The donor cell type controls anti-hapten (fluorescein isothiocyanate) primary antibody response to hapten-modified syngeneic cells

Immunology Letters, 5 (1982) 217-221

Elsevier BiomedicalPress

THE DONOR CELL TYPE CONTROLS ANTI-HAPTEN (FLUORESCEIN ISOTHIOCYANATE) PRIMARY ANTIBODY RESPONSE TO HAPTEN-MODIFIED SYNGENEIC CELLS K. SUZUKI, I. NAKASHIMA*, M. TAKASHI, F. NAGASE, N. KATO, K. ISOBE, K. MIZOGUCHI and M. SAITO Departments of Immunology and Bacteriology, Nagoya University, School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466, Japan

(Received 16 August 1982) (Accepted 20 August 1982)

1. Summary Hapten (fluorescein isothiocyanate, FITC)-sensitized syngeneic red blood cells (FITC-RBC) are exceptionally active for induction of anti-hapten primary antibody response, and FITC-modified syngeneic spleen cells depleted of RBC (FITC-SSC) are not immunogenic [4]. The present study has demonstrated that FITC-SSC injected simultaneously with FITCRBC inhibit partially the anti-FITC response to the latter. Either the immunogenicity of FITC-RBC or the response-inhibiting activity of FITC-SSC was increased as the concentration of hapten-sensitizing cells was raised from 0.005 mg/ml to 2 mg/ml. The inhibition of anti-FITC response by FITC-SSC strictly required live donor cells, but was not dependent on T-cell activity of either the donor or recipient. Neither FITC-thymocytes nor the FITC-T-cell-rich fraction of SSC showed a definite activity for inhibition, whereas the FITC-B-cell-rich fraction of SSC acted very effectively. These results suggest that the primary antihapten antibody response to hapten-modified syngeneic cells is primarily controlled by antigen-bearing live donor cells of different cell types. 2. Introduction Hapten-modified syngeneic cells can trigger pri* To whom correspondence should be addressed. Key words: hapten-modified syngeneiccell - fluorescein

isothiocynate-- B-ceUresponse - T-cellindependence donor cell type-dependent control

mary anti-hapten B-cell responses in mice independently of T-cell activity [ 1-3]. Our recent study has, however, indicated that the principal and probably the only antigenic cell type which triggers the antihapten response is the erythrocyte, and that haptenmodified syngeneic spleen cells (hapten-SSC) depleted of red blood cells (RBC) are not active [4]. On the other hand, earlier reports showed that intravenous (i.v.) injection of hapten-SSC [5-10] or hapten-sensitized syngeneic RBC (hapten-RBC) [9,11,12] into mice or rats results in hapten-specific tolerance of both humoral [5-8,11,12] and delayed-type hypersensitivity (DTH) [9,10]. In many experiments the tolerance involved the action of hapten-specific suppressor T-cells [6,9] and did not require live donor cells [5,8,9]. In the model of DTH, however, intact hapten-syngeneic cells of the restricted cell type also provoke an immediate state of unresponsiveness (clone elimination) [ 10], in addition to activation of suppressor T-cells. As far as we know, no one has ever clearly demonstrated that the anti-hapten antibody response to hapten-syngeneic cells is under control of live donor cells of the restricted cell type. Recently we found that the anti-H-2 alloantigen primary IgM antibody response was regulated by donor cells of different cell types [13,14]. The mechanism of this control was independent of T-cell activity and strictly required intact or live donor cells. The results to be presented in this report will show that adequately prepared hapten (fluorescein isothiocyanate (FITC))-intact syngeneic cells of restricted cell types do directly control the level of primary-antihapten antibody response.

0165-2478/82/0000-0000/$2.75 © 1982 ElsevierBiomedicalPress

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3. Materials and methods

4. Results

3.1. Mice Male and female SMA mice and athymic BALB/c nude mice, approximately 8 weeks old, were used. These mice were supplied from the Inbred Animal Breeding Laboratory of Nagoya University School of Medicine or purchased from Nippon CLEA, Osaka.

Table 1 (Expt. 1) shows that the anti-FITC PFC response to immunogenic FITC-RBC is inhibited in part by FITC-SSC which are not themselves immunogenic. FITC-unsensitized control SSC never inhibited the response. In parallel with the previous finding that the intact structure of FITC-RBC was obligatory for anti-FITC response [4], the inhibition of response by FITC-SSC required strictly live donor cells, and lightly heated (56°C, for 15 rain) cells were not active. Key findings in this study were that any preparations of SSC sensitized with FITC at a wide range of concentrations (0.005-2 mg/ml) never acted to augment the response, and that either the immunogenicity of FITC-RBC or the response-inhibiting activity of FITCSSC was increased as the concentration of hapten-sensitizing cells was raised (Expt. 2). This strongly suggests that the opposite actions of FITC-RBC and FITC-SSC for anti-FITC response cannot simply be explained by the effect of density of hapten on the cell surface, although the exact density of hapten on each cell surface remains to be determined. Further studies have demonstrated that inhibition of antiFITC response by FITC-SSC is independent of T-cell activity; the response of athymic nude mice to FITCnude RBC was definitely inhibited by FITC-nude SSC (Expt. 3). Lastly, FITC-thymocytes or the FITC-T-cellrich fraction of SSC showed little, if any, inhibitory effect on anti-FITC response, whereas the FITC-B-cellrich fraction acted highly inhibitorily. This suggests that the inhibition of anti-FITC response by FITC-SSC requires the action of cells of the restricted cell type.

3.2. Cell preparation Suspensions of cells in Eagle's modified minimum essential medium (MEM) were prepared from the spleen and thymus of mice by teasing the organs. To remove RBC the cell preparations were treated with 0.83% NH4C1 in the presence of 5% fetal calf serum (FCS) and 10 mM Hepes (pH 7.2) for 5 min at 4°C and then washed. Nylon wool-adherent (rich in B-cells) and non-adherent (rich in T-cells) spleen cell fractions were prepared as described elsewhere [15]. Peripheral blood cells were obtained by bleeding from the retroorbital venous plexus. 3.3. Modifications of cells by sensitizing with FITC Cells were modified by sensitizing with FITC principally following the technique of Ramos and M611er [3] as described previously [4]. In this study we took special care not to damage cells during hapten-sensitization; cells (10 X 107/ml) were incubated for 45 rain at 4°C in FITC solutions of various concentrations containing 5% FCS and 10 mM Hepes. The strength of fluorescent staining of FITC-cells under a fluorescent microscope has been described previously [4]. 3.4. Immunization Suspensions of FITC-cells were injected i.v. into syngeneic mice. To assay anti-FITC plaque-forming cell (PFC) response, the spleen was removed 4 days after immunization when the response peaked [3]. Each experimental group consisted of 4 - 6 mice. 3.5. Assay for anti-FITC PFC The anti-FITC PFC were detected using FITC-sheep red blood cells (SRBC) as targets as described previously [4]. The number of FITC-specific PFC was calculated by subtracting the number of PFC to SRBC from the number of PFC to FITC-SRBC. All PFC increased after injection of FITC-syngeneic cells were of IgM class [3]. 218

5. Discussion

The present study has demonstrated that anti-FITC primary antibody response to FITC-syngeneic cells is primarily regulated by intact or live donor cells of different cell types such as RBC and SSC. This new finding corresponds well with our recent observation that anti-H-2 d alloantigen primary PFC response is controlled by both donor H-2d+ RBC and H-2d+ B-lymphocytes (Ia ÷, Ig÷, FcR ÷, Thy-1 -) which directly give positive or negative signals to H-2d-specific recipient B-cells [ 13,14]. The inhibition of response observed in the present study was not, however, as strong as

Table 1 Competition of actions of FITC-RBC and FITC-SSC for induction of anti-FITC primary antibody response Expt. a Donor cells

PFC/spleen Nucleated cellsb

RBC Concentration of sensitizing FITC (mg/ml)

Dose (X 10 -6)

Source

1 1 1 1

20 0 20 20 20

+ + +

1

20

+

0.005 0.05 0.5 2 0.5 0.5 0.5 0.5

20 20 20 20 20 20 20 20

1 1

20 20

1 1 1 1 1

10 10 10 10 10

1 1

10 10

1

10

Mean S.E.) d Concentration of sensitizing FITC (mg/ml)

Dose (× 10 -6)

Unstimulated Coackground)

1 0 i 0

0 20 20 20 20

1

20

3240 (290)

0.005 0.05 0.5 2

0 0 0 0 20 20 20 20

+ Spleen (nude)

1

0 10

+ + + +

1 1 1 1

+ + + +

Spleen Spleen Spleen Spleen (heated) c Spleen (heated)

Spleen Spleen Spleen Spleen

Spleen Spleen Thymus Thymus

+ Spleen (B-cell-rich) + Spleen (T-cell-rich)

970 (190)

Stimulated

PFC over background

13,030 1020 13,360 4640 10,040

12,060 50 12,390 3670 9070

(3150) # (190) (3140) (1120)* (1480)

13,720 (2910)

12,750

2640 4100 17,140 30,800 14,240 14,140 9800 6740

(330) (860) (3130) # (4420) (2010) (2400) (1800) (2080)**

0 860 13,900 27,560 11,000 10,900 6560 3500

470 (180)

8550 (970) # 3150 (340)*

8080 2680

0 5 20 5 20

600 (310)

7530 2970 1960 8730 4480

6930 2370 1360 8130 3880

0 3

1340 (380)

1 1

3

(1030) # (450)*** (380)**** (1670) (1370)

5720 (560) # 3560 (450)**

4380 2220

6440 (950)

5100

aSMA mice (Expts. 1, 2, 4 and 5) or BALB/c nude mice (Expt. 3) were injected with FITC-modified or unmodified syngeneic donor cells. Spleens of mice were extracted 4 days after injection for anti-FITC PFC assay. bCells treated with 0.83% NH,C1 at 4°C for 5 min and then washed. CHeated at 56°C for 15 min. dThe difference between the value marked # a n d that marked *(P < 0.05); **(P < 0.02); ***(P < 0.01); or ****(P < 0.001) is statistically significant.

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that in anti-H-2 experiment. This is probably due to partial impairment of donor cells during hapten-modification in vitro, since live donor cells are obligatory for inhibition. In this study, we did not characterize the inhibitory cell type as strictly as we did in the anti-H-2 experiment [14]. But data suggest that the same cell type as identified in the previous experiment [14], the B-lymphocyte, is responsible for inhibition. We have not completely ruled out the role of macrophages in inhibition. However, FITC-macrophagedepleted SSC were no less inhibitory than conventional FITC-SSC (data not shown). Anyway, the present result has formally proved that the donor cell type-dependent control of B-cell response, first characterized in anti-H-2 alloantigen response [ 13,14], does work in interaction among syngeneic cells, and has promoted the idea that the control is mediated by the specialized function of each cell type. It has been shown that a prior injection of haptensyngeneic cells such as (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-RBC [ 11 ], 2,4,6-trinitrophenyl (TNP)-RBC [12], TNP-SSC [5,8] and FITC-SSC [7] into mice [7,8,11,12] or rats [5] inhibits anti-hapten antibody response to the subsequent immunogenic stimuli (induction of tolerance). Suppressor T-cells seem to play a critical role in the induction and maintenance of the tolerant state by TNP-SSC in vivo [ 10]. Either T-cells [5], lymphoid cells other than bone marrow ceils [8] or erythrocytes [ 11,12] acted effectively for induction of tolerance. Furthermore, the tolerance induction did not require live donor cells; heat-killed (56°C, for 30 rain) [5] or sonicated [8] TNP-SSC, or supernatants of TNP-SSC cultured in vitro for 24 h [5] did induce tolerance. These observations form a contrast to findings in the present study about requirements for inhibition of the primary response. The mechanism of control of primary response in the present study appears therefore different from that of tolerance induction in earlier ones. In line with this conclusion, the ordinary procedure used in earlier studies for sensitization of cells with TNP greatly impaired their activity to inhibit the primary response (data not shown). We should further compare our results with those reported by Ramos and M6Uer about immunogenicities of FITC-SSC [3,7]. In their experiments the hapten-concentration to sensitize cells was very critical; only cells sensitized with FITC at a concentration of 0.5 mg/ml (FITCo.s-SSC) 220

were active for induction of primary anti-FITC antibody response, and neither FITCo.0s-SSC nor FITCsSSC were immunogenic. These results do not agree with ours (ref. 4 and this study). Since Ramon and M611er did not state that they used RBC-depleted cells as a source of FITC-SSC, it might be possible that they estimated overall activities of two cell types with opposite activities such as FITC-RBC and FITC-B-lymphocytes as the activity of FITC-SSC. They also showed that either FITCo.os-SSC or FITCo.s-SSC induce tolerance [7]. It would certainly be important to determine whether or not the tolerance induced in their experiment includes the direct inhibition of B-cells by live donor cells of the restricted cell type. The exact mechanism of different actions of various donor cells remains to be clarified. It probably involves special activities of cell membranes to present antigens for B-cells, possibly related to different membrane rigidities, and distinct homing capacities of cells into lymphoid tissues to contact with recipient B-cells. We should remember the report of Scott and his collegues that TNP-SSC inhibit the anti-TNP response to immunogenic stimuli in vitro [ 16,17], and this inhibition had both T-cell-independent (direct B-cell blockade) and suppressor T-cell-mediated processes [6,17]. Only the latter process was found in vivo in their experiment. It might be that unimpaired donor cells which retain the physiological homing capacity could directly inhibit the B-cell response in vivo as partially impaired donor cells did in vitro. Further, the biological significance of possible B-cell (antigen-bearing donor cell)-B-cell (antigen-specific recipient cell) interaction for inhibition of the B-cell response has already been discussed elsewhere [14], as a model of B-B interaction through idiotype-anti-idiotype recognition [ 18]. Finally, whatever the mechanism is, the cell type-dependent control we demonstrated in the present study should logically operate in antihapten antibody response to free haptens introduced into animals, which would stick to cells or tissues of various types in vivo.

Acknowledgements This work was supported in part by Takeda Science Foundation. We thank Mrs. K. Ohashi for technical help.

References [1] Koskimies, S. and Makela, O. (1976) J. Exp. Med. 144, 467-475. [2] Naor, D., Saltoun, R. and Falkenberg, F. (1975) Eur. J. Immunol. 5,220-223. [3] Ramos, T. and M611er, G. (1978) Scand. J. Immunol. 8, 1-7. [4] Nakashima, I., Takashi, M., Nagase, F. and Kato, N. (1981) Eur. J. Immunol. 11,946-948. [5] Long, C. A. and Scott, D. W. (1977) Eur. J. Immunol. 7, 1-6. [6] Scott, D. W. (1978) Cell. Immunol. 37,327-335. [7] Ramos, T., M611er, E. and M611er, G. (1980) Eur. J. Immunol. 10, 100-104. [8] Moody, C. E., Siskind, G. W. and Weksler, M. E. (1980) Ceil. Immunol. 50, 432-439. [9] Claman, H. N., Miller, S. D. and Moorhead, J. W. (1977) Cold Spring Harbor Symp. Quant. Biol. 41,105-111.

[ 10 ] Conlon, P. J., M oorhead, J. W. alld Claman, H. N. ( 1980) Cell. Immunol. 51,360-369. [11] Hamilton, J. A. and Miller, J. F. A. P. (1973) Eur. J. Immunol. 3,457-460. [12] Moody, C. E., Innes, J. B., Siskind, G. W. and Weksler, M. E. (1978) J. Immunol. 120_, 844-849. [13] Nakashima, I., Clark, E. A., Lake, P., Kato, N., Nagase, F., Mizoguchi, K., Isobe, K. and Saito, M. (1982) Transplantation (in press). [14] Nakashima, I., Mizoguchi, K., Kato, N., Nagase, F., Isobe, K., Saito, M. and Suzuki, K. (1982) Eur. J. Immunol. (in press). [15] Nakashima, I. and Lake, P. (1979) Nature (London) 279,716-718. [16] Scott, D. W. and Long, C. A. (1976) J. Exp. Med. 144, 1369-1374. [17] Li, J. T. C. and Scott, D. W. (1980) J. Immunol. 125, 2380-2384. [18] Jerne, N. K. (1974) Ann. Immunol. (Paris) 125C, 373-389.

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