Immunogeneties 23: 351-356, 1986

© Springer-Verlag 1986

Delayed-Type Hypersensitivity to Allogeneic Mouse Epidermal Cell Antigens

I. Lyt- l+2 - T Cells Are Important for DTH

Norihisa IshiP, Hiroshi Nakajima', Ichiro Aoki 2, Takayo IshiP, Toyozoh Takahashi 4, and Kenji Okuda 4

Departments of Dermatology 1, Pathology 2, Oral Surgery 3, and Bacteriology 4, Yokohama City University School of Medicine, 3-46 Urafune-cho, Minami-ku, Yokohama 232, Japan

Abstract. Footpad swelling response was used to measure the alloantigenicity of epidermal cells (ECs) in delayed- type hypersensitivity (DTH). Strong footpad swelling was oberserved 3 h after the challenge, and it continued for 48 h after the challenge. Genetical incompatibility between the recipients and the ECs was required to induce significant footpad swelling. H-2 or non-H-2 incompati- bility between mice and ECs in the sensitization phase sufficed to develop significant footpad swelling. Incom- patibility caused by point mutation in the A region in- duced strong responses when B6.C-H-2 bin12 mice were immunized with B6/J ECs, but the disparity in immuno- globulin h (Igh) allotype genes was insufficient. H-Y anti- gen on ECs could also elicit the DTH response. Semiallo- geneic Fl-derived ECs sensitized the parental recipients. The responses were successfully transferred by immune lymph node cells, but not by immune sera. Treatment of these immune lymph node cells with monoclonal anti- bodies plus complement revealed that the cells respon- sible for DTH transfer were Lyt-1 + 2-, Ia- T cells.

Introduction

Skin has various components, and it is important to investigate the immune response against skin cells to ana- lyze the graft rejection response in skin transplantation. Although it is certain that allospecific cytotoxic T cells have an important function in graft rejection, the detailed mechanism of the cell-mediated process involved in allo- graft rejection is still unclear. Recent investigations sug- gested that a T-cell subpopulation (Lyt-1 + 2-) which me- diates DTH might be an important participant in allo- graft destruction (Ascher et al. 1981, Loveland et al.

Abbreviations used in this paper: DNFB, 2,4-dinitro-l-fluorobenzene; DTH, delayed-type hypersensitivity; ECs, epidermal cells; HBSS, Hanks' balanced salt solution; MHC, major histocompatibility complex; PBS, phosphate-buffered saline

1982). Smith and Miller demonstrated that alloantigen- specific DTH is induced by subcutaneous injection of genetically incompatible allogeneic spleen cells (Smith and Miller 1979a, b, c). Although the roles of both cyto- toxic T cells and DTH effector T cells in the skin graft rejection response have been studied (Mason et al. 1984), the antigenicity and cell interaction of the grafted ECs have not been well characterized due to the complicated anatomical phenomena of the transplanted mass. Thus, it seems important to investigate the immune response caused by ECs in vivo.

In the present study, footpad swelling response was induced by separated allogeneic ECs in order to analyze the alloantigenicity of ECs. We evaluated the genetically encoded antigens required for DTH induction. A large number of congenic mouse strains were employed to in- vestigate preferential response to antigens encoded within the H-2 regions. Finally, these footpad swelling responses were shown to be transferable by Lyt-1 +2-, Ia- T cells, suggesting that the responses were a measure of the DTH responses.

Materials and Methods

Mice. The congenic strains used in this study and their H-2 haplotypes are given in Table 1. All the mice were maintained in our colony at the Department of Bacteriology, Yokohama City University School of Medi- cine, Yokohama, Japan. Only female mice (8-15 weeks old) were used in the present investigation, except in certain experiments indicated below.

Antibodies. The ascites forms of hybridoma antibodies 13.4 (Ia.m7-specif- ic, E-specific; Lemke et al. 1979), 10.2.16 (Ia.17-specific, Ak-specific; Oi et al. 1978), and 16.3.22.5 (Kk-specific; Ozato et al. 1980) were used. Mono- clonal Thy-l.2-specific, Lyt-L2-specific, and Lyt-2.2-specific antibodies were purchased from Cedarlane Laboratories, Ltd. (Hornby, Ontario, Canada).

Preparation ofECs. ECs were prepared by modification of a previously described technique (Stingl et al. 1981). The dorsal surface of the mice was shaved with electric cfippers and then chemically depilated. After killing the animals, the dorsal skin was excised, subcutaneous tissue was mech-

352

Table 1. Fine specificity of EC alloantigen in inducing DTH response

N. Ishii et al.: DTH to Allogeneic Epidermal Cells

Mice used for ECs given for H-2 regions (1/2) Region of sensitization sensitization difference and challenge (1) and challenge (2) K Ap A a Ep E a S D

Footpad swelling ( x 10 -2 mm) (mean ± SE)

Sensitized mice Naive mice

A.BY C3H/HeN b b b b b b b Non-H-2, H-2 35 ___ 2.2* 3 + 2.4

k k k k k k k

B10.D2 BALB/cJ d d d d d d d Non-H-2 38 ± 3.1" 4 ± 3.0

d d d d d d d

C3H.SW C3H/HeN b b b b b b b H-2 50±4.9" 6 ± 2 . 4

k k k k k k k

C3H/HeN C3H.SW k k k k k k k H-2 38±0 .2" 2 ± 1.9

b b b b b b b

B10.AM B57BL/10 k k k k k k b K , A , E , S 2 7 ± 1.9" 3 ±0 .8

b b b b b b b

A.TH A.TL s s s s s s d A, E, S 25 ± 3.4* 5 ± 3.1

s k k k k k d

B10.S(7R) BI0.S(9R) s s s s s s d E, S 22 ± 4.0* 3 ± 3.0

s s s s k d d

s s s s k d d B10.S(9R) B10.S(7R) (E), S 5 ± 2.0 6 ± 1.3

s s s s s s d

B10.AM B10.BR k k k k k k b D 17± 1.2" 4 ± 2 . 2

k k k k k k k

B10.A(18R) B57BL/10 b b b b b b d D 11 ± 2.2* 0 ± 2.2

b b b b b b b

B6.C-H-2 bin12 B57BL/6J b b* b b b b b A (point mutation) 21 ± 1.4" 3 ± 2.9

b b b b b b b

b b b b b b b C57BL/6.Igh e B57BL/6J IgCH 5 ± 3.6 7 ± 2.4

b b b b b b b

b b b b b b b C57BL/6 C57BL/6 - 1 ± 1.7 2 ± 1.5

b b b b b b b

DTH reactivity was induced by s.c. sensitization, and the challenge was carried out 7 days later. Each group of experiments involved 4-6 mice. Naive mice were challenged without immunization * P < 0,001

anically removed, and the skin was floated, dermis side down, on 1% trypsin in PBS for 45 min at 37°C. Epidermal sheets were then mech- anically removed from the skin, washed twice in HBSS, minced, and stirred in RPMI 1640 medium for 1 h at 4°C with a magnetic stirrer. Then 0.05% DNase (type 1, Sigma Chemical Co., St. Louis, Missouri) in RPMI 1640 was added to the mixture to a final concentration of 0.025%. The mixture was stirred for an additional 20 min and then filtered through nylon mesh (Nytex, Tetko, Elmsford, New York). The filtrate was vigor- ously pipetted to break up clumps and washed three times in HBSS to yield a suspension of free ECs.

Sensitization and el±citation of footpad swelling. Mice were sensitized with 5 × 106 allogeneic ECs subcutaneously. Seven days after sensitization, the thickness of both hind footpads was measured. The mice were then

challenged in both hind footpads with 25 Ixl of filtrate containing i x 106 allogeneic ECs. The thickness of the same footpads was again measured at an appropriate time, and the response was calculated as the difference (in units of 10 - 2 ram) in thickness before and after the challenge.

Adoptive transfer. A suspension of 40 x 106 cells or 0.5 ml of serum from sensitized mice was injected intravenously into appropriate recipients.

Antibody plus complement treatment. One volume of cell suspension (107/ml) was incubated with one volume of antibody (1:10 dilution) at 4°C for 45 min. One volume of rabbit complement (diluted 1:4) was added without washing the suspension, and incubation was continued at 38°C for 1 h. The cells were then washed three times with HBSS and resuspen- ded in HBSS at an appropriate concentration.

N. Ishii et al.: DTH to Allogeneic Epidermal Cells 353

Statistics. The results were expressed as means and standard errors (SE). Differences between experimental and control groups were tested using

the Student's t test.

Results

Induction of footpad swelling against EC antigen. It has been reported by several investigators that allogeneic spleen cells can induce allospecific DTH. The initial series of experiments was designed to evaluate the ability of allogeneic ECs to induce allospecific DTH responses. After EC challenge, footpad swelling was measured at various times. When C57BL/6J (B6/J) mice were sensi- tized with allogeneic B10.AM ECs and challenged by B10.AM ECs, strong footpad swelling was observed 3 h after the challenge, and it continued for 48 h (Fig. 1). When B6/J mice were sensitized and challenged by the syngeneic B6/J ECs, strong footpad swelling was seen after 3 h, but it immediately decreased to around the zero level. Footpad swelling of nonsensitized B6/J mice chal- lenged by allogeneic ECs of B10.AM mice showed the same time course as that of the syngeneic sensitized con-

(XlO-2mm)

60

50

40

30

0 °

--10"

. . . .

Fig. 1. Time course of footpad swelling using allogeneic mouse EC antigens. B6/J mice were immunized with B10.AM ( 0 - - 0 ) or B6/J ( H ) ECs. After 7 days, footpad swellings were measured by the injection of the same mouse ECs as were used for immunization. B6/J mice were challenged with B 10.AM ( ~ - - - , ) ECs without immunizat ion

trols. Histological examination of the footpads 24-48 h after the challenge showed a severe inflammatory reac- tion with marked infiltration of mononuclear cells, while the histological reaction in the footpads of unsensitized mice was minimal (data not shown). On the basis of these results, we measured footpad swelling 24 h after the chal- lenge in the following experiments.

DTH-inducing ability of both H-2 and non-H-2 surface antigens of ECs. The next series of experiments was de- signed to evaluate the contribution of both H-2 and non- H-2 gene products of EC surface antigens to the induction of the DTH response. Mice were injected subcutaneously with 5 x 10 6 viable ECs and challenged 7 days later with 1 x 10 6 viable ECs in both hind footpads. As shown in Table 1, strong footpad swelling was seen in the A.BY mice sensitized with genetically (H-2- and non-H-2)- incompatible C3H/HeN ECs. The mice were also success- fully sensitized with non-H-2-incompatible and H-2-com- patible allogeneic ECs (B10.D2 vs BALB/cJ), and by non-H-2-compatible and H-2-incompatible allogeneic ECs (C3H.SW vs C3H/HeN). The contribution of the cell-surface antigens encoded by the H-2 subregion gene was also precisely analyzed. The results showed that class I or class II molecules alone served as antigens in the induction of DTH. Even a point mutation at the A mole- cule was sufficient to induce D T H (B6.C-H-2 bm12 vs B6/J). We could not observe any preferential response in multi- ple non-H-2 and H-2 genes, however. In addition, B6/J ECs failed to sensitize B6.Igh e mice (Igh determinant- incompatible). The Igh gene encodes the immunoglobulin allotypic determinant.

H-Y effect on allogeneic D TH response. The H-Y antigen is a strong non-H-2 antigen. The DTH response against the H-Y antigen using spleen cells has been reported. We investigated the ability of H-Y antigen on ECs to induce DTH using both fen~tale and male mice. As shown in Table 2, we observed a significant level of footpad swell- ing when we used a combination of female mice recipients and male ECs as the antigen. However, the reciprocal combination, in which H-Y antigen should be absent in female ECs, produced no response.

F1 effect of semiallogeneic DTH response. We tested the response of the semiallogeneic combination between (B6/J × DBA/2)F1 mice and their parents. As shown in Table 3, significant responses were not induced in F1 mice when parental ECs were used. Conversely, semiallogeneic F1 ECs successfully induced a significant response in pa- rental mice. It seemed that parent mice recognized semial- logeneic Fl ECs.

Alloantigen-mediated swelling caused by EC antigens is transferable by lymph node cells. The next experiment was

354 N. Ishii et al.: DTH to Allogeneic Epidermal Cells

Table 2. Effect of H-Y antigen in allogeneic DTH

Mice ECs Footpad swelling (mean -I- SE) (x 10-2 ram)

Sensitized mice Naive mice

footpad response (data not shown). But intravenous in- jection of immune lymph node cells successfully trans- ferred the response to naive mice (Table 4). We concluded that allogeneic EC-mediated footpad swelling was trans- ferable by immune lymph node cells.

C3H/HeN ~ C3H/HeN ~ 3 + 1.5 3 ___ 1.8 C3H/HeN 9 C3H/HeN ~ 21 _+ 1.4" 3 _ 2.6 C3H/HeN d' C3H/HeN ~ 3 _+ 3.2 4 -I- 1.3 C3H/HeN d ~ C3H/HeN c~ 2 + 2.7 0 -t- 2.9 C3H/HeN ? DBA/2 9 28 _ 1.3" 7 + 1.6

Naive mice were challenged with the indicated ECs without sensitization P < 0.001

Table 3. F 1 effect of semiallogeneic DTH response

Mice ECs Footpad swelling (x 10-2 mm)

Mean + SE

(B6/J x DBA/2)F 1 B6/J 4 _+ 2.2 F 1 DBA/2 5 _+ 1.4 F 1 B10.BR 16 _+ 2.0* F 1 (B6/J) t 3 -I- 2.6

B6/J DBA/2 16 _+ 1.4" B6/J (B6/J x DBA/2)F 1 17 _+ 2.2* B6/J (F1)t 3 _+ 1.6

See the footnote to Table 1 P < 0.001

t ECs were used for the challenge without sensitization

Table 4. Transfer of DTH*

Mice used for Transferred by ECs given for Footpad swelling sensitization sensitization ( x 10 -2 ram) and challenge and challenge

Mean + SE

C3H/HeN Serum (B6/J) ~" 4 _+ 1.5 C3H/HeN Lymph node cells (B6/J) 19 + 2.44: C3H/HeN - B6/J 21 __ 1.94: C3H/HeN - (B6/J) 5 -t- 1.4

C3H/HeN mice were immunized with B6/J EC. Seven days later, 4 x 107 lymph node cells or 0.5 ml serum from sensitized C3H/HeN mice were injected intravenously into naive C3H/HeN mice. Within 1 h, C3H/HeN mice were challenged with B6/J ECs

? ECs in brackets were given to nonimmunized recipients 4- p < 0.005

designed to investigate the component responsible for the response induced by ECs. Seven tenths of a milliliter of serum from the C3H/HeN mice sensitized by B6/J ECs was intravenously transferred to naive C3H/HeN mice. Within 1 h, the mice were challenged with B6/J ECs, and footpad swelling was measured after 24 h. No significant footpad swelling was observed (Table 4). Even an in- creased dose of serum failed to transfer a significant

Monoclonal antibody treatment of immune lymph node cells. Immune lymph node cells were treated with mono- clonal antibodies plus complement before being trans- ferred in order to investigate the phenotypes of the cells responsible for footpad swelling. As shown in Table 5, the treatment of sensitized B6/J lymph node cells with Thy-l.2-specific or Lyt-l.2-specific antibody plus comp- lement abolished the ability to transfer footpad swelling. But treatment of these lymph node cells with Ia-specific (A k, E) or Lyt-2.2-specific antibody plus complement failed to modify this response. Furthermore, the DTH activity of sensitized C57BL/10 lymph node cells against B 10.A(18 R)-derived E Cs was inhibited by Lyt-1.2-specif- ic antibody plus complement treatment but not by Lyt- 2.2-specific antibody plus complement treatment. These results indicated that Lyt-1 +2- and Ia - T cells are re- sponsible for the transfer of the footpad swelling.

Discussion

This study analyzed footpad swelling response induced by allogeneic ECs. The delayed kinetics of the responses was similar to that of contact sensitivity using DNFB antigen (Okuda et al. 1980), suggesting that these responses could be measures of DTH reactivity. Histological observation of the footpads supported this hypothesis.

The study using several strains of recombinant mice demonstrated that H-2 or non-H-2 incompatibility be- tween the recipients and the allogeneic ECs was enough to induce strong footpad swelling. Even incompatibility caused by point mutation of the A region (B6.C-H-2 bin12 vs B6/J) was critical in the induction of DTH (Table 1). Semiallogeneic ECs were able to sensitize the recipient. Concomitant EC antigens derived from B6/J parents had no discernible effect in the recognition of DBA/2-derived antigens on F1 ECs by B6/J recipients. Incompatibility in the serological component-encoding gene (B6.Igh e vs B6/J) was not critical in the induction of DTH. Moreover, H-Y antigen, which is a strong non-H-2 antigen, pro- duced strong DTH response. These findings imply that ECs express the same H-2 and non-H-2 transplantation antigens that have been detected in other tissues. These data are consistent with Smith and Miller's work (1979c), in which allogeneic spleen cells were used as antigens. Smith and Miller (1979c) reported class II (A) antigen preference in alloantigen-specific DTH induced by allo- geneic spleen cells, but we could not observe any preferen- tial response to non-H-2 and H-2 antigens. One possible

N. Ishii et al.: DTH to Allogeneic Epidermal Cells

Table 5. Antibody treatment of DTH effector lymph node cells with various monoclonal antibodies plus complement

355

Recipient ECs given for Transferred cells mice challenge

Footpad swelling ( x 10 -2 ram)

Cells Monoclonal antibody Mean _ SE treatment

C3H/HeN BALB/cJ BALB/cJ BALB/cJ

(BALB/cJ) +

B6/J B10.BR B10.BR B10.BR B10.BR

(B10.BR)

B57BL/10 B10.A(18R) B10.A(lSR) B10.A(18R) B10.A(18R) [B10.A(18R)]

C3H/HeN sensitized by BALBIcJ ECs Anti-A k + C 16 + 1.1 (5) t C3H/HeN sensitized by BALB/cJ ECs Anti-E k -I- C 14 +__ 2.4 (5) C3H/HeN sensitized by BALB/cJ ECs C 18 +__ 1.3 (5) - 2 + 2 . 4 ( 6 )

B6/J sensitized by B10.BR ECs Anti-Thy-l.2 + C 1 _+ 1.6 (I (5) B6/J sensitized by B10.BR ECs Anti-Lyt-l.2 + C -2 _+ 1.7 (I (5) B6/J sensitized by B10.BR ECs Anti-Lyt-2.2 + C 13 _+ 2.0 (5) B6/J sensitized by B10.BR ECs C 15 ± 2.1 (4)

C57BL/10 sensitized by B10.A(18R) ECs Anti-Thy-l.2 + C 1 _ 1.2 (I (6) C57BL/10 sensitized by B10.A(18R) ECs Anti-Lyt-l.2 + C 6 +_ 1.3 (I (5) C57BL/10 sensitized by B10.A(18R) ECs Anti-Lyt-2.2 + C 16 ± 1.1 (6) C57BL/10 sensitized by B10•A(18R) ECs C 19 + 1.0 (6) - - 2 + 1.6 ( 5 )

Lymph node cells from sensitized mice were incubated with the indicated monoclonal antibodies for 45 min at 37°C, then with complement (C) for another 30 min. After three washes, lymph node cells were injected intravenously into naive syngeneic mice. After challenging with the indicated ECs, footpad swellings were measured with a microdial thickness gauge ECs from all the strains of mice in brackets were given to nonimmunized recipients

t The numbers in parentheses indicate the number of mice used (I p < 0.001

explanation for this disparity is that the amount of anti- gens is different in ECs and spleen cells. It is known that ECs show the strongest type of graft rejection (Ikezawa et al. 1981). It is thus possible that ECs express the surface antigen more strongly than spleen cells.

Our DTH could be transferred to naive mice by im- mune lymph node cells but not by immune sera. The cells responsible for the transfer were T cells, since the activity of the cells was abrogated by treatment with Thy-l.2- specific antibody plus complement. Lyt-l+2 - and Ia - T cells were important in this DTH transfer. It has already been reported that Lyt-1 +2- T cells are responsible for DTH (Loveland and McKenzie 1982). Smith and Miller (1979c) stated that Lyt-1 +2- T cells were important for DTH against allogeneic spleen cell antigen assayed by footpad swelling. Similar cells have been identified in host-versus-graft responses to a minor antigen (H-Y) on spleen cells (Pole and Simpson 1983). Our results were consistent with these data and confirmed the hypothesis that these footpad swellings are a measure of DTH re- sponse.

Graft rejection is an alloantigen-specific immune re- sponse, and it is therefore important for manipulating the response against ECs. Our study provides a useful tool for analyzing the immune response against ECs in vivo.

Acknowledgments. We thank Prof. Ryukichi Nagai and Prof. Kazuaki Misugi for helpful discussions and Ms. Hiromi Akimoto for her help in the preparation of this manuscript.

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Received January 8, 1986; revised version received February 24, 1986