Cellular cooperation in lymphocyte activation

CELLULAR

IMMUNOLOGY

49,

Cellular IV. Requirement

TADASHI

142-

Cooperation

in Lymphocyte

Activation

of Cell-to-Cell Interaction for the Activation Lymphocytes by Protein A

MAMORU

of Medical

of Human Tand 6

KOHJIN KIN,? YOSHIHISA ITOH,? TADASHI MORITA,$ AND KOHEI SHIOIRI-NAKANO*

KASAHARA,*

Departments

153 (1980)

Biology and Parasitology, Jichi Medical School, Received

Klinical Tochigi-ken,

March

Pathology, Japan

KAWAI,~

and $Otolaryngology,

7, 1979

The stimulation of highly purified human T and B cells by soluble and insoluble protein A was studied. Insoluble protein A, such as protein A conjugated to Sepharose beads (S-pro A), orStaphylococcus aureus Cowan I strain bacteria (SpA CoI), markedly stimulated B cells, but did not affect T cells. SpA Co1 stimulated B cells independently of the presence of T cells. While soluble protein A failed to stimulate either T or B cells alone, it greatly stimulated the mixture of T and B cells. Mitomycin treatment revealed that the response to soluble protein A was ascribed mainly to the T-cell response with the B-cell helper effect, though partially to the B-cell response with the T-cell helper effect as well. The response of T cells to protein A was enhanced by both the adherent population and the nonadherent B-cell population. This T-B cooperation was mediated by direct cell-to-cell interaction rather than soluble mediators. The binding experiments also demonstrated that the amount of protein A bound to T cells was far less than that to B cells. These results point out the significance of B-cell participation in T-cell activation. The mechanism by which protein A activates T and B cells was also discussed.

INTRODUCTION It is generally known that protein A, a cell-wall protein from Staphylococcus aureus, binds specifically the Fc region of IgG from several species (1,2). There has been increasing interest in the finding that protein A displays strong mitogenic activity on human B lymphocytes (3), since lipopolysaccaride (LPS) or dextran sulfate, which were good mitogens for murine B lymphocytes, do not stimulate human B lymphocytes. There are several conflicting observations as to which type of lymphocytes are actually stimulated by protein A. In an earlier study by Rodey et al., protein A was reported to stimulate human T lymphocytes (4). Forsgren et al. indicated that while soluble protein A is a weak mitogen, protein A on an insoluble matrix, such as Staphylococcus aureus Cowan I bacteria (SpA CoI) or protein A conjugated to Sepharose, is a strong B-cell mitogen (3). Others have shown, on the other hand, that soluble protein A can stimulate both T and B lymphocytes (5,6). Romagnani et al. recently observed that while insoluble protein A is a B-cell mitogen, soluble protein A, in the presence of T cells, did exert mitogenicity on B cells (7). 142 0008-8749/80/010142-12$02.00/O Copyright All rights

0 1980 by Academic of reproduction in

Press,

any form

Inc. reserved.

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The present authors felt that apparent confusion observed above was possibly ascribable to the soluble or insoluble form of protein A used and the incomplete separation of T and B cells. To clarify these problems, an attempt was undertaken to analyze the responsiveness of highly purified T and B cells to soluble and insoluble protein A. It was clearly shown that while insoluble protein A was a potent T-cell-independent B-cell mitogen, soluble protein A stimulated neither purified T nor B cells alone. It was observed, however, that T cells in the presence of B cells or macrophages and B cells in the presence of B cells or macrophages and B cells in the presence of T cells responded effectively to soluble protein A. The significance of cell-to-cell interactions in T-cell activation described in previously (8-11) was ascertained in this paper. The mechanism of lymphocyte activation by protein A and the role of B cells as proliferation-supporting cells is discussed. MATERIALS

AND METHODS

Cell preparation procedure. Preparation procedure for T and B cells from human tonsil and peripheral blood has been described elsewhere (8- 11). In brief, cells from tonsil and peripheral blood were first depleted of phagocytic cells by carbonyl iron ingestion. Mononuclear cells were obtained by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) sedimentation. These cells were usually designated as “unfractionated” cells. They were then applied to a nylon fiber column (NC, Semidull nylon staple, DuPont, Del.), incubated at 37°C for 45 min, and eluted with warm Eagles’s minimum essential medium (MEM) supplemented with 2% fetal calf serum (FCS, Flow Laboratories, Md.). The NC-passed and NC-retained fractions were collected separately and allowed to form E-rosettes with sheep red blood cells (SRBC). E-Rosette-forming cells (E-RFC) and non-E-RFC were obtained from the bottom and the interface of Ficoll-Hypaque sedimentation, respectively. Both fractions were then washed and subjected to another cycle of E-rosette formation. Thus T cells as E-RFC from the NC-passed fraction and B cells as the non-E-RFC from NC-retained fraction were purified. Cell surface markers had been analyzed in detail previously (8). Depletion of adherent cell population from puri$ed B cells. To exclude the participation of monocytes and macrophages in the purified B-cell preparation, the nonadherent B-cell population (B . NAd) was further separated from the adherent cell population (B *Ad) by three or more incubations on plastic dishes (LUX No. 5220, LUX Scientific Co., Cal.) each at 37°C for 1 hr. The B .NAd population contained virtually no phagocytic cells or macrophages (less than 0.1%). The macrophage-rich fraction was also separated directly from peripheral blood by adhesion onto dishes, incubating at 37°C overnight. The plastic dishes had been previously treated with FCS. This macrophage-rich fraction contained over 40% macrophage-like cells. Lymphocyte culture. Cells were adjusted to 2.5 x lo5 cells/O.5 ml in RPM1 1640 culture medium (Nissui Seiyaku Co., Tokyo) containing 6% FCS and cultured for 72 hr, and 0.5 &i [3H]thymidine ([3H]TdR, 5 Wmmol, Radiochemical Center, Amersham, England) was added 16 hr before cell harvesting. Cells were harvested using a multiple automatic sample harvester (Labo Mash, Laboscience, Tokyo) and radioactivity incorporated was counted with a Packard liquid scintillation counter. The y-globulin-free calf serum was obtained from GIBCO (N.Y.) and y-

144

KASAHARA

ET AL.

globulin-free human serum was kindly donated by Dr. K. Kura, Saitama Medical School. Those contained less than 10 pg/ml of IgG as determined by single radial immunodiffusion. Mitomycin treatment. Cells were adjusted to 1 x 107/ml and incubated with 50 pg/ml of mitomycin C (mc, Sankyo Co., Tokyo) at 37°C for 30 min. They were then washed three times with MEM and resuspended at 5 x lo5 cells/ml. Mitogens. Concanavalin A (Con A, Boehringer-Mannheim), protein A from Staphylococcus aureus (Pharmacia), protein A conjugated to Sepharose (S-pro A, Pharmacia), and Con A conjugated to Sepharose (S-Con A, Pharmacia) were obtained commercially. Staphylococcus uureus Cowan I strain (SpA CoI) was proliferated, then killed with 0.5% formaldehyde and heat treatment (3, 8). All of these mitogens were used at the optimal concentration for mitogenicity as determined previously (8- 11). Iodinated protein A was either obtained commercially (‘251-labeled protein A, 30 &iIpg, Radiochemical Centre, Amersham) or iodinated using the enzymatic method employing lactoperoxidase (12), the specific activity being 4.2 &i/pg. FITC-Conjugated protein A was purchased from Pharmacia. RESULTS Purity

of T and B Cells Used in This Study

T cells were purified from human tonsil and peripheral blood by three steps; first, removal of phagocytic cells, next, passage through nylon fiber column, and, finally, obtaining as E-RFC after twice formation of E-rosette. B cells were obtained from the NC-retained fraction by twice removing E-RFC. Purified B cells were usually followed by depletion of plastic-dish-adherent cells. Purified T cells formed E-rosettes more than 98% and contained less than 1% surface Ig (SIg)-positive cells (Table 1). Purified B cells contained more than 95% SIg-positive cells and less than 1% E-RFC. Almost no monocytes and macrophages were detected in the purified B-cell population depleted of adherent cells. Stimulation

of Purijed

T and B Lymphocytes

by Soluble and Insoluble

Protein

A

We observed that the response of purified human T and B cells was quite different depending on whether protein A was presented in soluble or insoluble form. While insoluble protein A, such as SpA Co1 bacteria or S-pro A, stimulated B cells markedly, though it did not affect T cells at all (Table 2). Purified T cells were stimulated only slightly by soluble protein A, whose response was, if any, far smaller than the B-cell response to SpA CoI. Purified B cells were not stimulated by soluble protein A at all. Insoluble Con A (S-Con A), which was known to be agood stimulant for murine B cells, failed to stimulate human B cells for at least 3-day incubation. Synergistic

Response to Protein A in the Reconstituted

Culture of T and B Cells

Although the stimulation of T cells by soluble protein A was minimal, the response of T cells was greatly enhanced in the presence of autologous B cells (Fig. 1). In this experiment, purified T and B cells were so reconstituted that total cell number was always constant. The response of the reconstituted culture was

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Cell Membrane Marker Analysis of the Cells Used in This Study % of RFC Cell preparation

E

EAC”

% of SIg positive cells”

Peripheral blood, unfractionated Tonsillar lymphocytes, unfractionated Purified T cells Purified B cells Adherent population of purified B Nonadherent population of purified B

62-85 33-55 >98
7-36 28-45 o-7 81-94 NT NT

5-18 34-49
% of phagocytic cells’ 4-31 2-7 0 l-3 2-6
0 EAC was prepared according to the method of Ehlenberger and Nussensweig (8, 38). B SIg-Positive cells were detected by polyvalent fluorescein-conjugated goat anti-human Ig (1:6 dilution, Hyland, Los Angeles, Calif.). c Cells which phagocytosed latex particles (1.1 wm, Dow Chemical Co., Indianapolis, Ind.) during incubation at 3PC. for 2 hr.

maximal between the T/B ratios of 40160 to 80120. The response of SpA Co1 was, on the other hand, independent of the presence of T cells and increased according to the cell number of B cells present in the culture. Which of the T and B cells was actually responding to protein A was then determined through the mitomycin (mc) treatment of either T or B cells. As shown in Fig. 2, it was found that mainly T cells responded to protein A in the presence of mc-treated B cells, and that B cells responded similarly, though to a lesser extent, to protein A in the presence of mc-treated T cells. Overall response of the reconstituted culture was considered to be the mere sum of the response of T cells in the presence of B,, and that of B cells in the presence of T,,. And the contribution of T and B cells to the overall response was estimated approximately to 2: 1. Further Evidences for the Participation Response

of B Cells in the Enhancement

of T-Cell

It was demonstrated that purified T cells, which were otherwise unresponsive to protein A, responded to it markedly in the presence of B,, cells. It may be quite difficult to say whether B cells we employed did not contain other lymphoid cells, such as monocytes and macrophages, which might be actual participants in the T-cell enhancement. To exclude this possibility, B cells which were purified from the cell population initially depleted of phagocytic cells by carbonyl iron ingestion, were further separated into a large population of adherent (B .Ad) and a small population of nonadherent B cells (B +NAd). Again, the B .NAd population as well as the B-Ad population had a strong enhancing effect upon T-cell response (Fig. 3). The macrophage-rich fraction separated directly from peripheral blood also showed similar enhancement as comparable to purified B cells. It was thus clearly demonstrated that even a small percentage of B cells as well as macrophages could enhance T-cell response to protein A. Further evidence of the participation of B cells in the enhancement of T-cell response was presented in a previous paper showing that nonadherent B cells purified from a NC-passed fraction showed a strong helper activity upon T cells at

146

KASAHARA TABLE

ET AL. 2

Stimulation of Human T and B Lymphocytes by Soluble or Insoluble Protein A” [3HITdR Cdl preparation Unfractionated

Blank 1,602 ? 205 (1.0)

uptake

Con A 5 &ml

S-Con A 0.5%

Protein A 10 /L&nl

S-pro A 0.5%

SpA Co1 0.01%

58,493 f 3,127 (36.5)”

39,556 + 2,711 (24.7)

53,157 + 982 (33.2)

29.405 5 3,623 (18.4)

37,132 + 5,422 (23.2)

8,771 -t 1,609 (21.3)

1,861 + 298 (4.5)

Purified

T

412 -e_ 76 (1.0)

5,881 + (14.3)

525

Purified

B

984 -c 145 (1.0)

1,032 t (1.05)

169

644 i (0.65)

128

least comparable to that of purified some human B lymphoid cell lines similar enhancement (11). All these itself possesses a strong enhancing Cell-to-Cell

1,361 ? (3.3)

1,055 + I77 (1.01)

’ Results on the ~dls obtained from human peripheral blood were shown. Similar 1 Stimulation index = PHlTdR uptake @pm) in the presence of mitogen ?H]TdR uptake @pm) in the absence of mitogen

Requirement of Direct Cell Response

@pm)

126

45.518 -r 2,302 (46.3)

data were. obtained

336 f (0.82)

125

48,585 k 3,061 (49.4)

on the cells from palatine

tonsils.

B cells (11). It was indicated, moreover, that (B-cell-type acute lymphatic leukemia) showed results support the notion that at least the B cell capacity upon T-cell response to mitogens. Interaction

for the Enhancement

of the T-

Studies using the Marbrook-type culture chamber have shown that the enhancement of T-cell response to Con A was mediated by direct T-cell/B-cell interaction and not by soluble mediators (11). The present study included a similar

I

O-

T B

blank

!,~.~o~.~,\~ Cl

99

20 60

40 60

L

60 40

60 20

99 Cl

FIG. 1. Stimulation of purified T and B cells with soluble and insoluble protein A. T and B cells purified from palatine tonsil were reconstituted to make a total cell number 5 x 10” cells/ml at the various cell ratios indicated in the abscissa. [3H]TdR incorporation after 72-hr incubation was shown in the ordinate. The response of T and B cells from peripheral blood showed similarly. (0) Protein A (10 &ml), (A) SpA Co1 (O.Ol%), (0) blank control.

CELLULAR

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6-

4-

2-

OT 6


99

20 80

40 60

60 40

60 20

99
T B


20 80

40 60

60 40

FIG. 2. Effect of mitomycin treatment on the protein A responsiveness of reconstituted T or B cells from tonsil were treated with mitomycin and reconstituted at the indicated panel: purity of T cells was above 99%. Right panel: purity of T cells was 95%.

60 20

95 cl

culture. Either cell ratios. Left

experiment on the enhancement of T cells by protein A (Table 3). It was found that the response of T cells to protein A was greatly augmented by the presence of B,, cells in the same chamber. When responding T cells and helping B,, cells were separated by a Millipore filter, only nonsignificant augmentation was observed. When B cells were incubated with T,, cells in the same chamber, moreover, they

0

0.1 % of mc-

treated

05 calls

1.0 added

5

10

20

3. Effect of the addition of various mitomycin-treated cells on the purified T-cell response to protein A (peripheral blood). A varying number of mitomycin-treated cells in 0.2 ml was added to 1.5 x lo5 cells in 0.3 ml. (0) Nonadherent population from purified B, (W) purified B, (0) adherent population of purified B, (A) adherent cells directly taken from peripheral blood, (v) nonadherent cells from peripheral blood, (0) purified T.

FIG. soluble

148

KASAHARA

ET AL.

TABLE Requirement of Direct Cell-to-Cell

3

Interaction for the Enhancement of the T-Cell Response

Reconstitution0 Expt grow

[3H]TdR uptake (cpm)*

Upper

Lower

Blank

1

Till, B,,

T + Tmc T + B,, T T

132 295 103 172

c + 2 -I-

71 112 42 54

1,212 42,416 1,156 2,408

lr. 198 2 1,877 * 312 2 165

2

TltlC B me

B + T,, B + B,, B B

425 398 236 290

2 ” c 2

156 144 96 170

5,734 1,120 1,084 1,344

k 532 5 244 IT 165 k 625

Protein A (10 CLg/ml)

” Marbrook-type culture vessel in which two chambers were separated by a Millipore filter (0.45 pm) was used (11). The upper chamber contained 1 ml of medium only or cell suspensions (5 x 10J cells) and the lower 3 ml of cell suspensions (1.5 x lo6 cells). Mitogen was present in both chambers at the same concentration. b After cells were incubated for 60 hr in the Marbrook-culture vessel, they were placed in test tubes. Then 0.5 &i of [3H]TdR was added and 12 hr later they were harvested and the incorporated radioactivity was counted.

did respond sufficiently to protein A, but failed to respond in the separate chamber. These results indicate the necessity of direct cell-to-cell interaction for the optimal enhancement of T-cell (and also B-cell) response to protein A. Effect of Serum in Culture on the Mitogenicity

of Protein

A and SpA CoZ

To clarify the mechanisms of T- and B-cell activation by protein A, the dependency of the mitogenicity of protein A on serum in culture was first studied. This study was undertaken to determine whether direct binding of protein A or SpA Co1 to the cell surface suffices for stimulation or complex formation between protein A and serum components such as IgG is required. It was found that protein A and SpA Co1 could stimulate lymphocytes to some degree in culture without serum, while Con A could not do so (Table 4). A response to protein A comparable to that with FCS was observed, moreover, in a culture medium containing only bovine serum albumin, whereas, there was no response to Con A. Significant responses to Con A and protein A were obtained in culture medium containing yglobulin-free serum. These results suggest that extrinsic IgG, which forms a complex with protein A, is not prerequisite for its mitogenicity. Binding

Capacity of lz5Z-Labeled Protein

A to Purified

T and B Cells

In order to establish the correlation between stimulatory capacity and the binding properties of protein A, the binding of lz51-labeled protein A to purified T and B cells was examined. It was revealed that the binding of lz51-labeled protein A to B cells was far stronger than to T cells (Table 5). Furthermore, it was clearly shown that B cells but not T cells formed rosettes with SRBC conjugated with protein A (E-pro A). Similarly, B-ALL, a B lymphoid cell line (13), with almost 100% SIg, formed

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Effect of Serum in Culture on the Stimulatory Capacity of Con A, Protein A, and SpA COI” [3H]TdR uptake (cpm) Serum type*

Con A 5 &ml

Blank

Serum free

730 + (0.92)

790 * 112 (1.0)

Protein A 10 &ml 6,740 f

68

SpA Co1 0.01%

455

(8.3

7,520 r 433 (9.5)

y-Globulin-free human serum

601 k 45 (1.0)

6,517 A 942 (10.8)

9,868 k 530 (16.4)

3,256 k 246 (5.4)

y-Globulin-free calf serum

633 t 71 (1.0)

10,256 + 1,127 (16.2)

21,372 !z 1,792 (33.8)

17,838 + 1,504 (28.2)

30

28,305 ir 2,108 (24.2)

14,470 t 792 (12.4)

42,401 -c 3,761 (29.0)

43,100 k 2,729 (29.5)

39,125 + 3,369 (26.8)

Bovine serum albumin

1,170 + 128 (1.0)

FCS

1,460 + 225 (1.0)

967 + (0.83)

a Unfractionated cells obtained from palatine tonsils were used. b Each serum was added to comprise 6% in RPM1 1640 medium.

rosettes with E-pro A around almost every cell. It was also found that B-ALL fixed lz51-labeled protein A as strongly as tonsillar B cells did. It should be noted the percentage of rosette formation with E-pro A correlated well with the binding capacity of 1251-labeled protein A to the cells. The binding of protein A, especially TABLE

5

Selective Binding of Protein A to Tonsil T and B Cells and Some Lymphoid Cell Lines: Rosette Formation with E-Pro A and Binding of 1Z51-Labeled Protein A Cell surface marker (%o) Expt No.

Cell sources

1

Tonsil unfractionated Purified T Purified B

2

MOLT 4B Raji B-ALL” Saitohr

E

EAC”

SIgb

RFC with E-pro A” (P/o)

‘ZSI-protein A bound”

43 98 0

68 7 87

56 0.5 96

34 1 49

12,018 2,229 17,576

2.94 0.54 4.3

0 0 0 0

16 90 22 98

0 0 99 0

0 0 80 < 0

1,676 1,741 15,374 921

0.41 0.43 3.76 0.23

cpm

% of total count

a EAC was prepared according to the method of Ehlenberger and Nussensweig (38). b Enumerated with FITC-labeled rabbit anti-human polyvalent Ig (diluted to 1:6, Hyland). c Protein A fixed to SRBC. d Total count of 1251-labeled protein A (specific activity 4.2 &iI~g) used was 408,780 cpm (1 &i). e Established cell line from B-cell-type acute lymphatic leukemia (13). It possesses surface makers of B cell. ’ Established cell line from Ph’-positive acute lymphatic leukemia. It possesses the surface marker of complement (C3) receptor.

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ET AL.

that of SpA Co1 to B cells, was competitively inhibited by human IgG, and slightly inhibited by IgM, and therefore, the [3H]TdR incorporation with stimulation of SpA Co1 was completely inhibited by less than 10 &ml of IgG (data not shown). DISCUSSION This study has demonstrated first that soluble and insoluble protein A exerted different mitogenic activity on human T and B cells, and second that purified T cells, which were otherwise nonreactive to soluble protein A, could, in the presence of B cells or macrophages, be markedly stimulated by protein A, and, similarly, that purified B cells could respond to soluble protein A only in the presence of T cells. That cell-to-cell interaction is important for the stimulation of T and B cells by protein A has been stressed. It would seem definite that insoluble protein A, such as SpA Co1 bacteria or protein A conjugated to Sepharose, stimulates human B cells without the presence of T cells, as shown previously by Forsgren et al. (3), Romagnani et al. (7), and the present authors ((8) and this study). There have been conflicting observations, however, as to which it is T or B cells that are actually stimulated by soluble protein A. The observations that soluble protein A could stimulate T cells (2, 5, 6) are ascribed clearly from our data to the helper effect by a small number of B cells or macrophages contaminated in their T-cell preparations. Sakane and Green presented another different observation as to the proliferative response of T and B cells to protein A, showing that both T and B cells could be stimulated by protein A (5). The following reasons may be applicable to these apparent conflicts with us: (a) Their T-cell purification step was far more simple than that of this study. They obtained T cells by single E-rosette formation, wherein contamination with a few percentage of B cells and monocytes is inevitable. In the present study, on the other hand, T cells were purified by removal of phagocytic cells, passage through a nylon column, and two runs of E-rosette formation. (b) The different culture condition employed in these studies may have caused these results. In this study, the [3H]TdR uptake at day 3 was examined, while they did so on Day 5, when they observed the maximum response to protein A. With our experimental conditions, no increase of T-cell response to protein A was observed until the sixth day’s incubation. It is especially noteworthy that B cells as well as macrophages could exert a helper effect on the T-cell response to protein A. It has recently been demonstrated that highly purified T cells respond minimally to Con A or PHA and that their responses are markedly enhanced by interaction with other T-cell subsets or other lymphoid cell populations. Cooperation among T-cell subsets is found in synergistic interaction between murine thymus and lymph node cells in response to PHA (14), and interaction between thymus-cell subpopulations in response to Con A (15). The response of the Fc-negative T cells, which were unresponsive to Con A, was greatly increased by the presence of Fc-positive T cells (16). la-negative T cells, which were also unresponsive to Con A, responded significantly in the presence of Ia-positive T cells (17). The role of adherent cells in T-cell activation has been well documented (18-23). In these reports it is interesting to note that the Ia-positive macrophage plays an important role in restoring T-cell response (21). There are some data showing that B cells play a part as helper cells in the enhancement of T-cell response. B cells may permit a helper effect on mixed

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lymphocyte reaction between immunocompetent thymus cells (24). Delespesse et al. suggested that B cells may have some helper effect on the response of human T cells to Con A and PHA (25). Similarly, Romagnani et al. made an observation of T-cell enhancement by mc-treated B cells in response to protein A (7), although they didn’t confirm that the enhancement was actually mediated by B cells and not by macrophages. The present authors have definitely shown the B-cell participation in T-cell response to various T-cell mitogens (8- 11). All these observations have lead to the notion that the activation of T cells requires at least two signals (11,20, 2 1); one is mitogen binding to T-cell surface and the other is cell-to-cell interaction with some antigenic determinants absent on T cells. The nature of the latter signal has not been defined, but one of the candidates may be an Ia-like antigen. We propose these helper B or macrophages to be called as proliferation-supporting cells (PSC). These may be equivalent to the role of macrophages in antibody production as antigen-presenting cells. The results we and others have obtained are rather enigmatic as to the mechanisms of the mitogenicity of protein A. Purified T cells, which were nonreactive to protein A, could be stimulated markedly only in the presence of B cells (or macrophages). Other results showed that neither radioiodinated protein A bound to T cells nor E-pro A formed rosettes with T cells. Actually it was reported that protein A did bind specifically to IgG-positive B cells (26), and thus IgG-positive B cells could be separated from other lymphoid cells (unpublished data). Why is soluble protein A, which bound minimally to T cells, able to stimulate them effectively in the presence of B cells? And if the protein A bound to the B-cell surface was effective in the stimulation of T cells, why could not insoluble protein A stimulate them? This may be explained partly by the possibility that protein A bound to the B-cell surface, which might cause allosteric changes, acts quite differently from insoluble protein A. Alternatively, it may be that the presence of the second signal (such as B-cell helper effect) enables protein A with such low affinity to T cells to stimulate them. A similar puzzle is that the B cells were able to respond to soluble protein A only in the presence of T cells. If the insoluble form of protein A is important for the stimulation of B cells, why is soluble protein A, which must not fix sufficiently to T-cell surface, able to stimulate B cells effectively? It is well known that most T-cell mitogens, such as Con A, PWM, or PHA can stimulate B cells significantly only in the presence of T cells (8-10, 18, 27-31). PPD which was unable to stimulate B cells, could markedly activate B cells in the presence of T cells (32). Similarly, stimulation of B cells by protein A may require the presence of T cells. In this case, insolubilization of protein A by fixing it onto the T-cell surface might not be essential for the T-cell helper effect. It is believed that insoluble protein A would activate B cells by interacting specifically with the Fc-part of the IgG molecules on B cells (3). Preliminary experiments showed that rosette-forming cells of normal peripheral lymphocytes with E-pro A was 4- 11% (mean 7%), coinciding approximately with IgG-positive B cells (unpublished data), although there is yet data suggesting that B cells with intrinsic membrane IgG comprise less than 1% (33). Some IgM-positive B cells may be stimulated by protein A, since protein A binds certain IgM and IgA (34-36). It is unlikely that the cells with IgG passively bound to their Fc-receptors are triggered by protein A, since significant stimulation can be obtained in the culture medium

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without exogenous addition of serum. Moller and Landwall also confirmed that the two biological activities of protein A, to interact with Fc-portion of the Ig molecules and to act as polyclonal B-cell activator are different properties (37). Sakane and Green showed, moreover, that protein A could not stimulate L cells, Fc-receptorbearing lymphocytes. All these observations support the notion that protein A stimulates surface Ig-positive B cells directly. They do little, however, to reconcile the fact that protein A stimulates T cells. In a preliminary experiment, while stimulation of B cells by SpA Co1 was markedly inhibited by IgG of less than 10 CLglml, the inhibition of T-cell stimulation with protein A required more than 500 CLg/ml (unpublished data). This may suggest the possibility that protein A stimulates T cells in rather a different manner than B cells. Further study on the mechanism of T-cell activation by protein A will clarify this point. The present study on the mechanism of mitogenicity of protein A will provide fundamental bases for the immunopathological analysis in various immunodeficiencies and other immunological disorders. The simple measurement of proliferative response to SpA Co1 will make it possible to obtain direct information on the B-cell function, which is evaluated at present by the detection of plasma cells or immunoglobulin production induced by PWM. Through PWM stimulation, B-cell function is evaluated in relation to T-cell function, since PWM is a T-cell-dependent B-cell mitogen. In fact, in one patient of common variable immunodeficiency we observed that the lymphocytes responded normally to SpA CoI, although those from patients with some other agammaglobulinemia responded only poorly to SpA CoI. This suggests that the former B-cell function is not impaired (unpublished data). In other experiments, the proliferative response of cord blood lymphocytes to SpA Co1 were quite low compared to the response of healthy adults, suggesting that neonatal B cells are in immature states of differentiation (manuscript in preparation). Although the relationship between proliferative response to protein A (and also to SpA CoI) and the states of Staphylococcus aureu~ infection is yet unclear, it is highly probable that protein A or SpA Co1 acts as a nonspecific mitogen similar to PHA and Con A, as suggested by Sakane and Green (5). It should be taken into consideration in evaluation of responsiveness that the low responsiveness to various T-cell mitogens, including protein A, might be ascribable not only to the intrinsic defect of T-cell function, but also to other failures in proliferation-supporting cells such as the monocytes and B cells and in the interaction with these cells. Investigations on the response to protein A or SpA Co1 will be quite useful not only for the elucidation of mechanisms of T- and B-cell activation, but for the analysis of B-cell ontogeny and differentiation, and for the immunopathological approach to various immunological disorders. ACKNOWLEDGMENTS The authors wish to thankDr. K. Kura, Saitama Medical School, for supplyingy-globulin-free human serum and Dr. T. Matsuhashi, Institute of Medical Sciences, Tokyo University for supplying Staphylococcus aureus Cowan I strain. We are also in debt to Mr. H. Enomoto, Eikenkagaku Co., Nogi, for his help in iodination of protein A. This study was partly supported by (Grants 3206 1 and 377726) from the Ministry of Education, Science, and Culture of Japan.

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