674
Eur. J. Immunol. 1976.6: 674-679
LA. Ramshaw, P.A. Bretscher and C.R Parish
15 Kunkel, H.G., Agnello, V., Joslin, F.G., Winchester, R.J. and Capra, J.D., J. Exp. Med 1973.137: 331.
26 Weigert, M, Potter, M and Sachs, D.,Zmmunogenetics 1975.1: 511. 27 Eichmann, K., Eur. J. Zmmunol 1972.2: 301. 28 Pawlak, LL and Nisonoff, A., J. Exp. Med. 1973. 137: 855. 29 Eichmann, K.,Zmmunogenetics 1975. 2: 491. 30 Kindt, T. J., Thunberg, A.L, Mudgett, M. and Klapper, D.G., in Sercarz, E.E., Williamson, A.R and Fox, CF. (Eds) The Immune System: Genes, Receptors, Signals, Academic Press, New York 1974, p. 69.
16 Claflin, J.L. and Davie, J.M., J. Exp. Med. 1975.141: 1073.
31 Briles, D.E. and Krause, R.M., J. Zmmunol. 1974. 113: 522.
17 Murgita, R.A. andVas, S . L , J . Zmmunol. 1970. 104: 514. 18 Askonas, B.A., Williamson, A.R. and Wright, B.E.G., Proc. Nat. Acad Sci. US 1 9 7 0 . 6 7 1398.
32 Kuettner, MG., Wang, A. and Nisonoff, A.,J. Exp. Med 1972. 135: 579.
Briles, D.E. and Davie, J.M., J. Zmmunol. Methods 1975. 8: 363. Gamer, M. and Braun, D.G., J. Exp.Med. 1974. 139: 1513. Eichmann, K., Eur. J. Immunol. 1974.4: 296. Freedman, MH., Pincus, J.H., Yeger, H., McKenny, J.A. and Mage, R.A., Eur. J. Zmmunol. 1974.4: 553. 23 Klinman,N.R.andPress,J.L.,J. Exp. Med 1975. 141: 1133. 24 Kreth, HW. and Williamson, A.R., Eur. J. Zmmunol 1973.3: 141. 25 Apella, E. and Inman, J.K., in Reisfeld, R.A. and Mandy, W.T. (Eds) Contemporary Topics in Molecular Immunology, Plenum Press, New York 1973, p. 51.
34 Claflin, J.L., Eur. J. Zmmunol. 1976. 6 : 666.
10 Claflin, J.L. and Davie, J.M., J. Zmrnunol. 1975. 114: 70. 11 Gearhart, P.J., Sigal, N.H. and Klinman, N.R, J. Exp. Med. 1975. 141: 56. 12 Rudikoff, S. and Potter, M, Biochemistry 1974. 13: 4033. 1 3 Miller, F. and Metzger, H., J. Biol. Chem 1965. 240: 4740. 14 Williamson, A.R., Salaman, M R and Kreth, R.W., Ann. NYAcad. Sci 1973.209: 210.
19 20 21 22
1.A. Ramshaw', P.A. Bretscher and C.R. Parish Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra
33 Stankus, R.P. and Leslie, G.A., Zmmunogenetics 1976.3: 65. 35 Wu, T.T. and Kabat, E.A., J. Exp. Med. 1970. 132: 211. 36 Capra, J.D. andKindt, T.J., Zmmunogenetics 1976. I : 417. 37 Potter, M, Physiol Rev. 1972.52: 692. 38 Kluskens, L, Lee, W. and Kohler, H.,Eur. J. Zmmunol 1975. 5 : 489. 39 Cosenza, H., Quintfins, J. and Leflcovits, I.,J. Zmmunol. 1975. 5 : 343. 40 Sakato, N. andEisen, R,J. Exp. Med 1975. 141: 1411.
Regulation of the immune response
1. Suppression of delayed-type hypersensitivity by T cells from mice expressing humoral immunity The ability of horse red blood cell (HRBC)-specific T cells from mice expressing humoral immunity t o suppress the induction of HRBC-specific delayed-type hypersensitivity (DTH) was investigated. The transfer of Ignegative spleen cells, from mice injected 4 days previously with HRBC, completely suppressed the development of DTH in mice treated with cyclophosphamide and sensitized with HRBC. The suppressor cell was found to be lysed by treatment with anti-theta serum and complement. Furthermore, hemocyanin-specific immune T cells were able to suppress the DTH induced to HRBC, provided these two antigens were coupled together. These studies suggest that T cells present under conditions where humoral immunity is induced can suppress DTH and that such cells play an important role in the regulation of the immune response.
1. Introduction
In many instances humoral and cell-mediated immunity (CMI) are mutually antagonistic [ 11. For example, animals with humoral immunity are usually refractory t o the establishment of de[I 14111
Present address: Department of Microbiology and Immunology, Queens University, Kingston, Canada. Correspondence: CR. Parish, Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601, Australia Abbreviations: IYTH: Delayed-type hypersensitivity HRBC Horse red blood cells SRBC: Sheep red blood cells CRBC: Chicken red blood cells CP: Cyclophosphamide HCY: Hemocyanin ABA: Azobenzene arsonate CMI: Cell-mediated immunity FIA: Freund's incomplete adjuvant
layed-type hypersensitivity (DTH) [2]. Two possible explana tions for this refractory state are either that antibody or anti gen-antibody complexes block the activated T cells which mediate DTH [3] or that T cells present in animals with humoral immunity actively suppress the induction of cell-mediated immunity. Recent reports [4, 51 have shown that T cells, induced under certain conditions, can actively suppress the development and expression of DTH.
A theoretical scheme, which has been recently devised [ 61, suggests that precursor cells for different types of immunity require different amounts of help to be induced. Thus, CMI requires the least, IgM an intermediate amount, and IgG the most. It is also postulated that T cells can inhibit the induction of other classes of immunity; in particular, larger amounts of T cell help t h a ~required for inductior
Suppression of delayed hypersensitivity
Eur. I. Immunol. 1976.6: 674-679 suppress CMI responses, while CMI cells repress the induction of humoral immunity. The biological significance of these postulates has been discussed elsewhere [ 6 ] . The present studies were designed to test this scheme by determining whether the establishment of one type of immunity actively suppresses the development of other forms of immunity, and whether active suppression is the property of the T lymphocyte. The experiments described in this paper show that the transfer of T cells from mice with humoral immunity suppresses the development and expression of DTH.
2. Materialsand methods
2.1. Mice and antigens used Inbred CBA/H female mice 6-8 weeks old were used throughout these studies. Horse, sheep and chicken red blood cells (HRBC), (SRBC) and (CRBC) were collected and stored in Alsever‘s solution. Hemocyanin (HCY) was crystallized from the hemolymph of the crayfish, Jams Zulundii [7].
2.2. Test for DTH DTH was measured as footpad swelling. An eliciting dose of 1 x lo8 red blood cells was injected in 10 p1 into subcutaneous tissue on the surface of one hind footpad. The degree of swelling was measured routinely at 24 h with a dial-gauge caliper (“Schnelltaster”, H.C. Kroplin, Hessen, Germany). Each “unit” represents a swelling of 0.1 mm (+ 0.05 mm).
3. Results 3.1. General Several studies [12, 131 have shown that the treatment of animals with cyclophosphamide (CP), before an optimum sensitizing dose of antigen, causes a marked enhancement of the DTH reaction. Thus, mice injected with CP (200 mg/ kg) and 2 days later injected intravenously (i.v.) with 1O8 HRBC produce a large DTH reaction to HRBC injected into the footpad. DTH can first be demonstrated 4 days after sensitization with antigen and reaches peak levels between 5-6 days. To determine the effect of T cells from humoral immune mice on the development of DTH, normal mice were injected with 1O9 HRBC i.v., a dose which produces high levels of humoral immunity but no detectable DTH, and 4 days later the spleen cells were depleted of Ig-bearing cells. The Ig-negative (Ig-) spleen cells were then transferred to CP-treated mice 1 h before sensitization with HRBC. The DTH levels developed were determined 6 days later by injecting lo8 HRBC into the footpad and measuring the increase in footpad swelling 24 h later. The experimental protocol is shown in Fig. 1.
Cyclophorphomlde
I
IO(IHRBC I V
Test for D T H
IO’HRBC
I
2.3. Depletion of immunoglobulin-bearing cells The method of separation has been fully described elsewhere [8, 91. In summary, the procedure consisted of two stages: first, rosetting of Ig-bearing cells, and second, separation of the nonrosetting and rosetting lymphocytes on Isopaque/ Ficoll. The red cells and rosettes sank to the bottom of the tube while‘the remaining nonrosetting cells floated at the Isopaque/Ficoll interface. The latter cells were recovered and washed twice in medium.
675
In
Footpod
109HRBC I V
Figure 1. Determination of effect of transferring Ip- immune spleen cells on development of DTH to HRBC.
2.4. Anti-@ treatment of spleen cells Anti-@ (Thy-1.2) ascitic fluid was raised in AKR/J mice by multiple injections of CBA/H thymus cells as previously described [lo], and the spleen cell suspensions were treated with & a:@ serum and guinea pig complement as reported previously [ 101. 2.5. Coating of HCY onto red blood cells
A modification of the method of Gold and Fudenberg [ 1 11 was employed. A 0.1 % (w/v) solution of CrC13 in normal saline was prepared and adjusted to pH 5 with 1 N NaOH daily for one week. The standard protocol involved the addition of 100 pg HCY to 2 ml 10 % washed red blood cells followed by 0.2 ml of 0.1 % CrC13 solution and rapid mixing. The mixture was allowed t o stand at room temperature for 10 min before the cells were washed twice in phosphate buffered saline.
3.2. Suppression of DTH by spleen cells from mice with humoral immunity Mice treated with CP and sensitized with HRBC produced high levels (1 0.4 units) of DTH; this was not significantly altered by the transfer of spleen cells or Ig- spleen cells from normal mice (Fig. 2). However, the transfer of spleen cells or Ig- spleen cells from mice immunized t o produce a humoral response completely suppressed the development of DTH (Fig. 2), as corhpared with negative controls. The transfer of Ig-positive (Ig+) spleen cells from immune mice had no effect on the level of DTH obtained. The ability of these immune Ig- spleen cells to suppress an established DTH immunity is shown in Fig. 3. Ig- immune spleen cells transferred 24 h before footpad testing, that is, at a time when mice have developed high levels of DTH, reduced the DTH reaction from 11.8 units to 7.6 units.
Eur. J. Immunol. 1976.6: 674-679
LA. Ramshaw, P.A. Bretscher and C.R Parish
676
Cells Tranilerred
loB
HRBC
lo8
HRBC
loB
HRBC
narmo~
10'
HRBC
immune
10'
HRBC
10'
HRBC
NIL I 10 norm01 spleen cells
60
60 x
lo7
spleen
immune
cells
2 0 ~ 1 0 7isspleen cells
2
o
19-
L b
spleen cell*
4 0 ~ 1 0 Ig-~
immune
spleen cell3
NIL
NIL
0
20
40
F i Z ]
24h
60
80
100
120
Footpad rwellmg(0lmm)
Figure 2. Effect on development of DTH of transferring 6.0 x lo7 spleen cells or 2.0 x 107 Ig- spleen cells from normal mice or from mice immunized to produce a humoral response. Also shown is the effect on DTH of transferring 4.0 x 107 Ig- immune spleen cells. Statistical comparisons of the DTH reaction of mice receiving immune spleen cells or Ig- immune spleen cells to sensitized controls. or tho; receiving normal Ig- cells, yielded a P value of < 0.001. '
Cells Transferred
S e n m r i n y Anfcgen
The T cell dependence of DTH suppression was determined by treating Ig- immune spleen cells with anti-@ serum and complement or as a control normal serum and complement. The cells were then transferred t o CP-treated mice and the ability of the treated cells to suppress DTH determined. Fig. 4 shows that treatment with anti-@ serum prevented cells from suppressing the development of DTH.
3.3. The effect of transferring varying numbers of Igimmune spleen cells on the development of DTH Groups of CP-treated mice were given varying numbers of Ig- HRBC-immune spleen cells before sensitization with HRBC. Fig. 5 shows that maximum suppression of DTH occurred with the transfer of 2.5 x lo7 Ig- spleen cells. There was a linear decrease in the levels of DTH developed as the number of cells transferred was increased. Mice were bled and the anti-HRBC serum antibody levels determined before challenging with HRBC in the footpad for DTH. All groups showed a very weak anti-HRBC antibody response (maximum titer 1/24), which probably resulted from the selective effect of CP on the B cell compartment. Studies [ 14, 151 have shown that CP severely depletes the B cell areas while the T cell areas remain essentially intact. Slightly higher levels of antibody were present in groups of DTHsuppressed mice.
8 10 HRBC
NIL
lo8
180
HRBC
I
4I 140
8 10 HRBC
2 OX 1 0 ~ 1 g - immune 3pleen cells
120
I
.-:
;
9
NIL
NIL
Q
1.10 20
0
40
60
80
100
120
24h Footpad rwellhng(0lmm)
I5
Figure 3. Effect of transferring 2.0 x 107 Ig- spleen cells from normal mice or from mice immunized to produce a humoral response, on the expression of DTH. Ig- cells were transferred 24 h before testing for DTH, that is 5 days after sensitization. Statistical comparisons of the DTH reaction of mice receiving Igimmune cells to sensitized controls yielded a P value of 0.05.
~OXIO~IY-
immune cell3
Treated withAKRonti Theta serum
+
IO~HRBC
0
25
/:111151
12
Immune
015
03
06 IS-
cells
007
0
(x 10')
Figure 5. Effect of transferring varying numbers of Ig-immune spleen cells on the development of DTH. Serum antibody titers were measured by complement-mediated lysis of HRBC. (0- --9 represents footpad reaction Qf unsensitized mice. Statistical comparison of antibody titer of suppressed DTH mice and control, sensitized mice yielded a maximum P value of 0.1.
3.4. Kinetics of appearance of cells capable of suppressing the induction of DTH immunity
10BHRBC
C'
Treated wlfhAKRnormal serum
IO'HRBC
+c' NIL
z
NIL
0
40
20 24
60
80
100
120
h Footpod ~wellms(Olmm)
Figure 4 . Effect of anti-@ serum and complement treatment on ability of Ig- immune cells to suppress induction of DTH.
The previous experiments have shown that 4 day immune Ig- spleen cells can, on transfer, suppress the development of DTH. To determine when cells capable of suppressing DTH first appear in the spleen, mice were injected with lo9 HRBC; and 1, 2, 3, and 4 days later Ig- spleen cells were prepared and transferred to groups of CP-treated mice. Thest were then sensitized with HRBC and tested for DTH 6 days later. The results indicate (Fig. 6) that Ig- spleen cells from mice injected 1 day previously can partially suppress the de-
Suppression of delayed hypersensitivity
Eur. J. Immunol. 1976.6: 674-679
velopment of DTH. The development of DTH can be completely suppressed with Ig- cells from mice injected 3 days previously with HRBC. Immune Cell. Transferred Sensitizing Antlgen
2o
10' tgI
0 day
lo8 HRBC
I day
IO~HRBC
2 doy
lo8 HRBC
3 day
10' HRBC
4 day
lo8 HRBC
NIL
NIL
7 1 L
d
p 20
0
j'lL1151
40
60
80
100
120
24h Footpod rwellmg(0lmm)
Figure 6. Kinetics of appearance of cells capable of suppressing the induction of DTH. Mice were immunized with lo9 HRBC, and 0,1, 2, 3 and 4 days later 2.0 x 107 Ig- spleen cells were transferred to CP-treated mice which were then sensitized with HRBC. Statistical comparisons of the DTH reaction of control sensitized mice to mice receiving 1, 2, 3, and 4 day immune cells yielded P values of 0.05, 0.01, < 0.001 and < 0.001,respectively.
677
3.6. Suppression by linked associative recognition Bullock et al. [ 161 have shown that azobenzene arsonate (ABAtspecific suppressor T cells can inhibit the development of DTH t o other linked protein antigens, provided immunization is carried out with ABA conjugates of these antigens. The following experiment shows that HCY-specific immune T cells can suppress the DTH to HRBC when the two antigens are coupled together. Mice treated with CP and sensitized with HCY-coated HRBC develop high levels of DTH to the HRBC antigens. The DTH obtained is equivalent to that of mice sensitized with HRBC only. Mice immunized with HCY-coated autologous red cells produce high levels of anti-HCY antibody (results to be published). Ig- spleen cells from these HCY-immune mice were transferred t o CP-treated mice which were then sensitized with either HCY-coated HRBC, HRBC or mixture of HCYcoated mouse red cells and HRBC. Fig. 8 shows that DTH to the HRBC antigens was completely suppressed in mi-e given HCY-specific immune cells and sensitized with HCYcoated HRBC. All the control groups developed high levels of DTH to HRBC, demonstrating both the specificity of the suppression and the necessity for the determinants to be linked. Senlltlrlng ~~i~~~~
Hoemocyonm Immune Cells
I
NIL
HCY/HRBC
3.5. Specificity of suppression HRBC
To determine the specificity of suppression, Ig- spleen cells from mice immunized with HRBC were transferred t o groups of CP-treated mice and sensitized with either HRBC, SRBC, or CRBC. Fig. 7 shows that, as previously demonstrated, DTH to HRBC transferred with HRBC-immune Ig- spleen cells was completely suppressed in mice. In contrast, the DTH in mice sensitized with SRBC was partially suppressed while the DTH in mice sensitized with CRBC was unaffected.
NIL
HCY/HRBC
2 0 ~ 1 ig0 ~
HRBC
2 0x 1 ~ 7iy-
yc;$y
20
NIL
lo7 lo-
4.
I c.~~I
NIL
0
20
40
60
2lh Footpod swelling
NIL
2 0 ~ 1 0iY-~
NIL
2 o .lo7 lY-
IO'HRBC
IO8SRBC
I08SRBC
IO'CRBC
NIL
Nll
ID
100
120
HRBC(0lrnm)
Figure 8. Effect of HCY-specific Ig- cells on development of DTH to HRBC in mice sensitized with HCY-coated HRBC. 2.0 x 107 Igspleen cells from mice immunized to HCY were transferred to CPtreated mice which were then sensitized with either HCY-coated HRBC, HRBC or a mixture of HCY-coated mouse red blood cells and HRBC. Statistical comparisons of the DTH reaction of mice receiving HCY-immune Ig- cells and sensitized with HCY-coated HRBC to sensitized controls yielded a P value of < 0.001.
IO'HRBC
NIL
80
3.7. Role of antibody in the suppression of DTH 0
20
40 24 h
b0
80
100
120
Footpad ~ r e l l m g ( O 1mm]
Figure 7. Specificity of suppression. 2.0 x lo7 Ig- spleen cells from mice immunized with HRBC were transferred to groups of mice sensitized with either HRBC, SRBC or CRBC. Statistical comparisons of the DTH reactions of mice receiving Ig- immune cells to sensitized controls yielded P values of < 0.001 for HRBC, < 0.001 for SRBC and > 0.9 for CRBC.
It could be argued that antibody may mask or suppress DTH. In order to test this possibility mice were given either early (4 day) immune serum, hyperimmune serum or serum from mice whose DTH was suppressed by the transfer of immune T cells, before or after sensitization with HRBC. The results in Table 1 show that anti-HRBC antibody given to mice either before sensitization or 24 h before footpad testing failed to affect the level of DTH obtained.
678
Eur. J. Immunol. 1976.6: 674-679
LA. Ramshaw, P . k Bretscher and C R . Parish
Table 1. Effect of transferring various immune sera on the develop ment of D T H ~ ) Serum transferred
Time of transferb)
(0.2 ml)
Mean reciprocal serum antibody titer at time of footpad testing
24 h footpad swelling
Hyperimmune serum
BS AS BS AS
448 15 640
10.6 f 0.5 10.7 It 0.4 10.3 It 0.8 10.1 f 0.9
Serum from
BS
8 15
11.3 f 0.5 11.1 f 0.9
11
11.3 0.9
Early
serum
10
DTH-suppressed AS mice Nil
*
a) Mice were injected i.v. with serum either 24 h before sensitization or 24 h before footpad testing for DTH. b) BS: before sensitization; AS: after sensitization.
Evidence was also obtained to demonstrate that the DTH in mice given immune Ig- spleen cells is suppressed and not simply masked. Spleen cells from sensitized and suppressed mice were depleted of Ig-bearing cells and 5 x l o 6 spleen cells were suspended in 20 p1 of medium. These cells were injected into the footpad of normal mice, either with lo8 HRBC or alone, and the DTH reaction measured at 24 h. Table 2 shows that cells from sensitized mice, but not from suppressed mice, transferred high levels of DTH, indicating that T cells mediating DTH are not simply masked but are absent from the spleens of suppressed mice. Table 2. DTH reactions to HRBC obtained in normal mice following the transfer of Ig- spleen cells from either sensitized or suppressed micea) 1g.- Cells
HRBC
DTH reaction&)
transferred Sensitized
Sensitized Suppressed Suppressed Nil
++ +
10.4 It 0.6 1.9 f 0.8 3.0 It 0.9 2.1 f 0.3 1.4 It 0.2
a) Spleen cells from sensitized or suppressed mice were depleted of Ig-bearing cells and 5 x lo6 cells suspended in 20 pl of
medium with or without lo8 HRBC. These cells were then injected into the footpad of normal mice and the DTH reaction measured 24 h later. b) See Section 2.2.
4.Discussion The studies presented in this paper demonstrate that T cells from mice with humoral immunity can suppress the development of DTH responses. The regime used t o induce cells capable of suppressing DTH suggests that the cell responsible for suppression is the helper T cell. However, it cannot be formally excluded that another T cell, induced under the same conditions that favor humoral immunity, is the actual suppressor cell of DTH.
The suppression of DTH by immune T cells is specific since DTH responses to other noncross-reacting antigens are unaffected. However, the DTH to antigens that cross-react at the T cell level is partially suppressed (Fig. 7). Thus HRBCspecific T cells partially suppress the DTH induced against SRBC but not CRBC. HRBC and SRBC are known to crossreact at the helper T cell level [I 71, and DTH induced with HRBC can be elicited with SRBC but not CRBC (unpublished observations). Observations have also been reported [ 181 of cross-reactivity, at the level of suppressor T cells, between antigens that cross-react minimally at the B cell level.
CP was used in these studies t o obtain high levels of DTH to HRBC. The transfer of specific immune T cells can also suppress the DTH induced with low doses [2] or chemically modified [19] HRBC in normal mice (unpublished results). Three observations make it unlikely that antibody is involved in masking or suppression of DTH. First, only low levels of antibody were produced in suppressed mice, and in many cases the titers were not significantly higher than in sensitized animals (Fig. 5). Second, the administration of immune sera to sensitized animals failed, in all cases, to mask DTH. Finally, DTH could be transferred to normal mice with splenic T cells from sensitized animals but not with cells from suppressed mice. Other evidence ( [ I , 201) also renders improbable the importance of antibody in the suppression of DTH. The results of these transfer experiments suggest that CP may enhance DTH by initially restricting the development of T cells, which would normally suppress DTH, rather than directly affecting the B cell population. Furthermore, a stable established state of cell-mediated immunity may in turn repress any subsequent induction of helper T cells [ 6 ] . The relationship between the suppressor cells described here and other T cells, that have been reported to suppress CMI reactions [4, 5, 211, remains t o be determined. It is worth emphasizing, however, that helper T cells may be responsible for the suppression of DTH in other systems. Thus, Bullock et al. [20] were able t o suppress the development of DTH to ABA in Freund’s complete adjuvant by pretreating animals with ABA in Freund’s incomplete adjuvant (FIA). In this case anti-ABA antibody was not produced when ABA-FIA was given, but it is possible that the procedure used primed the T helper cell population. Such priming without a concomitant antibody response has been reported elsewhere [22,231. Similarly, the injection of picrylsulfonic acid [4] or dinitrochlorobenzene [5], shown to depress contact sensitivity due to the induction of T cell suppressors, may also represent priming of the T helper cell population. Splenic T cells from mice injected with HRBC 24 h previously were found to partially suppress the development of DTH when transferred to sensitized mice. This experiment does not take into account that further development of the transferred cells can occur within the recipient mice. However, this does demonstrate that within 24 h of contact with antigen, events have taken place so as to commit T cells t o a particular pathway of differentiation . The evidence showing that HCY-specific immune T cells are able to suppress the DTH to another unrelated antigen (HRBC), provided these are coupled together, has important
Induction of cellular immunity to vaccinia surface antigens
Eur. J. Immunol. 1976. 6 : 679-683
theoretical and practical implications. The fact that suppression can occur b y linked associative recognition argues against an anti-idiotype form of suppression in this system. Also, it should be possible t o suppress DTH t o an antigen A by immunizing with an appropriate antigen B and then reimmunizing with t h e AB conjugate. Preliminary results indicate that an established DTH t o HRBC can be suppressed by first immunizing with HCY and then with HCY coupled t o HRBC. This may have practical applications in situations where it would b e advantageous t o suppress a DTH response. Received April 8, 1976; in final revised form July 27, 1976.
5 . References 1 Parisli,C.R., Transplant.Rev. 1972. 13: 35.
2 Lagrange, P.H., Mackaness, G.B. and Miller, T.E., J. Exp. Med. 1974.139: 528. 3 Mackaness, G.B., Lagrange, P.H., Miller, T.E. and Ishibashi, T., J. Exp. Med. 1974.139: 543. 4 Zembala, M. and Asherson, G.L., Nature 1973. 244: 227. 5 Phanuphak, P., Moorhead, J.W. and Claman, H.N., J. Immunol. 1974.112: 849. 6 Bretscher, P.A., Cell. Immunol. 1974.13: 171.
U. Koszinowski and Hildegund Ertl Institute of Hygiene, University of Gott ingen, Gott ingen
679
7 Moore, C.H., Henderson, R.W. and Nichol, L.W., Biochemistry 1968. 7: 4075. 8 Parish, C.R. and Hayward, J.A., Proc. Roy. SOC.London Ser. B 1974.187: 65. 9 Parish, C.R., Kirov, S.M., Bowern, N. and Blanden, R.V., Eur. J. Immunol. 1974.4: 808. 10 Kirov, S.M., Eur. J. Immunol. 1974.4: 739. 11 Gold, E.R. and Fudenberg, H.H., J. Immunol, 1967.99: 859. 12 Turk, J.L. and Parker, D., Immunology 1973.24: 751. 13 Lagrange, P.H., Mackaness, G.B. and Miller, T.E., J. Exp. Med. 1974.139: 1529. 14 Poulter, L.W. and Turk, J.L., Nature-New Biol. 1972.238: 17. 15 Turk, J.L. and Poulter, L.W., Clin. Exp. Immunol. 1972. 10: 285. 16 Bullock, W.W., Katz, D.H. and Benacerraf, B., J. Exp. Med. 1975. 142: 275. 17 Hoffman, M. and Kappler, J.W., J. Exp. Med. 1973.137: 721. 18 Basten, A., Miller, J.F.A.P. and Johnson, P., Transplant.Rev. 1975. 26: 130. 19 Parish, C.R.,Eur. J. Immunol. 1972.2: 143. 20 Askenase, P.W., Hayden, B.J. and Gershon, R.K., J. Exp. Med. 1975.141 1 697. 21 Bullock, W.W., Katz, D. and Benacerraf, B., J. Exp. Med. 1975. 142: 261. 22 Kettman, J., Immunol. Commun. 1972. I : 289. 23 Liew, F.Y. and Parish, C.R., J. Exp. Med. 1974.139: 779.
Role of early viral surface antigens in cellular immune response t o vaccinia virus" Infection of mice with the vaccinia virus strain WR, Elstree o r DIs, a conditional lethal mutant of vaccinia virus, resulted in the generation of vaccinia virus-specific sensitized cytolytic T lymphocytes (CTL). It could be shown by cross-reactivity between the three strains and by inhibition experiments with specific antisera that early vaccinia surface antigens are sufficient for the generation of specific CTL in vivo and for the lysis of infected target cells in vitro.
1. Introduction
The specific activity of murine cytolytic T lymphocytes (CTL) sensitized against viruses [ 1-41, chemically modified cells [ 5 , 61, minor histocompatibility antigens [7] o r H-Y antigens [ 81 is restricted t o attacker and target cell homology of [I 14171
* This work was supported by the Deutsche Forschungsgemeinschaft, Grant KO 571/2. Correspondence: Ulrich Koszinowski, Department of Zoology, University College London, Gower Street, London WCIE 6BT, GB Abbreviations: ADCC: Antibody-dependent cell-mediated cytolysis CA: Cytolytic antibody assay CIA: Cytolysis inhibition assay CMC: Cell-mediated cytolysis CTL: Cytolytic T lymphocytes DAPI: 4,6Diamidino-2-phenyl-idol EVSA: Early viral surface antigens IF: Immunofluorescence LCM: Lymphocytic choriomeningitis LVSA: Late viral surface antigens TCID: Tissue culture infective dose VA: Antigens of the virus particle VSA: Viral surface antigens DIs: Dairen-I mutant strain of vaccinia virus CRBC: Chicken red blood cells
the K or D end of the H-2 complex. Killing of modified allogeneic cells is only possible in t h e tolerant situation of chimeric mice [9]. CTL activities therefore seem t o be specific for both H-2 and viral antigens. To obtain further information about possible physiological activities of CTL in the recovery from vaccinia virus infection, t h e specificity of t h e sensitizing antigenic structures induced by the virus has t o be investigated. Infection with vaccinia virus leads t o t h e destruction of t h e infected cell. During virus replication and propagation numerous virus-specific antigenic products are synthesized in t h e cytoplasm and on the surface of the infected cells. Some of these antigens are structural antigens of t h e virus while others d o not seem t o be antigenically related t o antigens found on the surface of t h e virus particle [lo]. Data reported here were obtained after infection of mice with different strains of vaccinia virus. These strains differed in the expression of viral surface antigens (VSA) o n infected cells. Early VSA were sufficient for induction of specific CTL in vivo and for lysis of infected cells in-vitro.
Eur. J. Immunol. 1976.6: 674-679
LA. Ramshaw, P.A. Bretscher and C.R Parish
15 Kunkel, H.G., Agnello, V., Joslin, F.G., Winchester, R.J. and Capra, J.D., J. Exp. Med 1973.137: 331.
26 Weigert, M, Potter, M and Sachs, D.,Zmmunogenetics 1975.1: 511. 27 Eichmann, K., Eur. J. Zmmunol 1972.2: 301. 28 Pawlak, LL and Nisonoff, A., J. Exp. Med. 1973. 137: 855. 29 Eichmann, K.,Zmmunogenetics 1975. 2: 491. 30 Kindt, T. J., Thunberg, A.L, Mudgett, M. and Klapper, D.G., in Sercarz, E.E., Williamson, A.R and Fox, CF. (Eds) The Immune System: Genes, Receptors, Signals, Academic Press, New York 1974, p. 69.
16 Claflin, J.L. and Davie, J.M., J. Exp. Med. 1975.141: 1073.
31 Briles, D.E. and Krause, R.M., J. Zmmunol. 1974. 113: 522.
17 Murgita, R.A. andVas, S . L , J . Zmmunol. 1970. 104: 514. 18 Askonas, B.A., Williamson, A.R. and Wright, B.E.G., Proc. Nat. Acad Sci. US 1 9 7 0 . 6 7 1398.
32 Kuettner, MG., Wang, A. and Nisonoff, A.,J. Exp. Med 1972. 135: 579.
Briles, D.E. and Davie, J.M., J. Zmmunol. Methods 1975. 8: 363. Gamer, M. and Braun, D.G., J. Exp.Med. 1974. 139: 1513. Eichmann, K., Eur. J. Immunol. 1974.4: 296. Freedman, MH., Pincus, J.H., Yeger, H., McKenny, J.A. and Mage, R.A., Eur. J. Zmmunol. 1974.4: 553. 23 Klinman,N.R.andPress,J.L.,J. Exp. Med 1975. 141: 1133. 24 Kreth, HW. and Williamson, A.R., Eur. J. Zmmunol 1973.3: 141. 25 Apella, E. and Inman, J.K., in Reisfeld, R.A. and Mandy, W.T. (Eds) Contemporary Topics in Molecular Immunology, Plenum Press, New York 1973, p. 51.
34 Claflin, J.L., Eur. J. Zmmunol. 1976. 6 : 666.
10 Claflin, J.L. and Davie, J.M., J. Zmrnunol. 1975. 114: 70. 11 Gearhart, P.J., Sigal, N.H. and Klinman, N.R, J. Exp. Med. 1975. 141: 56. 12 Rudikoff, S. and Potter, M, Biochemistry 1974. 13: 4033. 1 3 Miller, F. and Metzger, H., J. Biol. Chem 1965. 240: 4740. 14 Williamson, A.R., Salaman, M R and Kreth, R.W., Ann. NYAcad. Sci 1973.209: 210.
19 20 21 22
1.A. Ramshaw', P.A. Bretscher and C.R. Parish Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra
33 Stankus, R.P. and Leslie, G.A., Zmmunogenetics 1976.3: 65. 35 Wu, T.T. and Kabat, E.A., J. Exp. Med. 1970. 132: 211. 36 Capra, J.D. andKindt, T.J., Zmmunogenetics 1976. I : 417. 37 Potter, M, Physiol Rev. 1972.52: 692. 38 Kluskens, L, Lee, W. and Kohler, H.,Eur. J. Zmmunol 1975. 5 : 489. 39 Cosenza, H., Quintfins, J. and Leflcovits, I.,J. Zmmunol. 1975. 5 : 343. 40 Sakato, N. andEisen, R,J. Exp. Med 1975. 141: 1411.
Regulation of the immune response
1. Suppression of delayed-type hypersensitivity by T cells from mice expressing humoral immunity The ability of horse red blood cell (HRBC)-specific T cells from mice expressing humoral immunity t o suppress the induction of HRBC-specific delayed-type hypersensitivity (DTH) was investigated. The transfer of Ignegative spleen cells, from mice injected 4 days previously with HRBC, completely suppressed the development of DTH in mice treated with cyclophosphamide and sensitized with HRBC. The suppressor cell was found to be lysed by treatment with anti-theta serum and complement. Furthermore, hemocyanin-specific immune T cells were able to suppress the DTH induced to HRBC, provided these two antigens were coupled together. These studies suggest that T cells present under conditions where humoral immunity is induced can suppress DTH and that such cells play an important role in the regulation of the immune response.
1. Introduction
In many instances humoral and cell-mediated immunity (CMI) are mutually antagonistic [ 11. For example, animals with humoral immunity are usually refractory t o the establishment of de[I 14111
Present address: Department of Microbiology and Immunology, Queens University, Kingston, Canada. Correspondence: CR. Parish, Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601, Australia Abbreviations: IYTH: Delayed-type hypersensitivity HRBC Horse red blood cells SRBC: Sheep red blood cells CRBC: Chicken red blood cells CP: Cyclophosphamide HCY: Hemocyanin ABA: Azobenzene arsonate CMI: Cell-mediated immunity FIA: Freund's incomplete adjuvant
layed-type hypersensitivity (DTH) [2]. Two possible explana tions for this refractory state are either that antibody or anti gen-antibody complexes block the activated T cells which mediate DTH [3] or that T cells present in animals with humoral immunity actively suppress the induction of cell-mediated immunity. Recent reports [4, 51 have shown that T cells, induced under certain conditions, can actively suppress the development and expression of DTH.
A theoretical scheme, which has been recently devised [ 61, suggests that precursor cells for different types of immunity require different amounts of help to be induced. Thus, CMI requires the least, IgM an intermediate amount, and IgG the most. It is also postulated that T cells can inhibit the induction of other classes of immunity; in particular, larger amounts of T cell help t h a ~required for inductior
Suppression of delayed hypersensitivity
Eur. I. Immunol. 1976.6: 674-679 suppress CMI responses, while CMI cells repress the induction of humoral immunity. The biological significance of these postulates has been discussed elsewhere [ 6 ] . The present studies were designed to test this scheme by determining whether the establishment of one type of immunity actively suppresses the development of other forms of immunity, and whether active suppression is the property of the T lymphocyte. The experiments described in this paper show that the transfer of T cells from mice with humoral immunity suppresses the development and expression of DTH.
2. Materialsand methods
2.1. Mice and antigens used Inbred CBA/H female mice 6-8 weeks old were used throughout these studies. Horse, sheep and chicken red blood cells (HRBC), (SRBC) and (CRBC) were collected and stored in Alsever‘s solution. Hemocyanin (HCY) was crystallized from the hemolymph of the crayfish, Jams Zulundii [7].
2.2. Test for DTH DTH was measured as footpad swelling. An eliciting dose of 1 x lo8 red blood cells was injected in 10 p1 into subcutaneous tissue on the surface of one hind footpad. The degree of swelling was measured routinely at 24 h with a dial-gauge caliper (“Schnelltaster”, H.C. Kroplin, Hessen, Germany). Each “unit” represents a swelling of 0.1 mm (+ 0.05 mm).
3. Results 3.1. General Several studies [12, 131 have shown that the treatment of animals with cyclophosphamide (CP), before an optimum sensitizing dose of antigen, causes a marked enhancement of the DTH reaction. Thus, mice injected with CP (200 mg/ kg) and 2 days later injected intravenously (i.v.) with 1O8 HRBC produce a large DTH reaction to HRBC injected into the footpad. DTH can first be demonstrated 4 days after sensitization with antigen and reaches peak levels between 5-6 days. To determine the effect of T cells from humoral immune mice on the development of DTH, normal mice were injected with 1O9 HRBC i.v., a dose which produces high levels of humoral immunity but no detectable DTH, and 4 days later the spleen cells were depleted of Ig-bearing cells. The Ig-negative (Ig-) spleen cells were then transferred to CP-treated mice 1 h before sensitization with HRBC. The DTH levels developed were determined 6 days later by injecting lo8 HRBC into the footpad and measuring the increase in footpad swelling 24 h later. The experimental protocol is shown in Fig. 1.
Cyclophorphomlde
I
IO(IHRBC I V
Test for D T H
IO’HRBC
I
2.3. Depletion of immunoglobulin-bearing cells The method of separation has been fully described elsewhere [8, 91. In summary, the procedure consisted of two stages: first, rosetting of Ig-bearing cells, and second, separation of the nonrosetting and rosetting lymphocytes on Isopaque/ Ficoll. The red cells and rosettes sank to the bottom of the tube while‘the remaining nonrosetting cells floated at the Isopaque/Ficoll interface. The latter cells were recovered and washed twice in medium.
675
In
Footpod
109HRBC I V
Figure 1. Determination of effect of transferring Ip- immune spleen cells on development of DTH to HRBC.
2.4. Anti-@ treatment of spleen cells Anti-@ (Thy-1.2) ascitic fluid was raised in AKR/J mice by multiple injections of CBA/H thymus cells as previously described [lo], and the spleen cell suspensions were treated with & a:@ serum and guinea pig complement as reported previously [ 101. 2.5. Coating of HCY onto red blood cells
A modification of the method of Gold and Fudenberg [ 1 11 was employed. A 0.1 % (w/v) solution of CrC13 in normal saline was prepared and adjusted to pH 5 with 1 N NaOH daily for one week. The standard protocol involved the addition of 100 pg HCY to 2 ml 10 % washed red blood cells followed by 0.2 ml of 0.1 % CrC13 solution and rapid mixing. The mixture was allowed t o stand at room temperature for 10 min before the cells were washed twice in phosphate buffered saline.
3.2. Suppression of DTH by spleen cells from mice with humoral immunity Mice treated with CP and sensitized with HRBC produced high levels (1 0.4 units) of DTH; this was not significantly altered by the transfer of spleen cells or Ig- spleen cells from normal mice (Fig. 2). However, the transfer of spleen cells or Ig- spleen cells from mice immunized t o produce a humoral response completely suppressed the development of DTH (Fig. 2), as corhpared with negative controls. The transfer of Ig-positive (Ig+) spleen cells from immune mice had no effect on the level of DTH obtained. The ability of these immune Ig- spleen cells to suppress an established DTH immunity is shown in Fig. 3. Ig- immune spleen cells transferred 24 h before footpad testing, that is, at a time when mice have developed high levels of DTH, reduced the DTH reaction from 11.8 units to 7.6 units.
Eur. J. Immunol. 1976.6: 674-679
LA. Ramshaw, P.A. Bretscher and C.R Parish
676
Cells Tranilerred
loB
HRBC
lo8
HRBC
loB
HRBC
narmo~
10'
HRBC
immune
10'
HRBC
10'
HRBC
NIL I 10 norm01 spleen cells
60
60 x
lo7
spleen
immune
cells
2 0 ~ 1 0 7isspleen cells
2
o
19-
L b
spleen cell*
4 0 ~ 1 0 Ig-~
immune
spleen cell3
NIL
NIL
0
20
40
F i Z ]
24h
60
80
100
120
Footpad rwellmg(0lmm)
Figure 2. Effect on development of DTH of transferring 6.0 x lo7 spleen cells or 2.0 x 107 Ig- spleen cells from normal mice or from mice immunized to produce a humoral response. Also shown is the effect on DTH of transferring 4.0 x 107 Ig- immune spleen cells. Statistical comparisons of the DTH reaction of mice receiving immune spleen cells or Ig- immune spleen cells to sensitized controls. or tho; receiving normal Ig- cells, yielded a P value of < 0.001. '
Cells Transferred
S e n m r i n y Anfcgen
The T cell dependence of DTH suppression was determined by treating Ig- immune spleen cells with anti-@ serum and complement or as a control normal serum and complement. The cells were then transferred t o CP-treated mice and the ability of the treated cells to suppress DTH determined. Fig. 4 shows that treatment with anti-@ serum prevented cells from suppressing the development of DTH.
3.3. The effect of transferring varying numbers of Igimmune spleen cells on the development of DTH Groups of CP-treated mice were given varying numbers of Ig- HRBC-immune spleen cells before sensitization with HRBC. Fig. 5 shows that maximum suppression of DTH occurred with the transfer of 2.5 x lo7 Ig- spleen cells. There was a linear decrease in the levels of DTH developed as the number of cells transferred was increased. Mice were bled and the anti-HRBC serum antibody levels determined before challenging with HRBC in the footpad for DTH. All groups showed a very weak anti-HRBC antibody response (maximum titer 1/24), which probably resulted from the selective effect of CP on the B cell compartment. Studies [ 14, 151 have shown that CP severely depletes the B cell areas while the T cell areas remain essentially intact. Slightly higher levels of antibody were present in groups of DTHsuppressed mice.
8 10 HRBC
NIL
lo8
180
HRBC
I
4I 140
8 10 HRBC
2 OX 1 0 ~ 1 g - immune 3pleen cells
120
I
.-:
;
9
NIL
NIL
Q
1.10 20
0
40
60
80
100
120
24h Footpad rwellhng(0lmm)
I5
Figure 3. Effect of transferring 2.0 x 107 Ig- spleen cells from normal mice or from mice immunized to produce a humoral response, on the expression of DTH. Ig- cells were transferred 24 h before testing for DTH, that is 5 days after sensitization. Statistical comparisons of the DTH reaction of mice receiving Igimmune cells to sensitized controls yielded a P value of 0.05.
~OXIO~IY-
immune cell3
Treated withAKRonti Theta serum
+
IO~HRBC
0
25
/:111151
12
Immune
015
03
06 IS-
cells
007
0
(x 10')
Figure 5. Effect of transferring varying numbers of Ig-immune spleen cells on the development of DTH. Serum antibody titers were measured by complement-mediated lysis of HRBC. (0- --9 represents footpad reaction Qf unsensitized mice. Statistical comparison of antibody titer of suppressed DTH mice and control, sensitized mice yielded a maximum P value of 0.1.
3.4. Kinetics of appearance of cells capable of suppressing the induction of DTH immunity
10BHRBC
C'
Treated wlfhAKRnormal serum
IO'HRBC
+c' NIL
z
NIL
0
40
20 24
60
80
100
120
h Footpod ~wellms(Olmm)
Figure 4 . Effect of anti-@ serum and complement treatment on ability of Ig- immune cells to suppress induction of DTH.
The previous experiments have shown that 4 day immune Ig- spleen cells can, on transfer, suppress the development of DTH. To determine when cells capable of suppressing DTH first appear in the spleen, mice were injected with lo9 HRBC; and 1, 2, 3, and 4 days later Ig- spleen cells were prepared and transferred to groups of CP-treated mice. Thest were then sensitized with HRBC and tested for DTH 6 days later. The results indicate (Fig. 6) that Ig- spleen cells from mice injected 1 day previously can partially suppress the de-
Suppression of delayed hypersensitivity
Eur. J. Immunol. 1976.6: 674-679
velopment of DTH. The development of DTH can be completely suppressed with Ig- cells from mice injected 3 days previously with HRBC. Immune Cell. Transferred Sensitizing Antlgen
2o
10' tgI
0 day
lo8 HRBC
I day
IO~HRBC
2 doy
lo8 HRBC
3 day
10' HRBC
4 day
lo8 HRBC
NIL
NIL
7 1 L
d
p 20
0
j'lL1151
40
60
80
100
120
24h Footpod rwellmg(0lmm)
Figure 6. Kinetics of appearance of cells capable of suppressing the induction of DTH. Mice were immunized with lo9 HRBC, and 0,1, 2, 3 and 4 days later 2.0 x 107 Ig- spleen cells were transferred to CP-treated mice which were then sensitized with HRBC. Statistical comparisons of the DTH reaction of control sensitized mice to mice receiving 1, 2, 3, and 4 day immune cells yielded P values of 0.05, 0.01, < 0.001 and < 0.001,respectively.
677
3.6. Suppression by linked associative recognition Bullock et al. [ 161 have shown that azobenzene arsonate (ABAtspecific suppressor T cells can inhibit the development of DTH t o other linked protein antigens, provided immunization is carried out with ABA conjugates of these antigens. The following experiment shows that HCY-specific immune T cells can suppress the DTH to HRBC when the two antigens are coupled together. Mice treated with CP and sensitized with HCY-coated HRBC develop high levels of DTH to the HRBC antigens. The DTH obtained is equivalent to that of mice sensitized with HRBC only. Mice immunized with HCY-coated autologous red cells produce high levels of anti-HCY antibody (results to be published). Ig- spleen cells from these HCY-immune mice were transferred t o CP-treated mice which were then sensitized with either HCY-coated HRBC, HRBC or mixture of HCYcoated mouse red cells and HRBC. Fig. 8 shows that DTH to the HRBC antigens was completely suppressed in mi-e given HCY-specific immune cells and sensitized with HCYcoated HRBC. All the control groups developed high levels of DTH to HRBC, demonstrating both the specificity of the suppression and the necessity for the determinants to be linked. Senlltlrlng ~~i~~~~
Hoemocyonm Immune Cells
I
NIL
HCY/HRBC
3.5. Specificity of suppression HRBC
To determine the specificity of suppression, Ig- spleen cells from mice immunized with HRBC were transferred t o groups of CP-treated mice and sensitized with either HRBC, SRBC, or CRBC. Fig. 7 shows that, as previously demonstrated, DTH to HRBC transferred with HRBC-immune Ig- spleen cells was completely suppressed in mice. In contrast, the DTH in mice sensitized with SRBC was partially suppressed while the DTH in mice sensitized with CRBC was unaffected.
NIL
HCY/HRBC
2 0 ~ 1 ig0 ~
HRBC
2 0x 1 ~ 7iy-
yc;$y
20
NIL
lo7 lo-
4.
I c.~~I
NIL
0
20
40
60
2lh Footpod swelling
NIL
2 0 ~ 1 0iY-~
NIL
2 o .lo7 lY-
IO'HRBC
IO8SRBC
I08SRBC
IO'CRBC
NIL
Nll
ID
100
120
HRBC(0lrnm)
Figure 8. Effect of HCY-specific Ig- cells on development of DTH to HRBC in mice sensitized with HCY-coated HRBC. 2.0 x 107 Igspleen cells from mice immunized to HCY were transferred to CPtreated mice which were then sensitized with either HCY-coated HRBC, HRBC or a mixture of HCY-coated mouse red blood cells and HRBC. Statistical comparisons of the DTH reaction of mice receiving HCY-immune Ig- cells and sensitized with HCY-coated HRBC to sensitized controls yielded a P value of < 0.001.
IO'HRBC
NIL
80
3.7. Role of antibody in the suppression of DTH 0
20
40 24 h
b0
80
100
120
Footpad ~ r e l l m g ( O 1mm]
Figure 7. Specificity of suppression. 2.0 x lo7 Ig- spleen cells from mice immunized with HRBC were transferred to groups of mice sensitized with either HRBC, SRBC or CRBC. Statistical comparisons of the DTH reactions of mice receiving Ig- immune cells to sensitized controls yielded P values of < 0.001 for HRBC, < 0.001 for SRBC and > 0.9 for CRBC.
It could be argued that antibody may mask or suppress DTH. In order to test this possibility mice were given either early (4 day) immune serum, hyperimmune serum or serum from mice whose DTH was suppressed by the transfer of immune T cells, before or after sensitization with HRBC. The results in Table 1 show that anti-HRBC antibody given to mice either before sensitization or 24 h before footpad testing failed to affect the level of DTH obtained.
678
Eur. J. Immunol. 1976.6: 674-679
LA. Ramshaw, P . k Bretscher and C R . Parish
Table 1. Effect of transferring various immune sera on the develop ment of D T H ~ ) Serum transferred
Time of transferb)
(0.2 ml)
Mean reciprocal serum antibody titer at time of footpad testing
24 h footpad swelling
Hyperimmune serum
BS AS BS AS
448 15 640
10.6 f 0.5 10.7 It 0.4 10.3 It 0.8 10.1 f 0.9
Serum from
BS
8 15
11.3 f 0.5 11.1 f 0.9
11
11.3 0.9
Early
serum
10
DTH-suppressed AS mice Nil
*
a) Mice were injected i.v. with serum either 24 h before sensitization or 24 h before footpad testing for DTH. b) BS: before sensitization; AS: after sensitization.
Evidence was also obtained to demonstrate that the DTH in mice given immune Ig- spleen cells is suppressed and not simply masked. Spleen cells from sensitized and suppressed mice were depleted of Ig-bearing cells and 5 x l o 6 spleen cells were suspended in 20 p1 of medium. These cells were injected into the footpad of normal mice, either with lo8 HRBC or alone, and the DTH reaction measured at 24 h. Table 2 shows that cells from sensitized mice, but not from suppressed mice, transferred high levels of DTH, indicating that T cells mediating DTH are not simply masked but are absent from the spleens of suppressed mice. Table 2. DTH reactions to HRBC obtained in normal mice following the transfer of Ig- spleen cells from either sensitized or suppressed micea) 1g.- Cells
HRBC
DTH reaction&)
transferred Sensitized
Sensitized Suppressed Suppressed Nil
++ +
10.4 It 0.6 1.9 f 0.8 3.0 It 0.9 2.1 f 0.3 1.4 It 0.2
a) Spleen cells from sensitized or suppressed mice were depleted of Ig-bearing cells and 5 x lo6 cells suspended in 20 pl of
medium with or without lo8 HRBC. These cells were then injected into the footpad of normal mice and the DTH reaction measured 24 h later. b) See Section 2.2.
4.Discussion The studies presented in this paper demonstrate that T cells from mice with humoral immunity can suppress the development of DTH responses. The regime used t o induce cells capable of suppressing DTH suggests that the cell responsible for suppression is the helper T cell. However, it cannot be formally excluded that another T cell, induced under the same conditions that favor humoral immunity, is the actual suppressor cell of DTH.
The suppression of DTH by immune T cells is specific since DTH responses to other noncross-reacting antigens are unaffected. However, the DTH to antigens that cross-react at the T cell level is partially suppressed (Fig. 7). Thus HRBCspecific T cells partially suppress the DTH induced against SRBC but not CRBC. HRBC and SRBC are known to crossreact at the helper T cell level [I 71, and DTH induced with HRBC can be elicited with SRBC but not CRBC (unpublished observations). Observations have also been reported [ 181 of cross-reactivity, at the level of suppressor T cells, between antigens that cross-react minimally at the B cell level.
CP was used in these studies t o obtain high levels of DTH to HRBC. The transfer of specific immune T cells can also suppress the DTH induced with low doses [2] or chemically modified [19] HRBC in normal mice (unpublished results). Three observations make it unlikely that antibody is involved in masking or suppression of DTH. First, only low levels of antibody were produced in suppressed mice, and in many cases the titers were not significantly higher than in sensitized animals (Fig. 5). Second, the administration of immune sera to sensitized animals failed, in all cases, to mask DTH. Finally, DTH could be transferred to normal mice with splenic T cells from sensitized animals but not with cells from suppressed mice. Other evidence ( [ I , 201) also renders improbable the importance of antibody in the suppression of DTH. The results of these transfer experiments suggest that CP may enhance DTH by initially restricting the development of T cells, which would normally suppress DTH, rather than directly affecting the B cell population. Furthermore, a stable established state of cell-mediated immunity may in turn repress any subsequent induction of helper T cells [ 6 ] . The relationship between the suppressor cells described here and other T cells, that have been reported to suppress CMI reactions [4, 5, 211, remains t o be determined. It is worth emphasizing, however, that helper T cells may be responsible for the suppression of DTH in other systems. Thus, Bullock et al. [20] were able t o suppress the development of DTH to ABA in Freund’s complete adjuvant by pretreating animals with ABA in Freund’s incomplete adjuvant (FIA). In this case anti-ABA antibody was not produced when ABA-FIA was given, but it is possible that the procedure used primed the T helper cell population. Such priming without a concomitant antibody response has been reported elsewhere [22,231. Similarly, the injection of picrylsulfonic acid [4] or dinitrochlorobenzene [5], shown to depress contact sensitivity due to the induction of T cell suppressors, may also represent priming of the T helper cell population. Splenic T cells from mice injected with HRBC 24 h previously were found to partially suppress the development of DTH when transferred to sensitized mice. This experiment does not take into account that further development of the transferred cells can occur within the recipient mice. However, this does demonstrate that within 24 h of contact with antigen, events have taken place so as to commit T cells t o a particular pathway of differentiation . The evidence showing that HCY-specific immune T cells are able to suppress the DTH to another unrelated antigen (HRBC), provided these are coupled together, has important
Induction of cellular immunity to vaccinia surface antigens
Eur. J. Immunol. 1976. 6 : 679-683
theoretical and practical implications. The fact that suppression can occur b y linked associative recognition argues against an anti-idiotype form of suppression in this system. Also, it should be possible t o suppress DTH t o an antigen A by immunizing with an appropriate antigen B and then reimmunizing with t h e AB conjugate. Preliminary results indicate that an established DTH t o HRBC can be suppressed by first immunizing with HCY and then with HCY coupled t o HRBC. This may have practical applications in situations where it would b e advantageous t o suppress a DTH response. Received April 8, 1976; in final revised form July 27, 1976.
5 . References 1 Parisli,C.R., Transplant.Rev. 1972. 13: 35.
2 Lagrange, P.H., Mackaness, G.B. and Miller, T.E., J. Exp. Med. 1974.139: 528. 3 Mackaness, G.B., Lagrange, P.H., Miller, T.E. and Ishibashi, T., J. Exp. Med. 1974.139: 543. 4 Zembala, M. and Asherson, G.L., Nature 1973. 244: 227. 5 Phanuphak, P., Moorhead, J.W. and Claman, H.N., J. Immunol. 1974.112: 849. 6 Bretscher, P.A., Cell. Immunol. 1974.13: 171.
U. Koszinowski and Hildegund Ertl Institute of Hygiene, University of Gott ingen, Gott ingen
679
7 Moore, C.H., Henderson, R.W. and Nichol, L.W., Biochemistry 1968. 7: 4075. 8 Parish, C.R. and Hayward, J.A., Proc. Roy. SOC.London Ser. B 1974.187: 65. 9 Parish, C.R., Kirov, S.M., Bowern, N. and Blanden, R.V., Eur. J. Immunol. 1974.4: 808. 10 Kirov, S.M., Eur. J. Immunol. 1974.4: 739. 11 Gold, E.R. and Fudenberg, H.H., J. Immunol, 1967.99: 859. 12 Turk, J.L. and Parker, D., Immunology 1973.24: 751. 13 Lagrange, P.H., Mackaness, G.B. and Miller, T.E., J. Exp. Med. 1974.139: 1529. 14 Poulter, L.W. and Turk, J.L., Nature-New Biol. 1972.238: 17. 15 Turk, J.L. and Poulter, L.W., Clin. Exp. Immunol. 1972. 10: 285. 16 Bullock, W.W., Katz, D.H. and Benacerraf, B., J. Exp. Med. 1975. 142: 275. 17 Hoffman, M. and Kappler, J.W., J. Exp. Med. 1973.137: 721. 18 Basten, A., Miller, J.F.A.P. and Johnson, P., Transplant.Rev. 1975. 26: 130. 19 Parish, C.R.,Eur. J. Immunol. 1972.2: 143. 20 Askenase, P.W., Hayden, B.J. and Gershon, R.K., J. Exp. Med. 1975.141 1 697. 21 Bullock, W.W., Katz, D. and Benacerraf, B., J. Exp. Med. 1975. 142: 261. 22 Kettman, J., Immunol. Commun. 1972. I : 289. 23 Liew, F.Y. and Parish, C.R., J. Exp. Med. 1974.139: 779.
Role of early viral surface antigens in cellular immune response t o vaccinia virus" Infection of mice with the vaccinia virus strain WR, Elstree o r DIs, a conditional lethal mutant of vaccinia virus, resulted in the generation of vaccinia virus-specific sensitized cytolytic T lymphocytes (CTL). It could be shown by cross-reactivity between the three strains and by inhibition experiments with specific antisera that early vaccinia surface antigens are sufficient for the generation of specific CTL in vivo and for the lysis of infected target cells in vitro.
1. Introduction
The specific activity of murine cytolytic T lymphocytes (CTL) sensitized against viruses [ 1-41, chemically modified cells [ 5 , 61, minor histocompatibility antigens [7] o r H-Y antigens [ 81 is restricted t o attacker and target cell homology of [I 14171
* This work was supported by the Deutsche Forschungsgemeinschaft, Grant KO 571/2. Correspondence: Ulrich Koszinowski, Department of Zoology, University College London, Gower Street, London WCIE 6BT, GB Abbreviations: ADCC: Antibody-dependent cell-mediated cytolysis CA: Cytolytic antibody assay CIA: Cytolysis inhibition assay CMC: Cell-mediated cytolysis CTL: Cytolytic T lymphocytes DAPI: 4,6Diamidino-2-phenyl-idol EVSA: Early viral surface antigens IF: Immunofluorescence LCM: Lymphocytic choriomeningitis LVSA: Late viral surface antigens TCID: Tissue culture infective dose VA: Antigens of the virus particle VSA: Viral surface antigens DIs: Dairen-I mutant strain of vaccinia virus CRBC: Chicken red blood cells
the K or D end of the H-2 complex. Killing of modified allogeneic cells is only possible in t h e tolerant situation of chimeric mice [9]. CTL activities therefore seem t o be specific for both H-2 and viral antigens. To obtain further information about possible physiological activities of CTL in the recovery from vaccinia virus infection, t h e specificity of t h e sensitizing antigenic structures induced by the virus has t o be investigated. Infection with vaccinia virus leads t o t h e destruction of t h e infected cell. During virus replication and propagation numerous virus-specific antigenic products are synthesized in t h e cytoplasm and on the surface of the infected cells. Some of these antigens are structural antigens of t h e virus while others d o not seem t o be antigenically related t o antigens found on the surface of t h e virus particle [lo]. Data reported here were obtained after infection of mice with different strains of vaccinia virus. These strains differed in the expression of viral surface antigens (VSA) o n infected cells. Early VSA were sufficient for induction of specific CTL in vivo and for lysis of infected cells in-vitro.
