Immunogeneties 23: 351-356, 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.
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 18.104.22.168 (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-
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-2 504.9" 6 2 . 4
k k k k k k k
C3H/HeN C3H.SW k k k k k k k H-2 380 .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 37C. 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 4C 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 elcitation 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 4C for 45 min. One volume of rabbit complement (diluted 1:4) was added without washing the suspension, and incubation was continued at 38C 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.
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-
. . . .
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