- Email: [email protected]
Immunological properties of Fc receptor on lymphocytes
cm~u~m
39, 225-237
J~I~I~~~LOGY
Immunological
Properties
4. Fc Receptor JUNJI *The
Imtitute
(1978)
YODOI,* jar De~arfmc~kt
of Fc Receptor
of Con A Induced ARIMICHI
Suppressor
TAKABAYASHI,?
on Lymphocytes and Helper
AND
TOHRU
T Cells MASUDA
I~r~wzunology, Faculfy of Medicine, Kyoto Uniz~., and j-The of Suvgevy, Faculty of Mcdicircc, Kyoto Univ. Rcccived
Dcce~ral~cr
* Scco+td
14, 1977
When spleen cells or thymocytes of BALB/ c mice were cultured with 2 pg/tnl of concanavalin A (Con A) for 3 days, both helper and suppressor cells for the i+t vifro plaque forming cell (PFC) response to soluble and particulate antigens (DNP-Asc, SRBC, and HRBC) were induced. The helper cells were nonadherent (nonadh.) while the suppressor cells were adherent (adh.) to nylon wool (NW). As in the case of antigen-specific helpers, Con A-induced nonspecific help-r cells were devoid of Fc receptor (FcR-) . When FcRcells were fractionated into FcRNW adh. and non-adh. cells and cultured either with Con A or with allogeneic cells, marked generation of FcR’ T cells was observed from Days 3 to 5 only in the former cell population. Suppressor cells were also induced preferentially from the former subset. Depletion of the neogeneic FcR+ cells from the Con A-induced suppressor cell population resulted in the loss of the suppressive effect. This suppressor activity of FcR’ T cells is proved in primary as well as secondary responses to both particulate and soluble antigens.
IKTRODUCTION It is widely accepted that Fc receptors for IgG ( FcR) 1 are present on a subset of T lymphocytes (l-3). Fc receptors for IgM were also proved recently on another subset of T cells in human studies (46). It seemslikely that these classspecific Fc receptors on T cells play certain important roles in immune responses through the interaction with antibodies. Our recent studies (7, 8) as well as of others (1, 3), however, clearly demonstrated that the helper activity was associated with T cells devoid of FcR for IgG (FcR- T) rather than with the FcR-bearing T cells (FcR+ T). Because of the lability of FcR particularly on T cells (9-11) and of the intrinsic difficulty in handling FcR+ cells without disturbing their “resting” state 2 (12), relatively little is known about the function and differentiation of FcR+ T cells (2, 13). 1 Abbreviations used in this paper : FcR, Fc receptor fur IgG ; FcR- T and B, T and B cells devoid of FcR; FcR’ T and B, T, and B cells bearing FcR ; NW, nylon wool; Con A, concanavalin A ; KLH, keyhole limpet hemocyanin ; DNP, dinitrophenyl ; Asc, ascaris extract ; HRBC, horse red blood cells; and SRBC, sheep red blood cells; E.4, erythrocyte-antibody complex; MEM, Eagle’s mimimum essential medium; FCS, fetal calf serum; ZME, Zmercaptoethanol ; PFC, plaque forming cells ; MMC, mitomycin C. 2 Masuda, T., Miyama, M., Kuribayashi, K., Yodoi, J., Takabayashi, A. and Kyoizumi, S. Immunological properties of Fc receptor on lymphocytes. 5. Suppressive regulation of humoral immune response by Fc receptor bearing B lymphocytes. Cell. Immunol., 39, 1978. 225
All
OOOS-8749/78/0392-0225$02.00/O Copyright @ 1978 by Academic Press, Inc. rights of reproduction in any form reserved.
226
YODOI,
TAKARAYASHI,
AND
MASUDA
In the preceding paper (8)) it was reported that FcR+ B cells were generated in z&o in a short term culture of FcR- B cells, approximately 5070 of cultured cells being FcR+ after 3 days. However, the phenotypic conversion from nylon wool (NW) passed FcR- T into FcR+ T cells in vitro was not so remarkable, FcR+ cells being less than 20%. This appeared contradictory to the result of in tivo studies (14, 15) in which high proportions of adoptively transferred thymocytes developed FcR in the spleens of irradiated recipients. However, there is another T cell population relatively adherent to NW or glass wool; (adherent T cells) (16, 17). Interestingly, a close relationship between these T cells and suppressor T cells is often indicated (16, 18, 19). It is thus important to solve the question whether poor FcR+ neogenesis is a feature common to both adherent (adh.) and non-adherent (nonadh.) T cells, or alternatively the precursors of FcR+ T cells reside preferentially in the adh. T cells. If the latter is the case, it would be important to clarify the possible causal relationship between FcR+ T and suppressor T cells, both of which are intimately related to the adh. T cell population. To study the relationship among the expression of FcR, adhesiveness to NW and the appearance of suppressor T cells, we analyzed the differentiation process and the characteristics of Con A-induced suppressor and helper T cells, which were originally reported by Dutton et al. (20,21) . Results indicate that the helpers lack FcR and are least adherent to NW, while the suppressorsseemto possessFcR and to be NW-adherent. The precursors of the FcR+ suppressor cells are associated mainly with the adherent T cell population. These findings will be discussedin terms of T cell differentiation and heterogeneity. MATERIALS
AND
METHODS
Mice. Male and female C3H/He mice were supplied from the Institute of Experimental Animals, Faculty of Medicine, Kyoto University and BALB/c mice were from Shizuoka Agricultural Cooperative Association for Laboratory Animals. They were maintained under a specific pathogen-free condition (SPF) . Nude mice with the BALB/c background supplied from the Central Institute of Experimental Animals, Kawasaki, were also maintained under SPF and used 7 to 10 weeks of age. Antigens. Keyhole limpet hemocyanin (KLH) (Grade A) was purchased from Calbiochem. (San Diego, Calif., U.S.A.). Dinitrophenyl (DNP) -conjugated ascaris extract (DNP-Asc) was kindly provided by Dr. Hamaoka in Osaka University. DNP 10.8-KLH was prepared according to the method of Eisen (22). Horse and sheep red blood cells (HRBC and SRBC) were prepared by washing bloods with saline. Bloods, delivered by Nikken Animal Blood Center (Kyoto) , were stored in Alsever’s solution at 4°C until used within 3 weeks after bleeding. Immunization. Mice were injected intraperitoneally with 10 pg of alum-precipitated DNP-Asc or intravenously with 4 x lo8 SRBC. Spleen cells were obtained 4 weeks after immunization. Antisera. Anti-Thy 1.2 serum was prepared as previously described (7). IgG fraction of rabbit anti-SRBC antiserum was obtained by filtration through Sephadex G-200 (Pharmacia Fine Chemicals, Inc., Uppsala, Sweden) (7). Preparation
of erythrocyte-antibody
complex
(EA)
and
rosetting
procedure.
Briefly, EA was prepared by sensitizing SRBC with subagglutinating concentration of 7S fraction of rabbit anti-SRBC antiserum (7). An equal volume of lymphoid cells (5 x lo7 cells/ml) and EA (2 x lo9 cells/ml) were mixed, incubated at 37°C
FCR
OF
CON
A
INDUCED
SUPPRESSORS
AND
HELPERS
227
with gentle agitation for 15 min, centrifuged at 11Og for 5 min, and then incubated for further 15 min at 37°C. EA rosette-forming cells were counted using a hemocytometer. Lymphocytes, binding more than five sensitized red cells, were regarded as FcR+. Separation of rosetting (FcR+) and non-rosetting (FcR-) cells. The method in detail was described earlier (7). The rosetted cell mixture was centrifuged on a cushion of Ficoll-Isopaque, which consisted of 12 parts of 14% (W/V) Ficoll 400 (Pharmacia Fine Chemicals) in distilled water and five parts of 32.8% (W/V) sodium metrizoate (Nyegaard & Co. As. Oslo, Norway). After centrifugation at 12OOg for 12 min at 2O”C, FcR- cell fraction was collected from the interface. Rosetting cells (FcR+ cells) were sedimented to the bottom of tube. Cell fractions were treated with complement (C, fresh guinea pig serum absorbed with mouse spleen cells, at 4°C for 45 min) to lyse free and cell-bound EA. The purity of these two fractions, thus prepared, was on the average 97% for the FcR- and 60% for the FcR+, respectively as shown in our previous article (7). Culture medium Eagle’s minimum essential medium (MEM ; Nissui Seiyaku Co., Ltd., Tokyo) was used for the various treatments of the cells. For the fractionation of cells with NW columns, MEM was supplemented with 5% fetal calf serum (FCS, Microbiological Association, Bethesda, Md., lot 86918). For the cell culture, RPM1 1640 medium (Nissui Seiyaku Co., Ltd., Tokyo) supplemented with 10% FCS and 5 X 1O-5 2-mercaptoethanol (2ME) was used. Preparation of Con A stiwaulated cells. Spleen cells, thymocytes or fractionated cells were suspended in the culture medium at the concentration of 107/ml. One milliliter of cell suspension was put in the well of Limbro’s culture tray with 24 wells (FB16-24TC, Limbro Scientific Co., Inc., Mamden, Conn.) and cultured in the presence of 2 pg/ml Con A (Pharmacia Fine Chem., Sweden) under 5% CO2 and 95% air condition. After 3 days cells were harvested and washed three times with MEM. Separation of adherent (adh.) and non-adherent (non-adh.) cells with nylon wool (NW) coZu+tins. According to the method of Julius et al. (23), lymphoid cells (1 x 10R cells) were loaded onto the MEM-equilibrated NW (0.3 g) packed in a barrel of a disposable 20 ml plastic syringe. After standing at 37°C for 45 min, non-adh. cell population was eluted out with 30 ml of MEM. Adh. cells were released from NW by vigorous shaking of the NW in MEM. Recovery of cells was usually 60 to 70% (adh. plus non-adh. cells) of the applied cells. Culture for PFC response. Using the modified Mishell and Dutton’s system (24)) totally 2 x lo6 to 4 x lo6 of lymphoid cells were cultured with antigen in 2 ml of culture medium in Limbro’s culture tray with 24 wells. As the challenging antigens, 2 x lo6 per well of HRBC, SRBC, or 0.1 pg/ml (final concentration) of DNP-Asc. or DNP-KLH were added to the culture. On Day 5, the culture was terminated and the harvested cells from the duplicated culture were mixed. After washing of the cells, direct anti-HRBC or anti-SRBC PFC were assayed according to a modification of Jerne’s method (25) in agarose gel on slides. Direct anti-TNP PFC were assayed by a modification of the method of Rittenberg et al. (26). Data were the means of the number of PFC of triplicated assay slides, and were expressed as the PFC nmmber per lo7 initial cells. All experiments were performed at least two or three times and one typical result was presented in the text.
228
YODOI,
TAKABAYASHI,
AND
MASUDA
cells, depleted of FcR+ cells by E-4 rosetting, were cultured in 1 ml of culture medium. The culture was stimulated either with 2 pg/ml of Con A or with 1 million allogeneic cells (C3H/He) which had been incubated with 50 pg/ml of mitomycin C (MXC) for 30 min at 37°C and washed three times before addition to the culture. RESULTS Ejject of Con A-stiwm~lated spleen OY thyuaau cells on the in vitro primary response to HRBC. Spleen cells or thymocytes of BALB/ c mice were cultured for 3 days with 2 pg/ml of Con A to obtain Con A stimulated cells. Then the cultured cells were separated into T\;W adh. and non-adh. populations. In the primary anti-HRBC response (Fig. 1) , the indicator cell system was composed of 2 X lo6 nu/nu spleen cells and 5 x lo5 NW non-adh. normal BALB,/c splenic T cells. This combination usually provided more excellent primary IgM-PFC responsesthan spleen cells of oridinal BALB/c mice in our culture system. They were cultured with 2 X lo6 HRBC for 5 days. Various numbers of aclh. or non-adh. Con A stimulated cells (5 x 103to 5 x 105) were added to this system at the start of the culture, In control cultures the antigens was omitted. Without NW purified BALB/c T cells, nu/nu B cells showed no response (below background level). Supplementation with 5 X lo5 of the T cells raised the responseto 163s * 477 IgM PFC/107 cells. It was noted that the suppressive effect was conspicuous in adh. Con A-stimulated cells both from thymocytes and spleen cells; suppression was induced by as few as 5 X lo3 of adh. Con A stimulated cells. On the other hand, addition of 5 x lo3 to 5 x lo4 non-adh. Con -4 stimulated cells augmented the response to above 3000
5x103 Con-A
5x104 stimulated
5xin5 ceils
FIG. 1. Adherent T cells suppress, non-adherent T cells help. BA4LB/c spleen cells and thymocytes (lo7 cells/ml) were cultured with 2 pg/ml of Con A for 3 days. After washing three times, they were fractionated by NW columns into adherent (adh.) and non-adherent (nonadh.) cell populations. In the second culture, nu/nu spleen cells (2 X lo”), supplemented with 5 X 10” of NW purified normal BALB/c splenic T cells were challenged with 2 X 10” of horse red cells (HRBC). To these cultures various numbers (5 X lo”, 5 X 104, 5 X 10”) of non-adh. or adh. Con A stimulated cells induced from spleen cells (n-n, non-adh.; O-0, adh.) or thymtrcytes (A-A, non-adh. ; O-O, adh.) were added at the start of tllc culture. Anti-HRKC TghlPFC responses were assayed on Day 5. Arithmetic means * SE.
FCR
OF
CON
A
INDUCED
SUPPRESSORS
Con-A stimulated
spleen
\
2x104
FIG. 2. Adherent 5 X 105) of NW thymocytes were SRBC 1 month culture with 2 X
HELPERS
0-0
NW adh.
A--A
NW non-adh
0-o
oriqinal
-
NW adh.
A---).
NW non-adh.
229
cells
4 ,\
/ e,G,
AND
105
\
5x105
T
cells suppress the secondary response. Various numbers (2 X 104, 106, adh. or non-adh. Con A stimulated cells induced from BALB/c spleen cells or added to 2 X 10” of syngeneic spleen cells obtained from mice primed with before sacrifice. Anti-SRBC IgM PFC responses were tested after 5 day’s IO6 of SRBC. Arithmetic means * SE.
PFC/lO’T cells. At a higher dose (5 x 10s) than this, however, the response was suppressed to the background level (13s f 53) as in the case of adh. Con A blasts. Furthermore, non-adh. Con A stimulated cells either from spleen cells or from thymocytes could elicit a weak but difinite PFC response on nu/nu spleen cells even in the absence of NW purified T cells (63s f 159, blast from spleen cells ; 558 * 124, blasts from thymocytes). This result indicates that Con A stimulated spleen or thymus cells contain a cell population having an amplifying effect on the in vitro antibody production in addition to a suppressor population. Ejjfect of Con A-induced sz~p~~csso~s on the in zitro srcondq rcsponsc. Fig. 2 shows the effect of adh. and non-adh. Con ,4 stimulated cells on the in vitro secondary anti-SRBC IgM response of SRBC-primed BALB/c spleen cells. JVithout NW separation, both Con A stimulated thymocytes and spleen cells showed only a weak suppressive effect; 43y 0 suppression 1)~ Con A blasts from thymocytes and 37% suppression 1)~ those from spleen cells. In the case of Con A stimulated thymocytes, 2 X lo4 NW aclh. cells sufficed to induce a moderate degree of suppression. In the dose range between 2 x 10’ to lo”, NtV non-aclh. cells showed a weak sup-
230
YODOI,
TAKABAYASHI,
AND
MASUDA
pressive effect. Thus, the degree of suppression by Con A stimulated thymocytes was not remarkable in the secondary response. On the other hand, the NW adh. fraction of Con A stimulated spleen cells had a clear dose-dependent suppressive effect. The response was completely suppressedby 5 x 10” of NW adh. cells. The NW non-adh. fraction, on the contrary, had little suppressive activity. Thus, it seemslikely that the relatively weak suppressive activity of unseparated Con A stimulated spleen cells is due to the interaction or competition between suppressive adh. cells and non-suppressive non-adh. cells, though the possible existence of helper activity in the latter population as shown in Fig. 1 was not demonstrated in this system. Induction
of FcR+ T cells from
FcR-
NW
adherent
and non-a,dherent
T cells in
vitro. This experiment was designed to examine whether the rate of conversion from FcR- to FcR+ is different between NW adh. cells and non-adh. cells. BALB/c spleen cells were depleted of FcR+ cells by EA rosetting. FcR- cells, thus obtained, were fractionated into NW adh. and non-adh. FcR- cells by means of NW columns. Thy-1.2-positive cells in these two populations were 50 t 10% and 80 -t 2% respectively (mean of three independent experiments). Two million FcR- adh. or non-adh. cells were cultured in 1 ml of RPM1 1640 medium containing 10% FCS. NW-unfractionated FcR- cells treated either with anti-Thy-l.2 serum and C or (-1 Anti-Thy + ‘
b!LR Con A
(-)
MLP Con A
(-)
MLR Con A
1.2
NW non-adh. FCR-
NW adh. FcR‘ DAY 2
DAY 3
DAY 4
FIG. 3. Generationof FcR+ cells in the FcR- spleencell culture stimulatedby Con A and allogeneiccells.BALB/c spleencellswere depletedof FcR+ cellsby EA-rosetting. The FcRcellswere separated into FcR- adh.and FcR- non adh.populationsby NW columns.FcR- cells were treated with NMS or with anti-Thy-l.2 scruna+ C. Thesevariously treated FcR- cells (2 X 10’) were cultured in 1 ml of RPM1 1640mediumsupplemented with 10% FCS and stimulatedwith 2 pg/ml of Con A or 1 million mitomycin-treatedallogeneiccells (C3H/He) (MLR, mixed lymphocyte reaction). Reappearance of FcR’ cells and cell viability were checkeddaily. The columnrellresentsthe cell density,hatchedbar showsthe numberof neoqeneicFcR’ cells,andthe numberin the columnrepresents% FcR’ cellsin eachculture.
FCR
OF
CON
A
INDUCED
SUPPRESSORS
AND
HELPERS
231
I
2x104 Con A stimulated
6x104 2x105 cells / 2~10~ spl. cells
FIG. 4. Helper activity of FcR-‘cells in Con A stimulated cell culture. Con A stimulated cells induced from FcR- spleen cells were harvested after 72 hr incubation with Con A (2 pg/ml). They were fractionated again by EA rosetting into neogeneic FcR+ cells ( q --q , FcR-‘+) and FcR non-bearing cells ( w-n , FcR-+). Unfractionated FcR(A-A, FcR-) and these refractionated Con A blasts were added to 2 X 106 of DNP-Asc. primed cells and cultured with homologous antigen (0.1 #g/ml). After 5 days’ culture, anti-DNP IgM-PFC responses were assayed. Arithmetic means &SE.
normal mouse (AKR/J) serum (NMS) and C were also cultured as controls. Each culture was stimulated either with 2 &ml of Con A or with 1 X 10” MMC-treated allogeneic spleen cells (C3H/He). The numbers of total viable cells and FcR+ cells in stimulated cell culture were enumerated on Days 2, 3, and 4 to compare with those in non-stimulated cultures. Results, illustrated in Fig. 3, show that both allogeneic cells and Con A are effective in inducing FcR+ cells from FcR- cell population. The growth pattern of NW adh. FcR- cell population in culture was similar to those of NW non-adh. FcR- and NW untreated-NMS+C treated FcR- cell populations. Viable cell numbers decreasedtransiently but recovered by allogeneic or Con A stimulation within 3 days of culture up to approximately the initial level (2 x 106/ml). Generation of FcR+ cells from FcR- cell population was most remarkable in the culture of NW adh. FcR- cells (more than 30%), moderate in that of NW unfreated-NMS+C treafed FcR- cells (approximately 2070), and lowest in that of NW non-adh. FcRcells (less than 10%). Generation and proliferation of the FcR+ cells in the FcR- cell cultures seem to be dependent on T cell, since the number of cells does not increase in the culture of cells pretreated with anti-Thy-l.2 serum and C. Con A induced helpers and FcR. That antigen-specific helper T cells were NW non-adh. FcR- was shown in the preceding paper (S) . Furthermore, antigen nonspecific helper cells induced by Con A were also NW non-adh. as was shown in
232
YODOI,
TAKABAYASHI,
AND
MASUDA
Fig. 1 and Fig. 2. Thus, it was examined in this experiment whether Con A induced helpers also lack FcR or not. RAT,R/c spleen cells and their FcR+ and FcR- cell fractions were cultured in z~it~o with Con A for 3 days. Various doses (Z-20 x 104) of the stimulated cells were added to the culture of 2 x IO@ syngeneic DNPAsc. primed spleen cells and challenged with 0.1 pg/ml of DNP-Asc. However, no significant differences in the effect of Con A simulated cells on the anti-DNP response was found irrespective of the origin of Con A stimulated cells. As reported previously (8)) and shown in Fig. 3 in this paper, FcR+ cells appear in the culture of FcR- cells within 3 days in vitro. To see the real function of these coexisting FcR- and FcR’ cells, the Con A stimulated cells induced from FcR- cells were separated again into FcR bearing (FcR-“+) and non-bearing (FcR-‘-) cell fractions. As shown in Fig. 4, helper activity was proved only in FcR-+cell population. As few as 2 X 104/well of FcR-‘Con A blasts raised the anti-DNP response to above 2000 PFC/107. Unseparated blasts and FcR-++ cells had no significant helper activity. These results suggest that the poor helper effect of the Con TABLE
1
A Suppressors Induced from Adherent
Con
FcR-
Cells
on .%nti-SRBC
BALES/c spl.
SRBC
Con A-stimul.
cells
5 x 106
cells
Expk
Primed Primed Primed Primed Primed Primed
anti-SRBC” PFC/107
1
Unprimed Unprimed Unprimed Unprimed Unprimed Expt.
Responses
4 4 4 4 4
X X X X X
10Bo lo6 a lo6 a 106a 10fi a
C-1 (+) (+I (+I (+I
C-1 Original(-) FcRFcR-
non-adh.
4 x 105 4 x 105 4 x 105
774 1,926 882 828 2,381
f f f f •t
178 181 158 329 303
2 3 3 3 3 3 3
X X X X X X
10” lo6 lo6 10” lo6 lo6
b b b 6 h b
(-) (+I (+I (+I (+I (+I
(-1 C-1 FcRFcRnon-adh. FcRadh. FcRadh.c (EAd removed)
6 6 6 6
x x x X
105 105 105 105
1,995 28,639 19,507 31,122 18,443 31,122
f 133 f 3,173 f 467 f 487 f 600 + 2,008
a Four million unprimed BALB/c spleen cells were cultured with 5 X 10’ SRBC and 4 X 105 Con A stimulated cells. b BALB/c mice were immunized intravenously with 4 X lo* SRBC 1 month before sacrifice. Three million SRBC-primed spleen cells and 6 X lo5 Con A stimulated cells were cultured with 5 X 106 SRBC. The spleen cells as controls were cultured without either SRBC or Con A stimulated cells. c Con A stimulated cells were induced from unfractionated (Original) BALB/c spleen cells or from FcR-, NW non-adh. FcR(FcRnon-adh.), or NW adh. (FcRadh.) fraction of spleen cells by the culture with 2 pg/ml Con A for 3 days. The method to obtain FcRcell fraction and NW adh. and non-adh. cell fractions was described in Materials and Methods. d Neogencic FcR+ cells, which appeared in the culture of FcR- adh. cell population with Con A, were removed by Et\-rosetting method just before thr setting up of the sccontl culture for I’FC response. e On Day 5, direct anti-SRBC PFC responses were determined by modified Jerne’s method. Arithmetic means f SE.
FCR
DNP-Asc.
OF
CON
priwd
EALE/c spl.
DNP-KLH challenged
Orirlinal
DIIP-Asc.(O.l challenged
233
HELPERS
q/ml) anti-DHP
QM-PFC
illiD) (l/IO)
FcR- non-adherent
(lli'l)
II
(l/10)
II
FcR- adherent
!1/201
II
(l/10)
II
AND
(-)
II
8,
SUPPRESSORS
Can-A stiaulated
4, II
INDUCED
cells
3 x lo6 "
A
FcR-+adherent (FCR ren0ved) I/
(l/20) (l/10)
1
FIG. 5. Suppression of cell. Con A blasts were BALB/c spleen cells. A neogeneic FcR’ cells by (FcR’ removed). In the blasts were added to 3 X DNP-KLH. Anti-DNP (l/20 or l/IO) represent Arithmetic means -C SE.
2
E.G.
2
4
x 103/10'
cells
anti-DNP response by Con A stimulated cells from FcRNW adh. induced from FcRnon-adh., FcRadh., and unseparated (original) portion of Con A b!asts induced from FcRadh. cells was depleted of re-rosetting with EA after 3 days’ culture with mitogen; FcRadh. second culture, 1.5 X 10” or 3 X loj of the variously treated Con A 10’ of syngeneic spleen cells from the mice primed with DNP-Asc. or IgM PFC response were tested on Day 5. Numbers in parenthesis the ratio of supplemented cells to DNP-Asc. primed spleen cells.
A-stimulated FcR- cell fraction is perhaps due to the coexistence of newly formed FcR+ cells, and that Con A induced nonspecific helper cells are FcR--cells. Precursors
of Con
A induced-szrppwssor
cells in the priwmry
and secondary
PFC
Yesponsesto SRBC OY DNP. To the 4 x 10” normal BALB/c spleen cells were added 4 X lo5 syngeneic original spleen, FcR-, or S\V non-adh. FcR- cells, all of which were precultured with Con A for 3 days. They were cultured with 5 x lo6 SRBC for 5 more days. As shown in Table 1, Con a stimulated cells, induced from original or NW adh. FcR- cells9suppressedthe primary anti-SRBC responseto the basal level of the culture without antigen. On the other hand, Con ,4 stimulated cells obtained from NW non-adh. FcR- cells did not suppress the response (Expt. I). The result suggeststhat precursors of Con i2-induced suppressor cells are enriched in NW adh. FcR- cell fraction. It is also demonstrated in Table 1 (Expt. II) that the suppression occurs in virtue of FcR+ cells, which appear in the culture of NW adh. FcR- cells. In this experiment, 6 x 1Oj Con A-stimulated cells of different fractions were added to 3 X lo6 SRBC-primed spleencells and the mixtures were cultured for 5 more days with 5 x lo6 SRBC. The suppression of anti-SRBC responseswas noticed only in the groups, to which Con A-stimulated NM-untreated FcR- or ?;W adh. FcRcells were added. Furthermore, it is noteworthy that the suppressive activity is eliminated by the removal of FcR+ cells which are generated from FcR- cells by stimulation of Con A. The final experiment was designed to confirm that FcR+ T cells, generated from NW adh. FcR- cells by the stimulation of Con .4, are sulq)ressors in antibotl\ responseto a soluble antigen.
234
YODOI,
TAKABAYASHI,
AND
MASUDA
DNP-AX. primed spleen cells (3 x 106) were cultured either with 0.1 pg/ml of DNP-Asc. or with DNP-KLH to elicit anti-DNP PFC response. To these cultures were added 3 X lo5 or 1.5 X lo6 original spleen cells, NW non-adh. FcR- cells or adh. FcR- cells, all of which were precultured with Con A for 3 days, to see their suppressive effect on anti-DNP PFC response. In another group nf experiment, FcR-‘cells, which were prepared from the culture of the adh. FcR- cells with Con A, by removing FcR-‘+ cells by EA-rosetting method, were added to the culture of DNP-Asc primed spleen cells. In the control, original spleen cells was added. It is demonstrated in Fig. 5 that Con A-stimulated original spleen, and NW adh. FcR- cells suppress anti-DNP PFC responses, whereas the non-adh. FcRcells do not. Depletion of FcR+ cells from the adh. FcR’ cell fraction resulted in partial elimination of the suppression in the response when the cells were challenged with DNP-KLH. From these results, it would be postulated that precursors of Con A-induced, nonspecific suppressors are adhrent FcR- T cells and that they convert more readily into FcR+ T cells with suppressive function than the non-adh. FcR- T cell population which forms the pool of helpers. DISCUSSION Evidences are accumulating to indicate that the helper function in antibody response resides in FcR- T cells (2, 4). In the preceding report, we concluded that antigen-specific helper T cells neither bear FcRs nor convert to FcR+ T cells in vi&o (7, 8). Implications are that the FcR+ T cell population may be a subset having a different origin and function from helper T cells. To clarify the relationship between suppressor T cells for antibody response and FcR+ T cells, the functions of Con A stimulated T cells were investigated in the present studies. In the primary response in vitro (Fig. l), suppressor cells were induced both from spleen cells and thymocytes and were adherent to NW. Although helper T cells were also induced by Con A, these cells were non-adherent to NW. In the secondary anti-SRBC response, suppressor activity was demonstrated again in the NW-adherent cell fraction of Con A stimulated cells (Fig. 2). In this case, however, helper activity of non-adh. cells was not proved, possibly due to the abundance of the specific helper cells in the indicator system. The possible effects of a small amount of Con A which may be carried over into the indicator system (27) are perhaps of little significance, since both suppressor and helper cells were clearly separable in our system. In our previous paper (8)) it was shown that antigen-specific helper remained to be FcR- state even after being precultured for 7 days, though a few FcR+ cells newly appeared in the culture. No helper activity was observed in the generating FcR+ cells. The antigen-nonspecific helpers induced by Con A also lack FcR, while FcR+ cells generated from the FcR- cells by Con A (FcR-‘+) have no helper activity (Fig. 4). These results suggest that the helper activity in the Con A stimulated cells is masked by the suppressive effect of pre-existing as well as newly formed FcR+ cells in the stimulated cell culture. Con A induced helper cells are, thus, devoid of FcRs, as is the case of antigen-specific helper T cells (2, 3, 7, 8). Although the conversion rate of NW-purified FcR- T cells into FcR’ cells was significantly lower than that of FcR- 13 cells, the phenotypic conversion of NWadherent FcR- T cells into FcR+ cells was not analyzed in the preceding paper (8).
FCR
OF
CON
A
INDUCED
SUPPRESSORS
AIGD
HELPERS
235
Thus it seems particularly important to solve the problem whether the precursors of FcR’ T cells are really present within NW non-adh. FcR T cell population (28) or adh. FcR- T cell population. As shown in Fig. 3, FcR- adh. and FcR- non-adh. cells proliferate in parallel with each other in response to Con A or to allogeneic cells. During the proliferation of T cells in response to Con A or allogeneic cells, neogenesis of FcR+ cells was remarkable in the culture of NW-unfractionated FcRand NW-adh. FcR- cells (% FcR+; more than 30%). On the other hand, only a weak neogenesis of FcR+ cells was detected in the culture for FcR- non-adh. cells, despite marked proliferative response. It is difficult to demonstrate the T cell property of FcR+ cells directly in the Con A stimulated culture, since majority of Con A stimulated cells are resistant to anti-Thy-l.2 antiserum and C. This is probably due to the paucity of T cell specific antigens on the surface of blast cells (29). However, several circumstantial evidences described below seem to support the T cell origin of the neogeneic FcR+ cells in the culture stimulated by Con A or with allogeneic cells. ( 1) Pretreatment of the spleen cells with anti-Thy-l.2 + C almost totally abolished the proliferation (Fig. 3). (2) Majority of Con A stimulated cells (60 to 70%) were killed by rabbit anti-mouse thymocyte antiserum absorbed extensively with bone marrow cells in the presnce of guinea pig complement, although these cell were resistant to anti-Thy-l.2 serum and C. (3) Surface staining by fluorescinated rabbit anti-mouse Ig antibody showed that less than 5% of Con A stimulated cells bore easily detectable surface immunoglobulins. Besides, approximately 80% of the stimulated cells were killed by nnti-T1~31-1.2+C treatment when they were cultured one more day after washing to remove Con A. (manuscript in preparation) . It is unlikely that the FcR+ cells generated are macrophages because of the lack of phagocytic activity as reported previously (S) . Thus it is concluded that precursors of FcR+ T cells exist mainly in FcR- adh. T cell fraction, while they are few in the NW purified (non-adh.) T cell fraction. Suppressive activity of Con A blasts (20, 21, 30) induced from FcR- adh. and non-adh. cell fractions were compared in the primary and secondary in vitro PFC responses against particulate as well as soluble antigens (Table 1 and Fig. 5). In all cases, suppressive activity was found in the Con A stimulated cells from FcRadh. cell fraction, while it was absent in those from the FcR- NW non-adh. cell fraction. The result implies that neogeneic FcR+ T cells in the adh. cells are perhaps responsible for the suppressive phenomenon. This is supported by the fact that suppressor activity is removed by the depletion of neogeneic FcR+ T cells from the Con A blasts (Fig. 5 and Table 1). FcR’ T cells were also induced preferentially from FcR- adh. cell population of OVA-primed spleen cells by the challenge with homologous antigen. As was the case with Con A-induced suppressors, suppressor activity on the secondary anti-DNP response was proved in the OVA stimulated cells from FcR- adh. cell fraction and absent in those from FcR- non-adh. cell fraction (manuscript in preparation). Relevant to our result is the recent report by Moretta et al. (31), in which FcR+ T cells (binding IgG) in human peripheral blood are shown to suppress the B cell differentiation stimulated by pokeweed mitogen. However this suppressive activity was observed only after rosetting with EA. It should be noticed that, in our experimental system shown in Fig. 5 and Table 1, FcR+ T cells were not treated with EA.
236
YODOI,
TAKABAYASHI,
AND
MASLDA
Hence, the observed suppressnr activity possibly attributable to the neogeneic FcR+ T cells in the Con X stimulated culture is considered to be their inherent property and not the consequence of a nonspecific triggering event caused by the E,&rosetting procedure. Indeed, it was found recently that the manipulation of FcRs on FcR+ B cells either with EA? or insoluble antigen-antibody complex (32, 33) resulted in the non-specific suppression of B cell functions. Although it is unsettled whether this phenomenon reflects the physiological suppressive phenomenon or not, we must take this phenomenon into account in any treatment of FcR+ cells. It should be noticed that in normal spleens, the majority of FcR+ lymphocytes are B cells rather than T cells (7). In viva ( 14, 15) and in z~itro studies (28) indicate that antigeneic or mitogenic stimuli are required for the increase of FcR+ T cells. This implies that the regulatory influence exerted by FcR+ T cells are dependent on the two separable processes ; (1) phenotypic conversion from FcR- adh. T cells into FcR+ T cells and (2) triggering of the FcR+ T cells through FcRs. Either antigenic or mitogeneic stimulus will facilitate the conversion from the FcRT to FcR+ T cells. FcR+ T cells induced by Con A are considered to be already activated and thus manifest a suppressive activity without further triggering event via FcRs. The participation of suppressor T cells is now shown in various types of immune responses including in viva and in zritro antibody responses (18, 34, 35), DNA synthetic responses to mitogens and allogeneic cells (16), and also cell mediated cytotoxic reactions (19). In those studies, it is generally agreeable that suppressor T cells are adherent to NW (16, 18, 19, 35). Our results reported here confirmed these former studies, and further demonstrated that the FcR+ T cells were generated mainly from this adherent FcR- T cell population. In conclusion, in the spleen, there are at least two FcR- T cell subsets separable with the relative adhesiveness to SJV : ( 1) FcR- XW-non-adh. cells (FcR- NW purified T cells) which remain long in FcR- state and contain helper T cells (8) and (2) FcR- IYW-adh. cells which convert to FcR+ cells in high proportion and are responsible for suppressor activity. Both of these cell populations are reactive to Con A as well as to specific antigens. Specificity of the suppressor still remains to be investigated. Our results (Figs. 3 and 5) suggested an intimate relationship between the suppressor activity on NW adh. T cells and FcR+ T cells generating preferentially in this T cell population. However, further investigation is necessary to clarify the central problem whether FcR+ T cells are really suppressor T cells or not. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education of Japan. The helpful advice and discussion by Drs. M. Hanaoka (Virus Research Institute, Kyoto Univ.), S. Kyoto Univ.), M. Miyama and K. Muramatsu (Department of Zoology, Faculty of Science, Kuribayashi (Institute for Immunology, Faculty of Medicine, Kyoto Univ.) are gratefully acknowledged. We also wish to thank Miss hi. Nishimura and M. Fukuba for their skillful technical assistance.
REFERENCES 1. Basten, A., J. Miller, F. A. P., Warner, J. InzwmwJr. 115, 1159, 1975. 2. Stout, R. D., and Herzenberg, A., J. Exp.
W. Med.
L.,
Abraham,
142, 611, 1975.
R.,
Chia,
E.,
and
Gamble,
J.,
FCR
OF
CON
A
II\;Dt-CED
SUPPRESSORS
AND
IIELPI~RS
237
3. Ruhin, B., and Hertel-WTulff, B., Srtrrltl. J. Inrrrl~rjrol. 4, 451, 1975. 4. Moretta, I,., Ferrarini, M., Durante, ,\I. I,., and hliugari, 11. C., /:‘rfr. J. Z~~r~~rfrrrol. 5, 565, 1975. 117, 2171, 1976. 5. Moretta, L., Fcrrarini, M., Durantc, M. I,., and hlingari, ,22. C., J. Inr~rmnol. 6. Ferrarini, M., Moretta, L., Mingari, M. C., Tonda, I’., and Pernis, B., Eur. J. Imrnunol. 6, 520, 1976. 7. Miyama, M., Kuribayashi, K., Yodoi, J., Takabayashi, A., and Masuda, T., Cell. Irmrzunol., 35, 253, 1978. 8. Yodoi, J., Miyama, M., and Masuda, T., Cell. Ilr11rzr~r101., 35, 266, 1978. 9. Fridman, W. H., and Goldstein, P., Ccl/. Irrrnzzrnol. 11, 442, 1974. 10. Gisler, R. H., and Fridman, W. H., J. Esp. Xcd. 142, 507, 1975. 11. Grey, H. M., Anderson, C. L., Heusser, C. H., and Kurinick, J. T., In “Membrane Receptors of Lymphocytes” (M. Seligmann, J. L. Prend’homme, and F. M. Kourilsky, Eds.), p. 185. Am. Elsvier, New York, 1975. 12. Katz, D. H., Lymphocyte Differentiation, Recognition, and Regulation, p. 89. Academic Press, New York, 1977. 13. Stout, R. D., and Herzenbcrg, L. A., J. Bxp. Xrd. 142, 1041, 1975. 14. Yoshida, T. O., and Andersson, B., Scuxd. J. 1rnnrr4~101. 8, 401, 1972. 15. Krammer, P. H., Hudson, L., and Sprcnt, J., J. Exp. Med. 142, 1403, 1975. 16. Folch, H., and Waksman, B. H., J. Imnaunol. 113, 127, 1974. 17. Folch, H., and W’aksman, B. H., J. Zr~rrrzr(no1. 113, 140, 1974. 18. Basten, A., Miller, J. F. A. I’., and Johnson, P., 7‘rarxplan. Rev. 26, 130, 1975. 19. Hodes, R. J., and Hathcock, S., J. 1r~~nzz~&. 116, 167, 1976. 20. Dutton, R. W., J. E.cp. A/cd. 136, 1445, 1972. 21. Dutton, R. W., /. Exp. dfcd. 138, 1496, 1973. 22. Eisen, H. N., III “Methods in Medical Research” (H. N. Eisen, Ed.), pp. 94-102. Year Book Medical Publishers, Chicag-o, 1964. 23. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Z~wmur~oZ.3, 645, 1973. 24. Mishell, R. I., aud Dutton, R. W., J. Esp. ,lIrd. 126, 423, 1967. 25. Jerne, N. K., and Nordin, A. A., .Scirnrc 140, 305, 1963. 26. Rittenberg, M. I)., and Pratt, K. I,., I’loc. Sot. Exp. Biol. Afcd. 123, 575, 1969. 27. Tse, H., and Dutton, R. W., J. Er1. &Icd. 143, 1199, 1976. 28. Stout, R. D., WTaksal, S. D., and Hcrzenbcrg, L. iz., J. E-2-p. Xcd. 114, 54, 1976. 29. Greaves, M. I;., and Raff, M. C., iVtrture (New Biol.) 223, 239, 1971. 30. Dutton, R. W., ?‘rul~splurzt. Iirv. 26, 39, 1975. 31. Moretta, J., Webb, S. R., Grossi, C. E., Lydyard, P. M., and Cooper, M. D., J. Esp. :1fed. 146, 184, 1977. 32. Ryan, J. L., and He&art, P. A., J. Ex/J. Ned. 144, 768, 1976. 33. Sidman, C. L., and Unanue, E. R., J. E.z-p. Med. 144, 882, 1976. 34. Gershon, R. K., hlge, E. M., and Kondo, K., J. 1r~znzurzol. 112, 546, 1974. 35. Tada, T., Taniguchi, M., and Takemori, T., l‘ramplarlt. Rczf. 26, 106, 1975.
39, 225-237
J~I~I~~~LOGY
Immunological
Properties
4. Fc Receptor JUNJI *The
Imtitute
(1978)
YODOI,* jar De~arfmc~kt
of Fc Receptor
of Con A Induced ARIMICHI
Suppressor
TAKABAYASHI,?
on Lymphocytes and Helper
AND
TOHRU
T Cells MASUDA
I~r~wzunology, Faculfy of Medicine, Kyoto Uniz~., and j-The of Suvgevy, Faculty of Mcdicircc, Kyoto Univ. Rcccived
Dcce~ral~cr
* Scco+td
14, 1977
When spleen cells or thymocytes of BALB/ c mice were cultured with 2 pg/tnl of concanavalin A (Con A) for 3 days, both helper and suppressor cells for the i+t vifro plaque forming cell (PFC) response to soluble and particulate antigens (DNP-Asc, SRBC, and HRBC) were induced. The helper cells were nonadherent (nonadh.) while the suppressor cells were adherent (adh.) to nylon wool (NW). As in the case of antigen-specific helpers, Con A-induced nonspecific help-r cells were devoid of Fc receptor (FcR-) . When FcRcells were fractionated into FcRNW adh. and non-adh. cells and cultured either with Con A or with allogeneic cells, marked generation of FcR’ T cells was observed from Days 3 to 5 only in the former cell population. Suppressor cells were also induced preferentially from the former subset. Depletion of the neogeneic FcR+ cells from the Con A-induced suppressor cell population resulted in the loss of the suppressive effect. This suppressor activity of FcR’ T cells is proved in primary as well as secondary responses to both particulate and soluble antigens.
IKTRODUCTION It is widely accepted that Fc receptors for IgG ( FcR) 1 are present on a subset of T lymphocytes (l-3). Fc receptors for IgM were also proved recently on another subset of T cells in human studies (46). It seemslikely that these classspecific Fc receptors on T cells play certain important roles in immune responses through the interaction with antibodies. Our recent studies (7, 8) as well as of others (1, 3), however, clearly demonstrated that the helper activity was associated with T cells devoid of FcR for IgG (FcR- T) rather than with the FcR-bearing T cells (FcR+ T). Because of the lability of FcR particularly on T cells (9-11) and of the intrinsic difficulty in handling FcR+ cells without disturbing their “resting” state 2 (12), relatively little is known about the function and differentiation of FcR+ T cells (2, 13). 1 Abbreviations used in this paper : FcR, Fc receptor fur IgG ; FcR- T and B, T and B cells devoid of FcR; FcR’ T and B, T, and B cells bearing FcR ; NW, nylon wool; Con A, concanavalin A ; KLH, keyhole limpet hemocyanin ; DNP, dinitrophenyl ; Asc, ascaris extract ; HRBC, horse red blood cells; and SRBC, sheep red blood cells; E.4, erythrocyte-antibody complex; MEM, Eagle’s mimimum essential medium; FCS, fetal calf serum; ZME, Zmercaptoethanol ; PFC, plaque forming cells ; MMC, mitomycin C. 2 Masuda, T., Miyama, M., Kuribayashi, K., Yodoi, J., Takabayashi, A. and Kyoizumi, S. Immunological properties of Fc receptor on lymphocytes. 5. Suppressive regulation of humoral immune response by Fc receptor bearing B lymphocytes. Cell. Immunol., 39, 1978. 225
All
OOOS-8749/78/0392-0225$02.00/O Copyright @ 1978 by Academic Press, Inc. rights of reproduction in any form reserved.
226
YODOI,
TAKARAYASHI,
AND
MASUDA
In the preceding paper (8)) it was reported that FcR+ B cells were generated in z&o in a short term culture of FcR- B cells, approximately 5070 of cultured cells being FcR+ after 3 days. However, the phenotypic conversion from nylon wool (NW) passed FcR- T into FcR+ T cells in vitro was not so remarkable, FcR+ cells being less than 20%. This appeared contradictory to the result of in tivo studies (14, 15) in which high proportions of adoptively transferred thymocytes developed FcR in the spleens of irradiated recipients. However, there is another T cell population relatively adherent to NW or glass wool; (adherent T cells) (16, 17). Interestingly, a close relationship between these T cells and suppressor T cells is often indicated (16, 18, 19). It is thus important to solve the question whether poor FcR+ neogenesis is a feature common to both adherent (adh.) and non-adherent (nonadh.) T cells, or alternatively the precursors of FcR+ T cells reside preferentially in the adh. T cells. If the latter is the case, it would be important to clarify the possible causal relationship between FcR+ T and suppressor T cells, both of which are intimately related to the adh. T cell population. To study the relationship among the expression of FcR, adhesiveness to NW and the appearance of suppressor T cells, we analyzed the differentiation process and the characteristics of Con A-induced suppressor and helper T cells, which were originally reported by Dutton et al. (20,21) . Results indicate that the helpers lack FcR and are least adherent to NW, while the suppressorsseemto possessFcR and to be NW-adherent. The precursors of the FcR+ suppressor cells are associated mainly with the adherent T cell population. These findings will be discussedin terms of T cell differentiation and heterogeneity. MATERIALS
AND
METHODS
Mice. Male and female C3H/He mice were supplied from the Institute of Experimental Animals, Faculty of Medicine, Kyoto University and BALB/c mice were from Shizuoka Agricultural Cooperative Association for Laboratory Animals. They were maintained under a specific pathogen-free condition (SPF) . Nude mice with the BALB/c background supplied from the Central Institute of Experimental Animals, Kawasaki, were also maintained under SPF and used 7 to 10 weeks of age. Antigens. Keyhole limpet hemocyanin (KLH) (Grade A) was purchased from Calbiochem. (San Diego, Calif., U.S.A.). Dinitrophenyl (DNP) -conjugated ascaris extract (DNP-Asc) was kindly provided by Dr. Hamaoka in Osaka University. DNP 10.8-KLH was prepared according to the method of Eisen (22). Horse and sheep red blood cells (HRBC and SRBC) were prepared by washing bloods with saline. Bloods, delivered by Nikken Animal Blood Center (Kyoto) , were stored in Alsever’s solution at 4°C until used within 3 weeks after bleeding. Immunization. Mice were injected intraperitoneally with 10 pg of alum-precipitated DNP-Asc or intravenously with 4 x lo8 SRBC. Spleen cells were obtained 4 weeks after immunization. Antisera. Anti-Thy 1.2 serum was prepared as previously described (7). IgG fraction of rabbit anti-SRBC antiserum was obtained by filtration through Sephadex G-200 (Pharmacia Fine Chemicals, Inc., Uppsala, Sweden) (7). Preparation
of erythrocyte-antibody
complex
(EA)
and
rosetting
procedure.
Briefly, EA was prepared by sensitizing SRBC with subagglutinating concentration of 7S fraction of rabbit anti-SRBC antiserum (7). An equal volume of lymphoid cells (5 x lo7 cells/ml) and EA (2 x lo9 cells/ml) were mixed, incubated at 37°C
FCR
OF
CON
A
INDUCED
SUPPRESSORS
AND
HELPERS
227
with gentle agitation for 15 min, centrifuged at 11Og for 5 min, and then incubated for further 15 min at 37°C. EA rosette-forming cells were counted using a hemocytometer. Lymphocytes, binding more than five sensitized red cells, were regarded as FcR+. Separation of rosetting (FcR+) and non-rosetting (FcR-) cells. The method in detail was described earlier (7). The rosetted cell mixture was centrifuged on a cushion of Ficoll-Isopaque, which consisted of 12 parts of 14% (W/V) Ficoll 400 (Pharmacia Fine Chemicals) in distilled water and five parts of 32.8% (W/V) sodium metrizoate (Nyegaard & Co. As. Oslo, Norway). After centrifugation at 12OOg for 12 min at 2O”C, FcR- cell fraction was collected from the interface. Rosetting cells (FcR+ cells) were sedimented to the bottom of tube. Cell fractions were treated with complement (C, fresh guinea pig serum absorbed with mouse spleen cells, at 4°C for 45 min) to lyse free and cell-bound EA. The purity of these two fractions, thus prepared, was on the average 97% for the FcR- and 60% for the FcR+, respectively as shown in our previous article (7). Culture medium Eagle’s minimum essential medium (MEM ; Nissui Seiyaku Co., Ltd., Tokyo) was used for the various treatments of the cells. For the fractionation of cells with NW columns, MEM was supplemented with 5% fetal calf serum (FCS, Microbiological Association, Bethesda, Md., lot 86918). For the cell culture, RPM1 1640 medium (Nissui Seiyaku Co., Ltd., Tokyo) supplemented with 10% FCS and 5 X 1O-5 2-mercaptoethanol (2ME) was used. Preparation of Con A stiwaulated cells. Spleen cells, thymocytes or fractionated cells were suspended in the culture medium at the concentration of 107/ml. One milliliter of cell suspension was put in the well of Limbro’s culture tray with 24 wells (FB16-24TC, Limbro Scientific Co., Inc., Mamden, Conn.) and cultured in the presence of 2 pg/ml Con A (Pharmacia Fine Chem., Sweden) under 5% CO2 and 95% air condition. After 3 days cells were harvested and washed three times with MEM. Separation of adherent (adh.) and non-adherent (non-adh.) cells with nylon wool (NW) coZu+tins. According to the method of Julius et al. (23), lymphoid cells (1 x 10R cells) were loaded onto the MEM-equilibrated NW (0.3 g) packed in a barrel of a disposable 20 ml plastic syringe. After standing at 37°C for 45 min, non-adh. cell population was eluted out with 30 ml of MEM. Adh. cells were released from NW by vigorous shaking of the NW in MEM. Recovery of cells was usually 60 to 70% (adh. plus non-adh. cells) of the applied cells. Culture for PFC response. Using the modified Mishell and Dutton’s system (24)) totally 2 x lo6 to 4 x lo6 of lymphoid cells were cultured with antigen in 2 ml of culture medium in Limbro’s culture tray with 24 wells. As the challenging antigens, 2 x lo6 per well of HRBC, SRBC, or 0.1 pg/ml (final concentration) of DNP-Asc. or DNP-KLH were added to the culture. On Day 5, the culture was terminated and the harvested cells from the duplicated culture were mixed. After washing of the cells, direct anti-HRBC or anti-SRBC PFC were assayed according to a modification of Jerne’s method (25) in agarose gel on slides. Direct anti-TNP PFC were assayed by a modification of the method of Rittenberg et al. (26). Data were the means of the number of PFC of triplicated assay slides, and were expressed as the PFC nmmber per lo7 initial cells. All experiments were performed at least two or three times and one typical result was presented in the text.
228
YODOI,
TAKABAYASHI,
AND
MASUDA
cells, depleted of FcR+ cells by E-4 rosetting, were cultured in 1 ml of culture medium. The culture was stimulated either with 2 pg/ml of Con A or with 1 million allogeneic cells (C3H/He) which had been incubated with 50 pg/ml of mitomycin C (MXC) for 30 min at 37°C and washed three times before addition to the culture. RESULTS Ejject of Con A-stiwm~lated spleen OY thyuaau cells on the in vitro primary response to HRBC. Spleen cells or thymocytes of BALB/ c mice were cultured for 3 days with 2 pg/ml of Con A to obtain Con A stimulated cells. Then the cultured cells were separated into T\;W adh. and non-adh. populations. In the primary anti-HRBC response (Fig. 1) , the indicator cell system was composed of 2 X lo6 nu/nu spleen cells and 5 x lo5 NW non-adh. normal BALB,/c splenic T cells. This combination usually provided more excellent primary IgM-PFC responsesthan spleen cells of oridinal BALB/c mice in our culture system. They were cultured with 2 X lo6 HRBC for 5 days. Various numbers of aclh. or non-adh. Con A stimulated cells (5 x 103to 5 x 105) were added to this system at the start of the culture, In control cultures the antigens was omitted. Without NW purified BALB/c T cells, nu/nu B cells showed no response (below background level). Supplementation with 5 X lo5 of the T cells raised the responseto 163s * 477 IgM PFC/107 cells. It was noted that the suppressive effect was conspicuous in adh. Con A-stimulated cells both from thymocytes and spleen cells; suppression was induced by as few as 5 X lo3 of adh. Con A stimulated cells. On the other hand, addition of 5 x lo3 to 5 x lo4 non-adh. Con -4 stimulated cells augmented the response to above 3000
5x103 Con-A
5x104 stimulated
5xin5 ceils
FIG. 1. Adherent T cells suppress, non-adherent T cells help. BA4LB/c spleen cells and thymocytes (lo7 cells/ml) were cultured with 2 pg/ml of Con A for 3 days. After washing three times, they were fractionated by NW columns into adherent (adh.) and non-adherent (nonadh.) cell populations. In the second culture, nu/nu spleen cells (2 X lo”), supplemented with 5 X 10” of NW purified normal BALB/c splenic T cells were challenged with 2 X 10” of horse red cells (HRBC). To these cultures various numbers (5 X lo”, 5 X 104, 5 X 10”) of non-adh. or adh. Con A stimulated cells induced from spleen cells (n-n, non-adh.; O-0, adh.) or thymtrcytes (A-A, non-adh. ; O-O, adh.) were added at the start of tllc culture. Anti-HRKC TghlPFC responses were assayed on Day 5. Arithmetic means * SE.
FCR
OF
CON
A
INDUCED
SUPPRESSORS
Con-A stimulated
spleen
\
2x104
FIG. 2. Adherent 5 X 105) of NW thymocytes were SRBC 1 month culture with 2 X
HELPERS
0-0
NW adh.
A--A
NW non-adh
0-o
oriqinal
-
NW adh.
A---).
NW non-adh.
229
cells
4 ,\
/ e,G,
AND
105
\
5x105
T
cells suppress the secondary response. Various numbers (2 X 104, 106, adh. or non-adh. Con A stimulated cells induced from BALB/c spleen cells or added to 2 X 10” of syngeneic spleen cells obtained from mice primed with before sacrifice. Anti-SRBC IgM PFC responses were tested after 5 day’s IO6 of SRBC. Arithmetic means * SE.
PFC/lO’T cells. At a higher dose (5 x 10s) than this, however, the response was suppressed to the background level (13s f 53) as in the case of adh. Con A blasts. Furthermore, non-adh. Con A stimulated cells either from spleen cells or from thymocytes could elicit a weak but difinite PFC response on nu/nu spleen cells even in the absence of NW purified T cells (63s f 159, blast from spleen cells ; 558 * 124, blasts from thymocytes). This result indicates that Con A stimulated spleen or thymus cells contain a cell population having an amplifying effect on the in vitro antibody production in addition to a suppressor population. Ejjfect of Con A-induced sz~p~~csso~s on the in zitro srcondq rcsponsc. Fig. 2 shows the effect of adh. and non-adh. Con ,4 stimulated cells on the in vitro secondary anti-SRBC IgM response of SRBC-primed BALB/c spleen cells. JVithout NW separation, both Con A stimulated thymocytes and spleen cells showed only a weak suppressive effect; 43y 0 suppression 1)~ Con A blasts from thymocytes and 37% suppression 1)~ those from spleen cells. In the case of Con A stimulated thymocytes, 2 X lo4 NW aclh. cells sufficed to induce a moderate degree of suppression. In the dose range between 2 x 10’ to lo”, NtV non-aclh. cells showed a weak sup-
230
YODOI,
TAKABAYASHI,
AND
MASUDA
pressive effect. Thus, the degree of suppression by Con A stimulated thymocytes was not remarkable in the secondary response. On the other hand, the NW adh. fraction of Con A stimulated spleen cells had a clear dose-dependent suppressive effect. The response was completely suppressedby 5 x 10” of NW adh. cells. The NW non-adh. fraction, on the contrary, had little suppressive activity. Thus, it seemslikely that the relatively weak suppressive activity of unseparated Con A stimulated spleen cells is due to the interaction or competition between suppressive adh. cells and non-suppressive non-adh. cells, though the possible existence of helper activity in the latter population as shown in Fig. 1 was not demonstrated in this system. Induction
of FcR+ T cells from
FcR-
NW
adherent
and non-a,dherent
T cells in
vitro. This experiment was designed to examine whether the rate of conversion from FcR- to FcR+ is different between NW adh. cells and non-adh. cells. BALB/c spleen cells were depleted of FcR+ cells by EA rosetting. FcR- cells, thus obtained, were fractionated into NW adh. and non-adh. FcR- cells by means of NW columns. Thy-1.2-positive cells in these two populations were 50 t 10% and 80 -t 2% respectively (mean of three independent experiments). Two million FcR- adh. or non-adh. cells were cultured in 1 ml of RPM1 1640 medium containing 10% FCS. NW-unfractionated FcR- cells treated either with anti-Thy-l.2 serum and C or (-1 Anti-Thy + ‘
b!LR Con A
(-)
MLP Con A
(-)
MLR Con A
1.2
NW non-adh. FCR-
NW adh. FcR‘ DAY 2
DAY 3
DAY 4
FIG. 3. Generationof FcR+ cells in the FcR- spleencell culture stimulatedby Con A and allogeneiccells.BALB/c spleencellswere depletedof FcR+ cellsby EA-rosetting. The FcRcellswere separated into FcR- adh.and FcR- non adh.populationsby NW columns.FcR- cells were treated with NMS or with anti-Thy-l.2 scruna+ C. Thesevariously treated FcR- cells (2 X 10’) were cultured in 1 ml of RPM1 1640mediumsupplemented with 10% FCS and stimulatedwith 2 pg/ml of Con A or 1 million mitomycin-treatedallogeneiccells (C3H/He) (MLR, mixed lymphocyte reaction). Reappearance of FcR’ cells and cell viability were checkeddaily. The columnrellresentsthe cell density,hatchedbar showsthe numberof neoqeneicFcR’ cells,andthe numberin the columnrepresents% FcR’ cellsin eachculture.
FCR
OF
CON
A
INDUCED
SUPPRESSORS
AND
HELPERS
231
I
2x104 Con A stimulated
6x104 2x105 cells / 2~10~ spl. cells
FIG. 4. Helper activity of FcR-‘cells in Con A stimulated cell culture. Con A stimulated cells induced from FcR- spleen cells were harvested after 72 hr incubation with Con A (2 pg/ml). They were fractionated again by EA rosetting into neogeneic FcR+ cells ( q --q , FcR-‘+) and FcR non-bearing cells ( w-n , FcR-+). Unfractionated FcR(A-A, FcR-) and these refractionated Con A blasts were added to 2 X 106 of DNP-Asc. primed cells and cultured with homologous antigen (0.1 #g/ml). After 5 days’ culture, anti-DNP IgM-PFC responses were assayed. Arithmetic means &SE.
normal mouse (AKR/J) serum (NMS) and C were also cultured as controls. Each culture was stimulated either with 2 &ml of Con A or with 1 X 10” MMC-treated allogeneic spleen cells (C3H/He). The numbers of total viable cells and FcR+ cells in stimulated cell culture were enumerated on Days 2, 3, and 4 to compare with those in non-stimulated cultures. Results, illustrated in Fig. 3, show that both allogeneic cells and Con A are effective in inducing FcR+ cells from FcR- cell population. The growth pattern of NW adh. FcR- cell population in culture was similar to those of NW non-adh. FcR- and NW untreated-NMS+C treated FcR- cell populations. Viable cell numbers decreasedtransiently but recovered by allogeneic or Con A stimulation within 3 days of culture up to approximately the initial level (2 x 106/ml). Generation of FcR+ cells from FcR- cell population was most remarkable in the culture of NW adh. FcR- cells (more than 30%), moderate in that of NW unfreated-NMS+C treafed FcR- cells (approximately 2070), and lowest in that of NW non-adh. FcRcells (less than 10%). Generation and proliferation of the FcR+ cells in the FcR- cell cultures seem to be dependent on T cell, since the number of cells does not increase in the culture of cells pretreated with anti-Thy-l.2 serum and C. Con A induced helpers and FcR. That antigen-specific helper T cells were NW non-adh. FcR- was shown in the preceding paper (S) . Furthermore, antigen nonspecific helper cells induced by Con A were also NW non-adh. as was shown in
232
YODOI,
TAKABAYASHI,
AND
MASUDA
Fig. 1 and Fig. 2. Thus, it was examined in this experiment whether Con A induced helpers also lack FcR or not. RAT,R/c spleen cells and their FcR+ and FcR- cell fractions were cultured in z~it~o with Con A for 3 days. Various doses (Z-20 x 104) of the stimulated cells were added to the culture of 2 x IO@ syngeneic DNPAsc. primed spleen cells and challenged with 0.1 pg/ml of DNP-Asc. However, no significant differences in the effect of Con A simulated cells on the anti-DNP response was found irrespective of the origin of Con A stimulated cells. As reported previously (8)) and shown in Fig. 3 in this paper, FcR+ cells appear in the culture of FcR- cells within 3 days in vitro. To see the real function of these coexisting FcR- and FcR’ cells, the Con A stimulated cells induced from FcR- cells were separated again into FcR bearing (FcR-“+) and non-bearing (FcR-‘-) cell fractions. As shown in Fig. 4, helper activity was proved only in FcR-+cell population. As few as 2 X 104/well of FcR-‘Con A blasts raised the anti-DNP response to above 2000 PFC/107. Unseparated blasts and FcR-++ cells had no significant helper activity. These results suggest that the poor helper effect of the Con TABLE
1
A Suppressors Induced from Adherent
Con
FcR-
Cells
on .%nti-SRBC
BALES/c spl.
SRBC
Con A-stimul.
cells
5 x 106
cells
Expk
Primed Primed Primed Primed Primed Primed
anti-SRBC” PFC/107
1
Unprimed Unprimed Unprimed Unprimed Unprimed Expt.
Responses
4 4 4 4 4
X X X X X
10Bo lo6 a lo6 a 106a 10fi a
C-1 (+) (+I (+I (+I
C-1 Original(-) FcRFcR-
non-adh.
4 x 105 4 x 105 4 x 105
774 1,926 882 828 2,381
f f f f •t
178 181 158 329 303
2 3 3 3 3 3 3
X X X X X X
10” lo6 lo6 10” lo6 lo6
b b b 6 h b
(-) (+I (+I (+I (+I (+I
(-1 C-1 FcRFcRnon-adh. FcRadh. FcRadh.c (EAd removed)
6 6 6 6
x x x X
105 105 105 105
1,995 28,639 19,507 31,122 18,443 31,122
f 133 f 3,173 f 467 f 487 f 600 + 2,008
a Four million unprimed BALB/c spleen cells were cultured with 5 X 10’ SRBC and 4 X 105 Con A stimulated cells. b BALB/c mice were immunized intravenously with 4 X lo* SRBC 1 month before sacrifice. Three million SRBC-primed spleen cells and 6 X lo5 Con A stimulated cells were cultured with 5 X 106 SRBC. The spleen cells as controls were cultured without either SRBC or Con A stimulated cells. c Con A stimulated cells were induced from unfractionated (Original) BALB/c spleen cells or from FcR-, NW non-adh. FcR(FcRnon-adh.), or NW adh. (FcRadh.) fraction of spleen cells by the culture with 2 pg/ml Con A for 3 days. The method to obtain FcRcell fraction and NW adh. and non-adh. cell fractions was described in Materials and Methods. d Neogencic FcR+ cells, which appeared in the culture of FcR- adh. cell population with Con A, were removed by Et\-rosetting method just before thr setting up of the sccontl culture for I’FC response. e On Day 5, direct anti-SRBC PFC responses were determined by modified Jerne’s method. Arithmetic means f SE.
FCR
DNP-Asc.
OF
CON
priwd
EALE/c spl.
DNP-KLH challenged
Orirlinal
DIIP-Asc.(O.l challenged
233
HELPERS
q/ml) anti-DHP
QM-PFC
illiD) (l/IO)
FcR- non-adherent
(lli'l)
II
(l/10)
II
FcR- adherent
!1/201
II
(l/10)
II
AND
(-)
II
8,
SUPPRESSORS
Can-A stiaulated
4, II
INDUCED
cells
3 x lo6 "
A
FcR-+adherent (FCR ren0ved) I/
(l/20) (l/10)
1
FIG. 5. Suppression of cell. Con A blasts were BALB/c spleen cells. A neogeneic FcR’ cells by (FcR’ removed). In the blasts were added to 3 X DNP-KLH. Anti-DNP (l/20 or l/IO) represent Arithmetic means -C SE.
2
E.G.
2
4
x 103/10'
cells
anti-DNP response by Con A stimulated cells from FcRNW adh. induced from FcRnon-adh., FcRadh., and unseparated (original) portion of Con A b!asts induced from FcRadh. cells was depleted of re-rosetting with EA after 3 days’ culture with mitogen; FcRadh. second culture, 1.5 X 10” or 3 X loj of the variously treated Con A 10’ of syngeneic spleen cells from the mice primed with DNP-Asc. or IgM PFC response were tested on Day 5. Numbers in parenthesis the ratio of supplemented cells to DNP-Asc. primed spleen cells.
A-stimulated FcR- cell fraction is perhaps due to the coexistence of newly formed FcR+ cells, and that Con A induced nonspecific helper cells are FcR--cells. Precursors
of Con
A induced-szrppwssor
cells in the priwmry
and secondary
PFC
Yesponsesto SRBC OY DNP. To the 4 x 10” normal BALB/c spleen cells were added 4 X lo5 syngeneic original spleen, FcR-, or S\V non-adh. FcR- cells, all of which were precultured with Con A for 3 days. They were cultured with 5 x lo6 SRBC for 5 more days. As shown in Table 1, Con a stimulated cells, induced from original or NW adh. FcR- cells9suppressedthe primary anti-SRBC responseto the basal level of the culture without antigen. On the other hand, Con ,4 stimulated cells obtained from NW non-adh. FcR- cells did not suppress the response (Expt. I). The result suggeststhat precursors of Con i2-induced suppressor cells are enriched in NW adh. FcR- cell fraction. It is also demonstrated in Table 1 (Expt. II) that the suppression occurs in virtue of FcR+ cells, which appear in the culture of NW adh. FcR- cells. In this experiment, 6 x 1Oj Con A-stimulated cells of different fractions were added to 3 X lo6 SRBC-primed spleencells and the mixtures were cultured for 5 more days with 5 x lo6 SRBC. The suppression of anti-SRBC responseswas noticed only in the groups, to which Con A-stimulated NM-untreated FcR- or ?;W adh. FcRcells were added. Furthermore, it is noteworthy that the suppressive activity is eliminated by the removal of FcR+ cells which are generated from FcR- cells by stimulation of Con A. The final experiment was designed to confirm that FcR+ T cells, generated from NW adh. FcR- cells by the stimulation of Con .4, are sulq)ressors in antibotl\ responseto a soluble antigen.
234
YODOI,
TAKABAYASHI,
AND
MASUDA
DNP-AX. primed spleen cells (3 x 106) were cultured either with 0.1 pg/ml of DNP-Asc. or with DNP-KLH to elicit anti-DNP PFC response. To these cultures were added 3 X lo5 or 1.5 X lo6 original spleen cells, NW non-adh. FcR- cells or adh. FcR- cells, all of which were precultured with Con A for 3 days, to see their suppressive effect on anti-DNP PFC response. In another group nf experiment, FcR-‘cells, which were prepared from the culture of the adh. FcR- cells with Con A, by removing FcR-‘+ cells by EA-rosetting method, were added to the culture of DNP-Asc primed spleen cells. In the control, original spleen cells was added. It is demonstrated in Fig. 5 that Con A-stimulated original spleen, and NW adh. FcR- cells suppress anti-DNP PFC responses, whereas the non-adh. FcRcells do not. Depletion of FcR+ cells from the adh. FcR’ cell fraction resulted in partial elimination of the suppression in the response when the cells were challenged with DNP-KLH. From these results, it would be postulated that precursors of Con A-induced, nonspecific suppressors are adhrent FcR- T cells and that they convert more readily into FcR+ T cells with suppressive function than the non-adh. FcR- T cell population which forms the pool of helpers. DISCUSSION Evidences are accumulating to indicate that the helper function in antibody response resides in FcR- T cells (2, 4). In the preceding report, we concluded that antigen-specific helper T cells neither bear FcRs nor convert to FcR+ T cells in vi&o (7, 8). Implications are that the FcR+ T cell population may be a subset having a different origin and function from helper T cells. To clarify the relationship between suppressor T cells for antibody response and FcR+ T cells, the functions of Con A stimulated T cells were investigated in the present studies. In the primary response in vitro (Fig. l), suppressor cells were induced both from spleen cells and thymocytes and were adherent to NW. Although helper T cells were also induced by Con A, these cells were non-adherent to NW. In the secondary anti-SRBC response, suppressor activity was demonstrated again in the NW-adherent cell fraction of Con A stimulated cells (Fig. 2). In this case, however, helper activity of non-adh. cells was not proved, possibly due to the abundance of the specific helper cells in the indicator system. The possible effects of a small amount of Con A which may be carried over into the indicator system (27) are perhaps of little significance, since both suppressor and helper cells were clearly separable in our system. In our previous paper (8)) it was shown that antigen-specific helper remained to be FcR- state even after being precultured for 7 days, though a few FcR+ cells newly appeared in the culture. No helper activity was observed in the generating FcR+ cells. The antigen-nonspecific helpers induced by Con A also lack FcR, while FcR+ cells generated from the FcR- cells by Con A (FcR-‘+) have no helper activity (Fig. 4). These results suggest that the helper activity in the Con A stimulated cells is masked by the suppressive effect of pre-existing as well as newly formed FcR+ cells in the stimulated cell culture. Con A induced helper cells are, thus, devoid of FcRs, as is the case of antigen-specific helper T cells (2, 3, 7, 8). Although the conversion rate of NW-purified FcR- T cells into FcR’ cells was significantly lower than that of FcR- 13 cells, the phenotypic conversion of NWadherent FcR- T cells into FcR+ cells was not analyzed in the preceding paper (8).
FCR
OF
CON
A
INDUCED
SUPPRESSORS
AIGD
HELPERS
235
Thus it seems particularly important to solve the problem whether the precursors of FcR’ T cells are really present within NW non-adh. FcR T cell population (28) or adh. FcR- T cell population. As shown in Fig. 3, FcR- adh. and FcR- non-adh. cells proliferate in parallel with each other in response to Con A or to allogeneic cells. During the proliferation of T cells in response to Con A or allogeneic cells, neogenesis of FcR+ cells was remarkable in the culture of NW-unfractionated FcRand NW-adh. FcR- cells (% FcR+; more than 30%). On the other hand, only a weak neogenesis of FcR+ cells was detected in the culture for FcR- non-adh. cells, despite marked proliferative response. It is difficult to demonstrate the T cell property of FcR+ cells directly in the Con A stimulated culture, since majority of Con A stimulated cells are resistant to anti-Thy-l.2 antiserum and C. This is probably due to the paucity of T cell specific antigens on the surface of blast cells (29). However, several circumstantial evidences described below seem to support the T cell origin of the neogeneic FcR+ cells in the culture stimulated by Con A or with allogeneic cells. ( 1) Pretreatment of the spleen cells with anti-Thy-l.2 + C almost totally abolished the proliferation (Fig. 3). (2) Majority of Con A stimulated cells (60 to 70%) were killed by rabbit anti-mouse thymocyte antiserum absorbed extensively with bone marrow cells in the presnce of guinea pig complement, although these cell were resistant to anti-Thy-l.2 serum and C. (3) Surface staining by fluorescinated rabbit anti-mouse Ig antibody showed that less than 5% of Con A stimulated cells bore easily detectable surface immunoglobulins. Besides, approximately 80% of the stimulated cells were killed by nnti-T1~31-1.2+C treatment when they were cultured one more day after washing to remove Con A. (manuscript in preparation) . It is unlikely that the FcR+ cells generated are macrophages because of the lack of phagocytic activity as reported previously (S) . Thus it is concluded that precursors of FcR+ T cells exist mainly in FcR- adh. T cell fraction, while they are few in the NW purified (non-adh.) T cell fraction. Suppressive activity of Con A blasts (20, 21, 30) induced from FcR- adh. and non-adh. cell fractions were compared in the primary and secondary in vitro PFC responses against particulate as well as soluble antigens (Table 1 and Fig. 5). In all cases, suppressive activity was found in the Con A stimulated cells from FcRadh. cell fraction, while it was absent in those from the FcR- NW non-adh. cell fraction. The result implies that neogeneic FcR+ T cells in the adh. cells are perhaps responsible for the suppressive phenomenon. This is supported by the fact that suppressor activity is removed by the depletion of neogeneic FcR+ T cells from the Con A blasts (Fig. 5 and Table 1). FcR’ T cells were also induced preferentially from FcR- adh. cell population of OVA-primed spleen cells by the challenge with homologous antigen. As was the case with Con A-induced suppressors, suppressor activity on the secondary anti-DNP response was proved in the OVA stimulated cells from FcR- adh. cell fraction and absent in those from FcR- non-adh. cell fraction (manuscript in preparation). Relevant to our result is the recent report by Moretta et al. (31), in which FcR+ T cells (binding IgG) in human peripheral blood are shown to suppress the B cell differentiation stimulated by pokeweed mitogen. However this suppressive activity was observed only after rosetting with EA. It should be noticed that, in our experimental system shown in Fig. 5 and Table 1, FcR+ T cells were not treated with EA.
236
YODOI,
TAKABAYASHI,
AND
MASLDA
Hence, the observed suppressnr activity possibly attributable to the neogeneic FcR+ T cells in the Con X stimulated culture is considered to be their inherent property and not the consequence of a nonspecific triggering event caused by the E,&rosetting procedure. Indeed, it was found recently that the manipulation of FcRs on FcR+ B cells either with EA? or insoluble antigen-antibody complex (32, 33) resulted in the non-specific suppression of B cell functions. Although it is unsettled whether this phenomenon reflects the physiological suppressive phenomenon or not, we must take this phenomenon into account in any treatment of FcR+ cells. It should be noticed that in normal spleens, the majority of FcR+ lymphocytes are B cells rather than T cells (7). In viva ( 14, 15) and in z~itro studies (28) indicate that antigeneic or mitogenic stimuli are required for the increase of FcR+ T cells. This implies that the regulatory influence exerted by FcR+ T cells are dependent on the two separable processes ; (1) phenotypic conversion from FcR- adh. T cells into FcR+ T cells and (2) triggering of the FcR+ T cells through FcRs. Either antigenic or mitogeneic stimulus will facilitate the conversion from the FcRT to FcR+ T cells. FcR+ T cells induced by Con A are considered to be already activated and thus manifest a suppressive activity without further triggering event via FcRs. The participation of suppressor T cells is now shown in various types of immune responses including in viva and in zritro antibody responses (18, 34, 35), DNA synthetic responses to mitogens and allogeneic cells (16), and also cell mediated cytotoxic reactions (19). In those studies, it is generally agreeable that suppressor T cells are adherent to NW (16, 18, 19, 35). Our results reported here confirmed these former studies, and further demonstrated that the FcR+ T cells were generated mainly from this adherent FcR- T cell population. In conclusion, in the spleen, there are at least two FcR- T cell subsets separable with the relative adhesiveness to SJV : ( 1) FcR- XW-non-adh. cells (FcR- NW purified T cells) which remain long in FcR- state and contain helper T cells (8) and (2) FcR- IYW-adh. cells which convert to FcR+ cells in high proportion and are responsible for suppressor activity. Both of these cell populations are reactive to Con A as well as to specific antigens. Specificity of the suppressor still remains to be investigated. Our results (Figs. 3 and 5) suggested an intimate relationship between the suppressor activity on NW adh. T cells and FcR+ T cells generating preferentially in this T cell population. However, further investigation is necessary to clarify the central problem whether FcR+ T cells are really suppressor T cells or not. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education of Japan. The helpful advice and discussion by Drs. M. Hanaoka (Virus Research Institute, Kyoto Univ.), S. Kyoto Univ.), M. Miyama and K. Muramatsu (Department of Zoology, Faculty of Science, Kuribayashi (Institute for Immunology, Faculty of Medicine, Kyoto Univ.) are gratefully acknowledged. We also wish to thank Miss hi. Nishimura and M. Fukuba for their skillful technical assistance.
REFERENCES 1. Basten, A., J. Miller, F. A. P., Warner, J. InzwmwJr. 115, 1159, 1975. 2. Stout, R. D., and Herzenberg, A., J. Exp.
W. Med.
L.,
Abraham,
142, 611, 1975.
R.,
Chia,
E.,
and
Gamble,
J.,
FCR
OF
CON
A
II\;Dt-CED
SUPPRESSORS
AND
IIELPI~RS
237
3. Ruhin, B., and Hertel-WTulff, B., Srtrrltl. J. Inrrrl~rjrol. 4, 451, 1975. 4. Moretta, I,., Ferrarini, M., Durante, ,\I. I,., and hliugari, 11. C., /:‘rfr. J. Z~~r~~rfrrrol. 5, 565, 1975. 117, 2171, 1976. 5. Moretta, L., Fcrrarini, M., Durantc, M. I,., and hlingari, ,22. C., J. Inr~rmnol. 6. Ferrarini, M., Moretta, L., Mingari, M. C., Tonda, I’., and Pernis, B., Eur. J. Imrnunol. 6, 520, 1976. 7. Miyama, M., Kuribayashi, K., Yodoi, J., Takabayashi, A., and Masuda, T., Cell. Irmrzunol., 35, 253, 1978. 8. Yodoi, J., Miyama, M., and Masuda, T., Cell. Ilr11rzr~r101., 35, 266, 1978. 9. Fridman, W. H., and Goldstein, P., Ccl/. Irrrnzzrnol. 11, 442, 1974. 10. Gisler, R. H., and Fridman, W. H., J. Esp. Xcd. 142, 507, 1975. 11. Grey, H. M., Anderson, C. L., Heusser, C. H., and Kurinick, J. T., In “Membrane Receptors of Lymphocytes” (M. Seligmann, J. L. Prend’homme, and F. M. Kourilsky, Eds.), p. 185. Am. Elsvier, New York, 1975. 12. Katz, D. H., Lymphocyte Differentiation, Recognition, and Regulation, p. 89. Academic Press, New York, 1977. 13. Stout, R. D., and Herzenbcrg, L. A., J. Bxp. Xrd. 142, 1041, 1975. 14. Yoshida, T. O., and Andersson, B., Scuxd. J. 1rnnrr4~101. 8, 401, 1972. 15. Krammer, P. H., Hudson, L., and Sprcnt, J., J. Exp. Med. 142, 1403, 1975. 16. Folch, H., and Waksman, B. H., J. Imnaunol. 113, 127, 1974. 17. Folch, H., and W’aksman, B. H., J. Zr~rrrzr(no1. 113, 140, 1974. 18. Basten, A., Miller, J. F. A. I’., and Johnson, P., 7‘rarxplan. Rev. 26, 130, 1975. 19. Hodes, R. J., and Hathcock, S., J. 1r~~nzz~&. 116, 167, 1976. 20. Dutton, R. W., J. E.cp. A/cd. 136, 1445, 1972. 21. Dutton, R. W., /. Exp. dfcd. 138, 1496, 1973. 22. Eisen, H. N., III “Methods in Medical Research” (H. N. Eisen, Ed.), pp. 94-102. Year Book Medical Publishers, Chicag-o, 1964. 23. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Z~wmur~oZ.3, 645, 1973. 24. Mishell, R. I., aud Dutton, R. W., J. Esp. ,lIrd. 126, 423, 1967. 25. Jerne, N. K., and Nordin, A. A., .Scirnrc 140, 305, 1963. 26. Rittenberg, M. I)., and Pratt, K. I,., I’loc. Sot. Exp. Biol. Afcd. 123, 575, 1969. 27. Tse, H., and Dutton, R. W., J. Er1. &Icd. 143, 1199, 1976. 28. Stout, R. D., WTaksal, S. D., and Hcrzenbcrg, L. iz., J. E-2-p. Xcd. 114, 54, 1976. 29. Greaves, M. I;., and Raff, M. C., iVtrture (New Biol.) 223, 239, 1971. 30. Dutton, R. W., ?‘rul~splurzt. Iirv. 26, 39, 1975. 31. Moretta, J., Webb, S. R., Grossi, C. E., Lydyard, P. M., and Cooper, M. D., J. Esp. :1fed. 146, 184, 1977. 32. Ryan, J. L., and He&art, P. A., J. Ex/J. Ned. 144, 768, 1976. 33. Sidman, C. L., and Unanue, E. R., J. E.z-p. Med. 144, 882, 1976. 34. Gershon, R. K., hlge, E. M., and Kondo, K., J. 1r~znzurzol. 112, 546, 1974. 35. Tada, T., Taniguchi, M., and Takemori, T., l‘ramplarlt. Rczf. 26, 106, 1975.