Antigen-specific augmentation factor involved in murine delayed-type footpad reaction.III.Genetic restriction of delayed hypersensitivity augmentation factor (DAF)

Immunobiol., vol.

176, pp. 24-34 (1987)

Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

Antigen-Specific Augmentation Factor Involved in Murine Delayed-Type Footpad Reaction. IH. Genetic Restriction of Delayed Hypersensitivity Augmentation Factor (DAF) AKIRA Y AMADA, KUNISUKE HIMENO, SEIJI NAKAMURA, and KIKUO NOMOTO Received April 23, 1987· Accepted in Revised Form June 30,1987

Abstract We found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the culture supernatant of the mixture of immune T cells and specific antigen, or in the serum of mice immunized with xenogeneic erythrocytes and elicited for DTH footpad reaction. Previous experiments on the genetic restriction of this factor (DTH -augmentation factor; DAF) indicated that DAF activity was effective across the MHC-barrier in C3H/He (H-2 k ) - BALB/c (H-2 d) system. The genetic restriction between DAF and its acceptor cells was then investigated precisely using Igh (immunoglobulin heavy chain locus)-congeneic mice: 1) Expression of DAF activity was MHC-nonrestricted, 2) but was restricted by the Ighlinked gene on the 12th chromosome, 3) such Igh-linked gene restriction was also demonstrated by an absorption test with normal spleen cells. The acceptor cells for DAF were ThyI+, UT4+, Lyt-r T cells.

Introcluction The production of antibodies and the generation of cytotoxic T lymphocytes are regulated by helper T cells (Th) and suppressor T cells (Ts) through various types of interaction between macrophages and lymphocytes. The mechanisms of the interaction between such regulatory T cells and final effector cells may be divided into two types. One type is «cognate interaction» which requires a direct contact, and the other is «factormediated interaction», that is, mediated by a humoral factor(s). Such humoral factors are divided further into two groups, namely antigenspecific and non-specific factors. Antigen-specific factors are produced and Abbreviations: CFA = complete Freund's adjuvant; CRBC = chicken erythrocytes; CY = cyclophosphamide; DAF = DTH-augmentation factor; DFR = delayed-type footpad reaction; DTH = delayed-type hypersensitivity; Igh = immunoglobulin heavy chain locus; Ta = augmenting T cells; Th = helper T cells; Ts = suppressor T cells.

Delayed Hypersensitivity Augmentation Factor . 25 secreted by antigen-specific Ts, Th or augmenting T cells (Ta) and have an antigen-binding structure or anti-idiotypic structure in their molecules. Representatives of antigen-nonspecific factors are interleukin-2 (IL 2) (1, 2) and T cell-replacing factor (TRF) or B cell-differentiation factor (BCDF) (3). In the regulation of delayed-type hypersensitivity (DTH), the contribution of suppressor T cells or suppressor factors has been shown in many experimental systems (4-12). In contrast, there are only a few reports on Th in DTH. LEUNG and ADA (13) demonstrated that antigen-specific Th significantly enhances the primary in vitro induction of DTH to influenza virus. In the induction of DTH to xenogeneic erythrocytes, similarly Th were required for both in vitra (14) and in vivo (15) systems. The requirement of Th for in vivo induction of DTH to antigen-modified syngeneic cells was also reported by MILLER and BUTLER (16). In contact sensitivity, similar Th was also described (17). However, the corresponding helper or augmentation factor had not been reported. Recently, we found a humoral factor capable of augmenting DTH, antigen-specifically, in the culture supernatant of immune spleen cells and erythrocyte antigen (18, 19) or in the serum of mice immunized with xenogeneic erythrocytes to raise DTH and injected with the homologous antigen for elicitation (20, 21). In the present study, genetic restrictions of this factor (DTH-augmentation factor; DAF) and its acceptor cells were examined.

Materials and Methods Animals

Female C3H/He (H-Z k, Ighj ), DBA/2 (H-Z d, Igh'), C57BLl6 (H-Z b, Igh b), and BALB/c (H-2 d, Igh') mice were obtained from Shizuoka Laboratory Animal Center, Hamamatsu, Japan. Female AKR/J (H-2 k, Ighd) mice were obtained from the Seiwa Experimental Animal Institute, Fukuoka, Japan. Male CB-ZO (H-2 d, Igh b), SJLlJ (H-Z', Igh b), and SJA/9 (H-2', Igh') mice were obtained from Ohmura Laboratory Animals, Zama, Japan. Usually, mice were used at 8-1Z weeks of age, unless stated otherwise. Antibodies

Monoclonal anti-Thy 1.2 (F7D5) and monoclonal anti-Lyt 2.1 antibody were purchased from Serotee Ltd., Oxon, U.K., and Cedarlane Laboratories, Ontario, Canada, respectively. Anti-L3T4 (GK1.5) antibody was kindly provided by Dr. Y. KATSURA, Kyoto University, Japan. Preparation oE immune serum

Immune serum was prepared by the following procedure: Mice were immunized with 1 X 108 chicken erythrocytes (CRBC) emulsified with complete Freund's adjuvant (CFA, Difco, Detroit, MI, U.S.A.) supplemented with 5 mg/mi of heat-killed Mycobacterium tuberculosis Aoyama B. A 0.2-ml sampie of the emulsion was subcutaneously (s.c.) injected into a hind thigh. Cyclophosphamide (CY, Shionogi, Osaka, Japan) was dissolved in sterile water and administered intraperitoneally (i.p.) in a dose of 100 mg/kg, 2 days before the immunization. At 7 days after the immunization, mice were rechallenged with 0.05 ml of Z.5 x 107 CRBC into a hind footpad. One day later, the blood was collected and the serum prepared.

26 . AKIRA YAMADA, KUNISUKE HIMENO, SEIJI NAKAMURA, and KIKUO NOMOTO Assay of DAF actiyity DAF activity was assayed by the delayed-type footpad re action (DFR). One ml of the immune serum was transferred intravenously (i.v.) to mice pretreated by giving 100 mg/kg of CY i.p. 4 days before. Six hours later, serum-transferred or non-transferred control mice were injected with 2.5 X 106 CRBC into the right hind footpad. Three days after the serum transfer, DFR was elicited by an injection of 0.05 ml of CRBC suspension, containing 2.5 X 107 cells into the left hind footpad. The degree of swelling was measured 24 h later with a dial thickness gauge. Reactions were expressed as the difference in thickness between the left and right footpads. The results were expressed as the mean ± the standard error of the mean (SEM) of five animals. In this assay system, normal (non-immune) serum transfer had no effect, and the level of DFR did not differ from non-transferred controls as was previously demonstrated (20).

Absorption of immune serum by spleen cells Five ml of the immune serum from C3H/He mice containing DAF were incubated with 1 X 109 spleen cells from various Igh haplotype mice at 4 oe for 3 h. After incubation, the serum was collected by centrifugation at 1,300 x g for 10min.

Depletion of T cell subsets from spleen cells by antibody plus complement treatment In some experiments, absorption of immune serum was performed using T cell-depleted or T subset-depleted spleen cells. Depletion of T cells was carried out by the following procedure: Separate aliquots of C3H/He spleen cells (1 x 107/ml) were incubated at 4 oe for 60 min alone, or in a 1:1,000 dilution of monoclonal anti-Thy 1.2 antibody, and after washing, both aliquots were incubated subsequently at 37°e for 60 min in a 1:10 dilution of Low-tox rabbit complement (Cedarlane). After washing three times with Eagle's minimum essential medium (MEM), the cells were passed through a steel mesh (#300) to eliminate debris of dead cells and were used as T cell-depleted spleen cells. To eliminate L3T4+ or Lyt-2+ cells, 1 :10 diluted culture supernatant of GK1.5 or 1 :20 diluted anti-Lyt 2.1 antibody and complement were used.

Results

Genetic restrietion oi DTH-augmentation iactor When the serum from immunized mice was transferred to normal recipients and xenogeneic erythrocytes homologous to the immunogen were subsequently injected, DFR became positive 3 days later. On the contrary, DFR elicited at 3 days after the immunization was negative in mice not given such an immune serum (20). In this serum transfer system, we demonstrated that DAF activity was effective across the MHC (H-2)barrier, i.e., DAF from C3H/He mice augments the DFR in BALB/c recipient, or vice versa (21). Therefore, experiments were carried out to confirm this phenomenon in various mouse strains. As shown in T able 1, DAF from C3H/He mice immunized with CRBC augmented the CRBCspecific DFR in syngeneic C3H/He and H-2 different BALB/c, DBAI2 recipients. This result confirmed the above-mentioned phenomenon. However, DAF from C3H/He mice did not express activity in H-2 identical AKR/J and non-identical C57BLl6 recipient mice, although C57BLl6 recipient mice responded to DAF from syngeneic donors. These results

Delayed Hypersensitivity Augmentation Factor . 27 Table 1. Genetic restriction of DAF from CRBC-immune serum DAF donor 1

Footpad swellini (X 0.1 rnrn)

Recipients Strain

C3H/He C3H/He AKR/J BALB/c DBAI2 C57BLl6 C57BLl6 C57BLl6

H-2

Igh

k k d d b b

J

d a c b b

Non -transferred controls

Serum -transferred

Increment

0.8 ± 0.2 0.4 ± 0.2 4.7 ± 0.2 0.7 ± 0.2 0.9 ± 0.4 1.5 ± 0.5

4.6 ± 0.6 3 1.1 ± 0.4 4 7.5 ± 0.73 4.0 ± 0.6 3 1.3 ± 0.4 4 8.6 ± 0.9 3

3.8 0.7 2.8 3.3 0.4 7.1

DAF-containing serum was obtained frorn mice immunized with CRBC and elicited for DFR. 2 DAF-containing serum was i.v. transferred to CY-pretreated recipient mice. Six hours later, mice were imrnunized with 2.5 x 106 CRBe. Three days after immunization, DFR was elicited by an injection of 2.5 x 10 7 CRBC into the footpad. Twenty-four hours later, footpad swelling was measured. Values are means ± SEM of five animals. 3 Values that differ significantly from non-transferred controls, p < 0.005. 4 Not significant. 1

suggest that DAF is effective across the H-2 barrier, but there may exist another type of genetic restriction between DAF and recipient mice.

Immunoglobulin heavy chain locus (Igh)-linked gene restrietion oE DAF In addition to MHC-restriction, immunoglobulin heavy chain locus (Igh)-linked gene restriction is also known to be involved in ceH to ceH interaction by other systems. The next experiments were performed to determine the genetic restriction linked to Igh in this system. As shown in Table 2, DAF from BALB/c (Igh a) mice augmented the DFR in syngeneic BALB/c mice, but did not augment the DFR in CB-20 (Ighb), congeneic strain from BALB/c. Similarly, such DAF (from BALB/c) augmented the Table 2. Immunoglobulin heavy chain (Igh)-linked gene restrietion of DAF from BALB/c (H_2 d , Igh') mice Recipients

Footpad swelling (x 0.1 mrn)

Strain

H-2

Igh

Non-transferred controls

Serum-transferred

Increment

BALB/c CB-20 SJA/9 S]LI]

d d

a b a b

0.7 ± 0.6 0.5 ± 0.2 0.6 ± 0.2 0.2 ± 0.2

9.1 ± 0.9 1 0.3 ± 0.2 2 3.2 ± 0.3 1 0.2 ± 0.1 2

8.4 -0.2 2.6 0

1

2

Values that differ significantly from non-transferred controls, p < 0.005. Not significant.

28 . AKIRA YAMADA, KUNISUKE HIMENO, SEI]I NAKAMURA, and KIKUO NOMOTO

DFR in Igh-identical SJA/9 (Igh a) mice which were Igh-congeneic strain of SJLlJ (Ighb) background. In contrast, in SJLlJ mice, DFR was not augmented by such transfer. These results suggest that the expression of DAF activity is genetically restricted by the Igh-linked gene.

Absorption of DAF activity by spleen cells from various strains Experiments were carried out to determine whether the DAF activity was abolished by absorption with normal spleen cells from various mouse strains. DAF-containing serum from C3H/He (Ighi) mice was incubated with spleen cells at 4 oe, and DAF activity was assessed using C3H/He mice as the recipients. As shown in Table 3, DAF activity was absorbed with spleen cells from normal (non-immune) syngeneic C3H/He mice and Igh semi-identical BALB/c (Igh a) mice. However, such activity was not absorbed with spleen cells from Igh distinct CB-20 (Ighb) or C57BLl6 (Igh b) mice. These results suggest that acceptor cells for DAF are present in normal spleen cells and that the binding of DAF to the acceptor cells is restricted by the Igh-linked gene.

T cell-dependent absorption of DAF by spleen cells The next experiments were performed to determine what kind of cells in normal spleens participate in the absorption of DAF. DAF-containing serum from C3H/He was incubated with syngeneic spleen cells treated with anti-Thy 1.2 antibody and complement or complement alone, and DAF activity was assessed (Table 4). Spleen cells treated with complement alone effectively absorbed DAF activity, similar to the results shown in Table 3. However, spleen cells treated with anti-Thy 1.2 plus complement Table 3. Absorption of DAF activity by spleen cells from various strains 1 Exp.

2

3

Transferred serum absorbed with

Footpad swelling (x 0.1 mm)

Non-transferred controls Unabsorbed C3H/He (Ighi) spleen cells Non-transferred controls Unabsorbed BALB/ c (Igh') spleen cells CB-20 (Igh b) spleen cells Non-transferred controls Unabsorbed C57BLl6 (Igh b) spleen cells

0.3 ± 0.2 5.8 ± 0.6 0.8 ± 0.3 0.2 ± 0.1

2.4 ± 0.3 0.6 ± 0.3 2.2 ± 0.3 0.7 ± 0.3 3.5 ± 0.2 3.7 ± 0.3

Y ]3 ]2

Y

Increment

5.5 0.5

2.2 0.4 2.0 2.8 3.0

Five ml of DAF-containing serum from C3H/He (Igh i) mice were absorbed with 1 x 109 spleen cells from various strains of mice at 4 °C for 3 h, and the DAF activity was assessed in recipient C3H/He mice. 2 Values that differ significantly from unabsorbed group, p < 0.005. 3 Not significant. 1

Delayed Hypersensitivity Augmentation Factor . 29 Table 4. T cell-dependent absorption of DAF by spleen cells Transferred serum absorbed with!

Footpad swelling (x 0.1 mm)

Non-transferred controls Unabsorbed C' treated spleen cells Anti-Thyl. 2 plus C' treated spleen cells

1.0 ± 0.2 4.6 ± 0.3 0.6 ± 0.3 5.0 ± 0.4

Increment

3.6 -0.4 4.0

Y

! Number of cells used for absorption was adjusted before treatment with antibody and complement. Five ml of DAF-containing serum from C3H/He mice were absorbed with complement alone- or anti-Thy 1.2 and complement-treated spleen cells (1 x 109 of original cdl number) at 4 °C for 3 h, and the DAF activity was assessed. 2 Values that differs significantly from C'-treated spleen cell-absorbed group, p < 0.005.

could not absorb the DAF activity. Thus, the acceptor cells for DAF are composed of Thy-l-positive T cells.

DAF was absorbed by L3T4+ but not Lyt-2+ T cells To determine the T cell subset of the acceptor cells, further experiments were carried out. DAF-containing serum was incubated with syngeneic spleen cells treated with anti-UT4 plus complement, anti-Lyt-2 plus complement, or complement alone, and the DAF activity was assessed. As shown in Table 5, DAF activity was absorbed by both spleen cells treated with complement alone or anti-Lyt-2 plus complement, whereas by an incubation with anti-UT4 plus complement-treated spleen cells, DAF activity was not absorbed. These results suggest that the acceptor cells für DAF are composed of a T cell subset which bears UT4 but not Lyt-2 antIgen.

Table 5. DAF is absorbed by L3T4+ but not Lyt-2+ T cells Transferred serum absorbed with

Footpad swelling (x 0.1 mm)

Non-transferred controls Unabsorbed C' treated spleen cells Anti-L3T4 + C' treated spleen cells Anti-Lyt-2 + C' treated spleen cells

0.3 ± 0.2 5.6 ± 0.7 2.3 ± 0.7]3 5.7 ± 0.8 2.7 ± 0.3

]2

Increment (% absorption)!

5.3 2.0 5.4 2.4

(0)

(62.3) (-1.9) (54.7)

! % absorption was calculated by the following formula: . Increment of unabsorbed group - increment of absorbed group % absorption = . x 100 mcrement of unabsorbed group 2 Value that differs significantly from C'-treated spleen cell-absorbed group, p < 0.01. 3 Not significant.

30 . AKIRA YAMADA, KUNISUKE HIMENO, SEIJI NAKAMURA, and KIKUO NOMOTO

Discussion In this series of studies, we found an augmentation factor involved in DTH (DTH-augmentation factor; DAF) in the culture supernatant of the mixture of immune spleen cells and specific antigen (18), or in the serum of mice immunized with xenogeneic erythrocytes and elicited for DFR (20). DAF acts antigen-specifically and has an antigen-binding capacity, yet differs from ordinary immunoglobulin molecules. The molecular weight of this factor from the serum is about 200 K to 450 K daltons, determined by gel filtration. We also found that DAF was produced and secreted by T cells (19), the subset of which required the presence of the thymus only for a very short period during ontogenie development (21). Previous experiments on the genetic restriction of DAF suggested that the DAF activity was effective across the major histocompatibility complex (MHC)-barrier (21), despite various reports that antigen-specific factors derived from T cells were generally restricted by the MHC gene cluster and had la or I-J molecules (22). We found that DAF was a MHC-nonrestricted factor but was restricted by another type of genetic control (Table 1). Analogously to the MHC gene cluster, the Igh-linked gene is also known to restrict cell to cell or factor to cell interactions, as reported (10, 11, 23-39). C3H/He and BALB/c mice, both of which are able to accept DAF from C3H/He, have Ighi and Igh" haplotype, respectively. It is weIl known that Ighi and Igh" haplotype show cross-reactivity in Igh-l allotype of IgG2a (40) and T cell alloantigen (41). DBA/2 (lgh C) was also augmented by DAF from C3H/He (Table 1), and such a cross-reaction has been demonstrated in the Igh-l allotype system caused by commonly shared public determinants (40). Therefore, the next experiments were performed using Igh-congeneic mice, and the same tendency of genetic restriction was observed in the system with Ighcongeneic strains (Table 2). Moreover, absorption tests by syngeneic or allogeneic spleen cells demonstrated that the acceptor cells for DAF were present in normal spleen cells from Igh-identical or semi-identical strains (Table 3), and were Thy-l+, UT4+, Lyt-T T cells (Tables 4 and 5). Thus, the binding of DAF to the acceptor T cells was restricted, in an Igh-linked manner. By an in vitra induction system of DTH, the presence of the acceptor cells was further confirmed, i.e., addition of DAF-adsorbed T cells augmented induction of effector cells for DTH, whereas DAF-exposed B cells did not (42). The Igh-linked manner in the binding between DAF and the acceptor cells suggested that DAF has an Igh-linked gene product in its moleeule. Whether or not DAF has an Igh-linked gene product was tested using a monoclonal SJL anti-SJA/9 (anti-lgh") antibody, HA27.13. This antibody recognizes Igh"-linked allotypic determinants expressed on antigen (KLH)-induced proliferative T cells and effector T cells for DTH induced by BCG-PPD system (41, 43). The absorption of DAF by such antibody (HA27.13)-coupled Sepharose was unsuccessful (data not shown).

Delayed Hypersensitivity Augmentation Factor . 31 This result suggests at least three possibilities; first, different Igh-linked determinants are expressed on DAF. Second, DAF has a complementary structure which is an «internal image» for the Igh-linked allotypic determinant, and the third, DAF is a receptor for Igh-linked allotypic determinant. The Igh-linked gene restriction between the T-T interaction has been demonstrated, and this interaction is mediated by the product of Igh-linked gene on the 12th chromosome expressed on T cells (29, 41, 43-50) or T cell factors (10, 23-25, 31-35). OWEN et al. (49) demonstrated that Igh-linked determinants expressed on T cells, namely Tpre, Tthy, Tind, and Tsu were encoded by a gene cluster closely located between Igh-C and Pre-l on the 12th chromosome. The strain combinations of mouse used in the present study do not distinguish between Igh locus and such loci described by OWEN et al. In contact sensitivity, the requirement of Th was described as well as delayed-type footpad reactions (17). COLIZZI et al. (51) reported an antigen-specific T-helper factor involved in contact sensitivity. Their factor was detected in the culture supernatant of lymph node T cells from contactsensitized mice, and augmented an induction phase of contact sensitivity antigen-specifically. However, in contrast to DAF mentioned above, their helper factor had I-A determinant, and expression of helper activity was restricted by MHC but not Igh-linked gene. ZEMBALA et al. (11, 12) reported an antigen-specific suppressor factor involved in contact hypersensitivity. Their factor binds to antigen-nonspecific acceptor cells and reacts to specific antigen as an antigen receptor on the acceptor cells. Antigen-activated acceptor cells secrete the final effector molecules in a similar manner to the secretion of vasoactive amines by IgEsensitized mast cells (11, 12, 52). DAF with specific-antigen binding capacity also may bind to acceptor cells, antigen-nonspecifically, and ren der them reactive to specific antigen, as shown previously with suppressor factor by ZEMBALA's group. In the present study, we demonstrated the presence of acceptor T cells for DAF. It remains to be clarified whether acceptor T cells which have bound DAF differentiate to functional effector cells in response to antigen-specific stimulation and secrete lymphokines or other final effector molecules in response to further antigenic stimulation. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. We thank Dr. K. OKUMURA and Dr. Y. KATSURA for providing monoclonal antibodies, Ms. M. OHARA for her comments on the manuscript, and Ms. S. UEDA, C. NAWADA, 1. YOSHIMATSU, and A. YAMADA for technical and secretarial assistance. References 1.

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