Human Nonspecific Suppressor Factor (hNSF): Cell Source and Effects on T and B Lymphocytes

Immunobiol., vol. 192, pp. 262-271 (1995) © 1995 by Gustav Fischer Verlag, Stuttgart

lThe Third Division of Internal Medicin, 2Department of Biochemistry, 3Division of Transfusion, Shimane Medical University, Izumo, Japan

Human Nonspecific Suppressor Factor (hNSF): Cell Source and Effects on T and B Lymphocytes

RICARDO XAVIER 1 , MORIHIKONAKAMURA2

, SHOTAIKOBAYASHI1, HIROTO ISHIKURA3

, and YOSHINORITANIGAWA2

Received October 5, 1994· Accepted in revised form November 21, 1994

Abstract

The human nonspecific suppressor factor (hNSF), the probable counterpart of the murine monoclonal nonspecific suppressor factor (MNSF), has been isolated from the ascitic fluid of a patient with systemic lupus erythematosus and characterized. hNSF presents an inhibitory activity on the proliferation and IgG production of mitogen stimulated human PBMC. In the present study, we demonstrate that hNSF can be isolated from the supernatants of Con A-activated T cells, but not from CDS-depleted T cells, indicating the CDS+ T cells are the major source of the factor. We also studied the effects of hNSF on purified human Band T cells; hNSF strongly inhibited the proliferation and Ig secretion by highly purified B cells induced by SAC plus IL-2, as well as the proliferation of T cells activated by Con A plus IL-2. These results indicate that hNSF is a CDS+ T cell product with strong antigen-nonspecific immunoregulatory action on both lymphocyte populations.

Introduction

The monoclonal nonspecific suppressor factor (MNSF), a product of a murine T cell hybridoma that suppresses Band T lymphocyte responses to mitogens, has been isolated and characterized (1-3). Using an anti-MNSF monoclonal antibody (4) we have recently isolated a suppressive activity from the ascitic fluid of a patient suffering from systemic lupus erythematosus (SLE) (5). This putative human counterpart of MNSF, denominated human nonspecific suppressor factor (hNSF), migrates as a

Abbreviations: hNSF=human nonspecific suppressor factor, MNSF=monoclonal nonspecific suppressor factor, PBMC = peripheral blood mononuclear cells, TCRa = T cell receptor a chain

hNSF: source and action on T and B cells . 263

band of about 65 kD, what probably represents an aggregate of smaller hydrophobic protomers (25 and 35 kD bands) (5). An anti-hNSF monoclonal antibody (P2) was produced which can neutralize the suppressive activity of hNSF and does not cross-react with a number of human recombinant cytokines such as TGF~2, IL-2, IL-4, IL-IO and IFN-y (5). Interestingly, hNSF was found to be antigenically related to the a chain of the T cell receptor, as has been described for a number of antigen-specific suppressor factors (6, 7).

hNSF can be isolated from the supernatant of Con A-activated normal PBMC cultures with the use of an affinity column containing P2 mAb. In an attempt to identify the major cell source of the factor, we investigated the presence of hNSF activity in the culture supernatants of isolated T cells or CD4- and CDS-depleted T cell populations after non-cognate activation. Functionally, affinity purified hNSF strongly suppresses IgG production by pokeweed mitogen (PWM-)stimulated PBMC (5). However, it was not clear whether this inhibition was a direct effect on B cells or whether hNSF also has an effect on T cells. Therefore, in order to determine more precisely the target cells of hNSF suppression, we examined the effects of hNSF on highly purified T and B cell populations.

Materials and Methods

Affinity chromatography

hNSF was purified from the ascitic fluid of a SLE patient or from the indicated culture supernatants by affinity columns constructed by coupling anti-hNSF (P2) monoclonal antibody to Sepharose 4B (Pharmacia Fine Chemicals, Sweden), as described (5). Specific activity of purified hNSF was 4.5 x 105 U/mg and it resolved as a single 65 kDa band on SDS-PAGE.

Cell separation

PBMC were obtained from healthy donors by separation over a lymphocyte separation solution (Nacalai Tesque Inc., Kyoto, Japan). Monocytes and natural killer cells were depleted by incubating them for 45 min at room temperature with 5 mM I-leucine methyl ester (Sigma, St. Louis, MO, USA) in serum-free medium (8). After washing, T cells were separated by rosetting (2 x) with 2-aminoethylisothiouronium bromide-treated sheep erythrocytes (E+ -rosetting T cells) (9). B cells were further positively separated with antiCD19 coated Dynabeads (Dynal, Norway) and detached with Detachabeads (Dynal) according to the maker's instructions; 96-98 % CD19 positive cells were obtained. The CD4- and CD8-depleted T cell populations were obtained by negative panning with anti-CD4 and anti-CD8 mAb (Becton Dickinson, CA, USA), as described (10); populations were> 95 % CD4+CD8- or CD4-CD8+ as determined by indirect flow cytometry.

Culture conditions/bioassays

Human PBMC or purified B or T cells were cultured for the indicated time periods at 37°C in RPM I 1640 medium supplemented with 1 mM glutamine, penicillin/streptomycin and 10 % fetal calf serum. Affinity purified hNSF was added in various concentrations at the beginning of mitogen stimulated cultures (1 U of hNSF is defined as the

264 . R. XAVIER, M. NAKAMURA, S. KOBAYASHI, H. ISHIKURA, and Y. TANIGAWA

amount capable of 50 % suppression of IgG production by PWM-stimulated PBMC). For T cell proliferation assays, T cells (rosetting cells, followed by depletion of CD19+ cells with Dynabeads) were cultured with Con A (5 [tg/ml) for 12h, washed with RPM I and recultured in the presence of human recombinant IL-2 (25 U/ml) (Toyobo, Osaka, Japan) as described (11). For B cell studies highly purified B cells were cultured in the presence of Staphylococcus aureus Cowan I (SAC) (Calbiochem, La Jolla, CA, USA; 1:60,000 v/v) plus IL-2 (50 U/ml (12) or phorbol myristic acetate (PMA) (10 ng/ml) (Sigma) plus ionomycin (50 ng/ml) (Calbiochem) (13), and proliferation or Ig production was assessed.

Analysis of secreted 19

IgG concentration in culture supernatants was measured by a sandwich ELISA as described (5). A similar method was used for IgM measuring; briefly, anti-human IgM (Nichirei, Tokyo, Japan) was coated on 96-well plates (Nunc, Roskilde, Denmark), followed by the addition of diluted supernatant or standard concentrations of human IgM Oackson Lab., West Grove, PA, USA); horseradish peroxidase-conjugated goatanti-human IgM (Tago, Burlingame, CA, USA) was the second antibody, and color detection was performed as for IgG ELISA.

Proliferation assays

Inhibition of proliferative responses was examined; cells were cultured in the presence or absence of hNSF for the indicated times, after which they were harvested. CH]thymidine (1 [tCi/well) was pulsed in the last 16 hours, and the incorporation was measured by standard liquid scintillation counting technique.

Statistical analysis

Means of three independent experiments for controls and hNSF addition in various concentrations were compared by two-tail Student's t test and p:S 0.01 was considered as significant.

Results

Isolation of hNSF from T cell culture supernatants

We investigated the presence of hNSF suppressive activity in the culture supernatants of purified T cells and CD4- or CDS-depleted T cells. As shown in Figure 1, suppressive activity, as measured by the inhibition of IgG production by PWM-activated PBMC, was present in the P2-column eluate fraction from the supernatant of Con A-activated T cells (35 % suppression as compared to the control), but not from the supernatant of unstimulated cells. Higher activity could be eluted from the supernatant of CD4-depleted T cells (56 % suppression), whereas none could be recovered from the CDS-depleted T cells, suggesting that CDS+ cells are the major source of hNSF. No significant suppressive activity was detected in the supernatants from the effluent affinity fractions or from B cell culture supernatants (not shown).


Control

Whole T cells

CDS-depleted

CD4-depleted

500 1000 1500

IgG {ng/ml}

Figure 1. hNSF activity on the culture supernatants of T cells and T cell subsets. Total T cells, CD4-depleted and CDS-depleted T cell populations were prepared as in «Materials and Methods» and cultured (1 x 106 cells/ml) in the absence or presence of Con A (5 [!g/ml) for 4S h. Then, culture supernatants were harvested and fractionated in an affinity column containing the P2 anti-hNSF mAb, and the eluate fraction was harvested, dialized against PBS and concentrated to 10 x original volume. Pokeweed mitogen (1 % )-stimulated human PBMC (2 x 105

) were cultured with no addition (D) or with addition of the P2-eluate from the supernatant of unstimulated (IWJ!) or Con A-activated (.) T cell populations. After 6 days, the IgG concentration in the supernatants was estimated by ELISA. Results are expressed as mean ± SEM of three independent experiments. Statistically significant differences (Student's t test: p < 0.001) are indicated by (*).

hNSF effects on purified T cells

To investigate the effects of the hNSF purified from the aSCitiC fluid obtained from a SLE patient on E+ -rosetting T cells, DNA synthesis of cells activated by Con A for 12 h, washed and subsequently stimulated with IL-2 was measured in the absence or presence of the factor. This duration of Con A-stimulation is sufficient for T cell activation and expression of IL-2 receptor, but not for subsequent proliferation in the absence of exogenous IL-2 (14, 15). As shown in Figure 2, optimal concentrations of hNSF inhibited up to about 70 % the proliferation of T cells as measured by the incorporation of tritiated thymidine. Therefore, hNSF is produced by T cells and has a potent inhibitory effect on these cells.

hNSF inhibits B cell proliferation and Ig secretion

Because hNSF strongly suppressed the IgG production by PWM-stimulated PBMC (5), we attempted to resolve whether this is a direct action on B cells or a T cell-dependent response. Highly purified B cell populations were stimulated to proliferate by two kinds of stimuli and the effect of the


Table 1. Inhibition of B Lymphocyte Proliferation by hNSP.

hNSF eH]TdR Incorporation (cpm)b (Vlml)

Nil SAC + IL-2 PMA + Ionomycin

0 214 6,412 7,159 0.1 218 5,922 (7) 7,103 «1) 1 224 3,231 (49)" 4,312 (42)"

10 211 2,110 (67)" 3,118 (56)" 100 210 2,128 (66)" 2,892 (59)" 10 + P2 mAbd 218 6,204 (3) 7,101 «1)

a Purified B lymphocytes (1 X10s) were cultured without stimulus or with SAC (1:60,000 v/v) plus IL-2 (5 D/ml) or phorbol myristic acetate (10 ng/ml) plus ionomycin (50 ng/ml) in the presence of various concentrations of affinity purified hNSF.

b 3H-thymidine incorporation was measured as in Materials and Methods after 5 days of culture. In parentheses is the percentage of suppression as compared to no addition of the factor.

C Significantly different from controls in the absence of hNSF addition (p:s 0.001, Student's t test).

d Neutralizing concentrations of the anti-hNSF mAb P2 were added at the beginning of culture.

0

a: '0 I- 25 ~ M~

';:ff!. o~

c: § 50 .2 :;: In (II T In ...

T Q) 0 ... a. a. ... 1 a. 0 75 1 ::J 0

en,E

0.1 10 100

hNSF (Ulml)

Figure 2. Suppression of T cell proliferation by hNSF. Rosetting E+ T cells were cultured with Con A (5 Itg/ml) for 12 h, washed and recultured with IL-2 (25 D/ml) and various concentrations of affinity purified hNSF. On day 3, cells were harvested after 16-h pulse with eH]thymidine. Percent suppression is calculated as (l-[cpm in presence of hNSF][background])/([cpm in abscence of hNSF] - [background]). Mean of percent suppression ± SEM for three independent experiments is shown. Proliferation in the absence of hNSF addition was 43,215 ± 6,482 cpm/10s cells.


o

25

50

75

100

0.1 10 100

hNSF (U/ml)

Figure 3. hNSF suppresses Ig production by highly purified B cells. Purified peripheral human B lymphocytes (> 98 % CD19\ 1 x 105 cells/well) were stimulated with SAC (1:60,000 v/v) plus IL-2 (50 V/ml) in the absence or presence of various concentrations of affinity purified hNSF. After 5 days, supernatants were harvested and secreted IgG and IgM concentrations were determined by ELISA. Results are expressed as percent of Ig suppression over the control response (IgG: 2420 ng/ml; IgM: 4560 ng/ml); mean ± standard deviation bars for three independent experiments are shown.

concomitant presence of affinity purified hNSF was studied (Table 1). The addition of hNSF could partially suppress B cell proliferation induced by SAC plus IL-2, with a maximum inhibition of about 65 % of the control

Table 2. Effects of IL-2 on hNSF-induced suppression of IgG secretion by B cellsa.

IL-2 (Vlml) IgG (ng/ml)b Suppression (% t No addition hNSF (5 Vlml)

0 ND ND 103 ± 14 ND

10 253 ± 26 100 ± 20d 60 30 650 ± 76 203 ± 28d 68

100 1316 ± 72 716 ± 39d 45 300 1450 ± 86 1333 ± 66 8

1000 1516 ± 92 1400 ± 86 7

a Purified human B lymphocytes (1 x105) were cultured with SAC (1:60,000 v/v) and

cultured in the presence of various concentrations of human recombinant IL-2 in the absence or presence of 5 V/ml of affinity purified hNSF. Supernatant IgG production was measured 5 days later.

b Mean ± SEM of three independent experiments. ND indicates IgG levels below those detectable by the ELISA assay (about 50 ng/ml).

C Suppression of IgG production in the presence of hNSF as compared to no addition. d Significantly different from no addition controls (p :s 0.001, Student's t test).


response. A similar value of maximum suppression was observed when B cells were activated by phorbol myristic acetate and ionomycin. In both cases the degree of inhibition was dose-dependent and could be blocked by the addition of the P2 mAb.

Next we examined the effects of hNSF on Ig secretion by B lymphocytes. Purified B cells were stimulated by SAC plus IL-2 in the presence of varying concentrations of hNSF. The factor profoundly suppressed both IgG and IgM production (Fig. 3). In terms of time course, the maximal effects could only be seen when the factor was added during the first 2 days of culture, with little suppressive activity (about 10 % ) in very late addition (data not shown).

To determine if increasing concentrations of IL-2 would overcome the suppressive effects of hNSF, SAC-activated B cells were cultured with 5 U of hNSF and increasing concentrations of IL-2, and the IgG production was measured and compared with results from similar cultures without hNSF. As can be seen in Table 2, the suppressive effect could be partially reverted by high concentrations of IL-2, an evidence that T cell cytokines can modulate the biologic activity of hNSF.

Discussion

We have previously described the isolation and characterization of a novel suppressive cytokine with suppressive activity, the hNSF. In the present work we have extended our knowledge about hNSF, determining that CD8+ T cells are the main source of the factor and that it has direct inhibitory effects on mitogen induced proliferation of purified T cells and on proliferation and Ig production by highly purified B cells. Careful determination of viable cells at the end of the culture periods demonstrates that hNSF effects are not due to cytotoxicity. It is also unlikely that other contaminant cytokines are responsible for the suppressive activity observed in our assays, because this activity could be specifically neutralized by the addition of the P2 mAb.

The dominant role of CD8+ T cells in mediating suppression is well recognized in various experimental settings, and because of the antigennonspecific nature of hNSF action, it is likely to be produced by effector suppressor cells (16, 17). Although still contested by some investigators (18), the concept of T cell mediated suppression has gained support with some recent experimental findings, such as the definition of surface cell markers of T suppressor cells (19, 20), the demonstration that these cells use aB TCR for antigen recognition (21, 22) and the molecular characterization of a T cell derived factor with antigen-specific suppressive activity (23).

Few cytokines, such as IL-4 and TGFB, have been found to have a direct inhibitory effect on purified B cells (24). At present it is unclear at precisely which stage( s) of the B cell growth and differentiation pathway hNSF exerts its inhibitory action. With the above data it is not possible to discern


whether hNSF can suppress B cell differentiation to IgG secreting cells independent of its effect on proliferation because stimulation of B cells with SAC + IL-2 can induce both proliferation and IgG secretion. However, the kinetics analysis, showing low levels of suppression with late additions, suggests that hNSF acts at an early stage of the B cell activation process. Increasing concentrations of IL-2 could also partly revert the hNSF suppression, and this is an evidence that other T cell derived cytokines may be important modulators of hNSF action on B lymphocytes.

The similarity of the observed effects of hNSF with the effects described for the murine MNSF (1-3) reinforces the idea that hNSF is the human counterpart of MNSF. Labeled MNSF binds to a single class receptor on the surface of mitogen-activated Band T cells (25). MNSF also interacts with non-lymphocytic cells. Inhibition of IL-4 production by sensitized bone marrow-derived mast cells exposed to antigen (25) and of TNFa synthesis by a LPS-stimulated macrophage cell line (unpublished data) have been observed. These data indicate that the MNSF/hNSF system may have a broad role in the regulation of a number of cells involved in the inflammatory response.

In conclusion, hNSF is a novel cytokine produced by CDg+ T cells and has direct antigen-nonspecific inhibitory action on T and B cells. Because of its potential immunoregulatory role and the observation of hNSF in the ascitic fluid of a SLE patient, the study of this suppressive factor and its molecular characterization may provide important information for a better understanding of immunological tolerance and the autoimmune process.

Acknowledgments

Supported in part by a Grant-in-Aid (05770316) for scientific research from the Ministry of Education, Science and Culture, Japan. We are grateful to Ms. Y. NISHIKORI and T. UNSHIMA, whose technical help was invaluable to the completion of the present work.

References

1. NAKAMURA, M., H. OGAWA, and T. TSUNEMATSU. 1986. Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma. J. Immunol. 136: 2904.

2. NAKAMURA, M., H. OGAWA, and T. TSUNEMATSU. 1988. Mode of action of monoclonal-nonspecific suppressor factor (MNSF) produced by murine hybridoma. Cell Immunol. 116: 230.

3. NAKAMURA, M., H. OGAWA, and T. TSUNEMATSU. 1992. Characterization of Nterminal amino acid sequence of monoclonal nonspecific suppressor factor (MNSF). Cell Immunol. 139: 139.

4. NAKAMURA, M., H. OGAWA, and T. TSUNEMATSU. 1987. Characterization of monoclonal nonspecific suppressor factor (MNSF) with the use of a monoclonal antibody. J. Immunol. 138: 1799.

5. XAVIER, R. M., M. NAKAMURA, and T. TSUNEMATSU. 1994. Isolation and characterization of a human nonspecific suppressor factor from ascitic fluid of systemic lupus


erythematosus. Evidence for a human counterpart of the monoclonal nonspecific suppressor factor and relationship to the T cell receptor a-chain. J. Immunol. 152:2624.

6. GREEN, D. R., R. BISSONETE, H. ZHENG, T. ONDA, F. ECHEVERRI, R. J. MOGIL, J. K. STEELE, M. VORALIA, and A. FOTEDAR. 1991. Immunoregulatory activity of the Tcell receptor a chain demonstrated by retroviral gene transfer. Proc. Nat!' Acad. Sci. USA 88: 8475.

7. KUCHROO, V. K., M. C. BYRNE, Y. ATSUMI, E. GREENFIELD, J. B. CONNOLLY, M. J. WHITTERS, R. M. JR. O'HARA, M. COLLINS, and M. E. DORF. 1991. T-cell receptor a chain plays a critical role in antigen-specific suppressor cell function. Proc. Nat!. Acad. Sci. USA 88: 8700.

8. THIELE, D. L., M. KUROSAKA, and P. LIPSKY. 1983. Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood. delineation by its sensitivity to the lysosomotropic agent L-leucine methyl ester. J. Immuno!. 1311:2282.

9. FALKOFF, R. J. M., L. P. ZHU, and A. S. FAUCI. 1982. Separate signals for human B cell proliferation and differentiation in response to staphylococcus aureus: evidence for a two-signal model of B cell activation. J. Immunol. 129: 97.

10. VIA, C. S., G. C. TSOKOS, N. 1. STOCKS, M. CLERIC!, and G. M. SHEARER. 1990. Human in vitro allogenic responses: demonstration of three pathways of T helper cell activation. J. Immuno!. 144: 2524.

11. KEHR,J. H., L. M. WAKEFIELD, A. B. ROBERTS, S.JAKOWLEW, M. ALVAREZ-MoN, R. DERYNCK, M. B. SPORN, and A. S. FAUCI. 1986. Production of transforming growth factor f3 by human T lymphocytes and its potential role in the regulation of T cell growth. J. Exp. Med. 163: 1037.

12. FALKOFF, R. J. M., L. P. ZHU, and A. S. FAUCI. 1982. Separate signals for human B cell proliferation and differentiation in response to staphylococcus aureus: evidence for a two-signal model of B cell activation. J. Immunol. 129: 97.

13. KEHRL, J. H., A. B. ROBERTS, L. M. WAKEFIELD, S. JAKOWLEW, M. B. SPORN, and A. S. FAUCI. 1986. Transforming growth factor f3 is an important immunomodulatory protein for human B lymphocytes. J. Immuno!. 137: 3855.

14. LARSSON, E. L., and A. COUTINHO. 1979. The role of mitogenic lectin in T-cell triggering. Nature 280: 239.

15. LARSSON, E. L. 1981. Mechanism of T cell activation. II. Antigen- and lectindependent acquisition of responsiveness to TCGF is a nonmitogenic, active response of resting T cells. J. Immuno!. 126: 1323.

16. CANTOR, H., F. W. SHEN, and E. A. BOYSE. 1976. Separation of helper T cells from suppressor T cells expressing different Ly components. II. Activation by antigen: after immunization, antigen-specific suppressor and helper activities are mediated by distinct T-cell subclasses. J. Exp. Med. 143: 1391.

17. DORF, M. E., and B. BENACERRAF. 1984. Suppressor cells and immunoregulation. Ann. Rev. Immuno!. 2: 127.

18. MOLLER, G. 1988. Do suppressor T cells exist? Scand. J. Immunol. 27: 247. 19. INOUE, T., Y. ASANO, S. MATSUOKA, M. FURUTANI-SEIKI, S. AIZAWA, H. NISHI

MURA, T. SHIRAI, and T. TADA. 1993. Distinction of mouse CD8+ suppressor effector T cell clones from cytotoxic T cell clones by cytokine production and CD45 isoforms. J. Immunol. 150: 2121.

20. DEVENS, B. H., A. W. KOONTZ, J. A. KAPP, C. W. PIERCE, and D. R. WEBB. 1991. Involvement of two distinct regulatory T cell populations in the antigen-specific suppression of cytolytic T cell generation. J. Immunol. 146: 1394.

21. TAKATA, M., P. K. MAlTI, R. T. KUBO, Y. CHEN, V. HOLFORD-STREVENS, E. S. RECTOR, and A. SEHON. 1990. Cloned suppressor T cells derived from mice tolerized with conjugates of antigen and monomethoxypolyethylene glycol: relationship


between monoclonal T suppressor factor and the T cell receptor. J. Immunol. 145: 2846.

22. FAIRCHILD, R. L., R. T. KUBO, and J. W. MOORHEAD. 1990. DNP-specific/class I MHC-restricted suppressor molecules bear determinants of the T cell receptor u- and f3-chains: the Vf38+ chain dictates restriction to either K or D. J. Immunol. 145: 200l.

23. MIKAYAMA, T., T. NAKANO, H. GOMI, Y. NAKAGAWA, Y.-C. LIU, M. SATO, A. IWAMATSU, Y. ISHII, W. Y. WEISER, and K. ISHIZAKA. 1993. Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor. Proc. Nat!. Acad. Sci. USA 88: 8700.

24. LIPSKY, P. E. 1989. The control of antibody production by immunomodulatory molecules. Arthritis Rheum. 32: 1345.

25. NAKAMURA, M., H. OGAWA, and T. TSUNEMATSU. 1990. Characterization of cellsurface receptors for monoclonal nonspecific suppressor factor (MNSF). Cell Immunol. 130: 28l.

26. NAKAMURA, M., R. M. XAVIER, and Y. TANIGAWA. 1994. Monoclonal nonspecific suppressor factor (MNSF) inhibits the IL4 secretion by bone marrow-derived mast cell (BMMC). FEBS. Lett. 339: 239.

Dr. MORIHIKO NAKAMURA, Department of Biochemistry, Shimane Medical University, Izumo-693,Japan

Human Nonspecific Suppressor Factor (hNSF): Cell Source and Effects on T and B Lymphocytes

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