature on the binding of FITC-conjugated O K T 8 to P B M C as illustrated by binding curves (A) and Hill plots (B). The points are the m e a n s of nine determinations from three independent experiments.

The binding reactions were completed within 10 min and the fluorescence intensities remained unchanged for at least 60 min at all temperatures. The fluorescence intensity of PBMC reacted with FITC-conjugated OKT3 at 1 2 ° C was not influenced by the preceding binding reaction at 36 o C, and this was also true of the reverse incubation from 12°C to 36 o C. As shown in Figs. 2, 3 and 4, the binding of FITC-conjugated OKT3, OKT4 and OKT8 exhibited saturation kinetics at all temperatures. The mAb saturation curves did not always follow the hyperbolic relationship characteristic of Michaelis-Menten kinetics but suggested a sigmoid relationship. For kinetic analysis, the binding curves were plotted using the Hill equation (Hill, 1910; Wyman, 1964; Triggle, 1971): log X / ( 1 - X ) = n H log X -- log Kd, where X represents fractional saturation of lymphocytes with the antibody at a concentration x, K d is a dissociation constant and n H is the Hill coefficient (Fig. 2, Fig. 3, Fig. 4). A plot of log X / ( 1 - X ) against log x, the so-called

Hill plot, is found to provide a straight fine, particularly for the 20-80% saturation area, and provides a simple method for evaluating K d and n H•

The binding of FITC-conjugated OKT3 was inhibited by unconjugated OKT3, and the Hill plots of the binding curves in the absence or presence of unconjugated OKT3 were parallel to each other (Fig. 5), indicating that inhibition was competitive. Similar results were obtained for OKT4 and OKT8. It was evident that the binding of the various FITC-mAb was specific for the ligand-receptor interaction. Kinetic parameters obtained from the Hill plots are summarized in Table I. The maximum binding value for OKT3 decreased as the temperature rose from 12°C to 36 ° C (Fig. 3). On the other hand, the maximum binding for OKT8 increased as the temperature was raised (Fig. 4). As regards the Hill coefficient, the values obtained with OKT4 and OKT8 were the same (nr~ = 1.8) and did not vary with temperature change (Fig. 2, Fig. 4), whereas that ob-

164 TABLE I KINETIC PARAMETERS OF OKT3, OKT4 AND OKT8 BINDING OKT3

12 ° C 36 ° C

OKT4

Max. bind. value

nH

12.9 7.8

1.8 1.0

OKT8

Kd (Xl0-gM)

Max. bind. value

nH

1.56 2.01

6.5 6.5

1.8 1.8

Kd (x10-9M)

Max. bind. value

1.97 1.30

22.9 29.9

n H

gd

(×10 9M) 1.8 1.8

3.05 1.71

Max. bind. value, maximum binding value expressed as the number of antibody molecules bound per lymphocyte ( × 104 molecules/cell), which were calculated from the fluorescence intensity of lymphocyte (see materials and methods section); nil, Hill coefficient; K d, dissociation constant. Key:

tained with OKT3 decreased from 1.8 to 1.0 as the temperature was raised from 1 2 ° C to 36 ° C (Fig.

3).

To obtain thermodynamic functions, standard enthalpy ( A H °) was estimated from the Van't Hoff's equation: In K a 2 / K a l = - A H ° / R ( I / T 2 1 / T 1 ) , where R is the gas constant, and K a l and Ka2 a r e association constants at temperature T 1

log X

TABLE II THERMODYNAMIC FUNCTIONS OF OKT3, OKT4 AND OKT8 BINDING AH °

OKT3 OKT4 OKT8 Key:

1-X

A S o,

1.2-

AG °

AS °

(K cal.mo1-1) (K cal.mo1-1)

(cal. K - l . m o l --1 )

309K-285K

309K

285K

309K-285 K

-1.83 +3.20 +4.12

-12.30 -12.57 -12.40

-11.49 -11.36 -11.11

--+33.89 --+51.07 =+53.46

standard enthalpy; AG °, standard free energy; standard entropy.

AH °,

1 . 0 -o

0.8-

o

~

0.6log Ka 9.0

0.2-

0

---'//2!0

8.9

o o/'~V'/

3.0

log x

-0.2 --

./"/

-0.4-

8.8

"~- I I ~ .

8.7

~e&/.'/

--

8.5

-0.8

--

8.4

5. Competitive inhibition of FITC-conjugated OKT3 binding by unconjugated OKT3. Hill plots of the binding of FITC-conjugated OKT3 at 36°C in the absence (©) or presence of unconjugated OKT3 used at a final concentration of 50 ng/ml (O) or 100 ng/ml (A). The points are the means of 3 determinations.

OKT 3

"i'm OKT4

8.6

-0.6

Fig.

0 ,~. 0 ,~" II

~" i 3.20

i 3.30

I 3.40

1/~ x 10

i 3.50

OKT 8 i 3.60

~3

Fig. 6. Van't Hoff plots of OKT3, OKT4 and OKT8 binding. The logarithms of K a value (M -1 ) were plotted against the reciprocals of temperature. The slopes equal - A H ° / 2 . 3 0 3 R (see results section).

165 and T2 respectively. The slope of a plot of log K~ versus 1 / T equals - A H ° / 2 . 3 0 3 R (Fig. 6). The K a value was obtained from a reciprocal of K d, which was evaluated from the Hill plot as the concentration of antibodies at half maximum binding. Standard free energy (AG °) and entropy (AS ° ) were calculated using the thermodynamic equations: - A G ° = 2.303R × T × log Ka, and AS ° = ( A H ° - A G ° ) / T . As shown in Table II, the binding of OKT3 was exothermic whilst OKT4 and OKT8 binding was endothermic. Discussion

One of the greatest advantages of the flow cytometer in investigating ligand-receptor interactions is its capacity to precisely measure a cell bound ligand in a cell suspension that includes unbound ligand. This permits measurements which avoid the risk of perturbing the integrity of the cell membrane or of interfering with the ligand-receptor interaction by washing or similar procedures. Therefore, the cells can be kept in the same environment throughout the experiments. This distinct property of the flow cytometer has been remarked upon by Bohn (1976) and is reconfirmed in this experiment (Fig. 1). In order to perform quantitative flow cytometry, we have developed a new standard fluorescence microsphere (Oonishi et al., 1985). Using this, the number of FITC-conjugated ligand molecules bound to cells was easily estimated on a flow cytometer. Considerable care was taken in the present experiment to perform the flow cytometric assay at a constant temperature. A small volume of lymphocyte suspension, such as is used in flow cytometry, may change its temperature momentarily unless the sample cup, sample line and sheath stream are kept at a constant temperature. In fact, as shown in Fig. 2, Fig. 3 and Fig. 4, the binding reactions of mAb are affected considerably by temperature, so thermostatic control during the assay is indispensable if unequivocal results are to be obtained. In the present experiments the binding of OKT3, OKT4 and OKT8 to human T cells has been examined. As shown in Table I, the maximum binding values varied with the temperature. A rise in temperature led to a decrease in the

maximum binding value for OKT3 and to an increase for OKT8. Assuming that the maximum binding value of mAb can be related to the total number of active binding sites of the surface molecules, the number of active sites of T3 at 36 ° C was 60% smaller than at 12°C, and that of T8 at 3 6 ° C was 30% greater than at 12°C. In order to evaluate the effect of temperature on the binding reaction, the value of the Hill coefficient (n H) was estimated. When the temperature was raised from 12°C to 36°C, the value of nH for OKT3 changed from 1.8 to 1.0, whereas the values (1.8) for OKT4 and OKT8 did not vary with temperature. The Hill coefficient does not generally represent the number of interacting ligand binding sites but rather is a measure of the interaction between these sites (Wyman, 1968; Triggle, 1971). According to Wyman's thermodynamic theory of the binding of ligands to macromolecules with interacting sites (Wyman, 1964, 1968), the Hill coefficient (which may be viewed as the interaction coefficient or the cooperativity index) is closely related to the average free energy of interaction of the sites, and the value of n u greater than unity means that the interaction is stabilizing. For mAb binding to the cell surface molecule, it can be concluded that, in the case of OKT3, the interaction of receptor sites at 12°C is relatively more stabilizing than that at 36 ° C, and that the interaction of receptor sites with OKT4 and OKT8 is more stabilizing than is the reaction with OKT3. Since OKT3, OKT4 and OKT8 are mouse IgG2 antibodies considered to be of identical binding valency, the change in n r~ value of OKT3 with temperature suggests that profound changes in the state of the lymphocyte membrane a n d / o r binding sites occur. Furthermore, thermodynamic analysis using the Van't Hoff's equation reveals that the binding of OKT3 is exothermic, whereas binding of OKT4 and OKT8 is endothermic, and that the standard entropy (AS °) for OKT4 and OKT8 are larger than that for OKT3 (Table II). Thus, T3 is clearly contrasted with T4 and T8 in both kinetic and thermodynamic data. It has been reported that the binding of mAb to T3 at 3 6 ° C induces a loss of T3 and T cell receptor complex with subsequent activation of the cell (Van Wauwe et al., 1980; Meuer et al., 1983b; Weiss et al., 1984). On the other hand, the

166

binding of mAb to T4 or to T8 does not induce modulation of these molecules (Meuer et al., 1984). Taking into consideration the fact that the binding of OKT3 to T3 at 36 ° C can induce a loss of the T3-OKT3 complex, it seems probable that the temperature-dependent loss of OKT3 from the cell surface can obscure or distort the measurement of binding and this may be related to the apparent difference in the binding characteristics between OKT3 and other mAb. However, the amount of bound OKT3 molecules at 12°C was not influenced by a preceding binding reaction at 36 o C. Thus it can be concluded that the loss of OKT3 molecules from the cell surface at 36 ° C is negligibly small under the conditions employed, and that the difference in the binding characteristics between OKT3 and the other mAb is due mainly to their intrinsic binding characteristics. It is worth noting that we were not able to obtain these results until we established quantitative flow cytometry as an analytical tool for binding studies. It is known, however, that thermodynamic functions derived by an indirect kinetics approach do not always agree with data obtained by direct calorimetric measurement. More detailed studies of the thermodynamic characteristics of the T cell recognition system are being carried out using direct microcalorimetric measurements. The analytical methods described in this report will permit investigations of lymphocytes in diseases associated with abnormal immune responses, in viral infections and in neoplasia. In such conditions, the surface molecules on the lymphocytes may be expected to alter their kinetic and thermodynamic nature following binding of the mAb. From this point of view, the behaviour of the T4 molecule in aquired immune deficiency syndrome (AIDS) is of interest. The essental immunologic feature of AIDS is that the AIDS virus infects, replicates within and ultimately depletes T4 ÷ cells numerically and functionally (Fauci et al., 1985). Since the T4 molecule serves as a receptor for the AIDS virus (Dalgleish et al., 1984; Klatzman et al., 1984; McDougal et al., 1986), the T4 on virusinfected lymphocytes would be expected to demonstrate altered binding characteristics with mAb. A quantitative approach to studying the binding of mAb with T4 may be useful in the investigation of virus pathogenicity in AIDS.

Acknowledgements We are very grateful to Mrs. Vicki Kurasige for pertinent advice on the manuscript and to Miss Emi Tajiri for her excellent typing of the manuscript. This work was partly supported by the Medical Information Institute, The Kanto Teishin Hospital and by a Grant-in-Aid for Scientific Research (Grant 60570045 to N. Uyesaka) from The Ministry of Education, Science and Culture of Japan.

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Van den Elsen, P., Shepley, B.A., Cho, M. and Terhorst, C. (1985) Isolation and characterization of a cDNA clone encoding the murine homologue of the human 20 Kd T3/T-cell receptor glycoprotein. Nature 314, 542 Van Wauwe, J.P., DeMey, J.R. and Goossens, J.G. (1980) OKT3: a monoclonal anti-human T lymphocyte antibody with potent mitogenic properties. J. Immunol. 124, 2708. Weiss, A., Wiskocil, R.L. and Stobo, J.D. (1984) The role of T3 surface molecules in the activation of human T ceils: a two-stimulus requirement for IL2 production reflects events occurring at a pre-translational level. J. Immunol. 133, 123. Wyman, J. (1964) Linked functions and reciprocal effects in hemoglobin: A second look, Adv. Protein Chem. 19, 223 Wyman, J. (1968) Regulation in macromolecules as illustrated by hemoglobin. Quart. Rev. Biophys. 1, 35