Int. Archs Allergy appl. Immun. 56: 57-64 (1978)
Experimental Models for Prevention of Graft-versus-Host Reaction in Bone Marrow Transfusion III. Reversible and Irreversible Differentiation of Lymphocytes Destined for Cytotoxicity to Effector Cells for Splenomegaly Sumio Miyazaki, Kikuo Nomoto, Ataru Kuroiwa, Nagahide Goya and Kenji Takeya Departments of Pediatrics and Microbiology, Faculty of Medicine, Kyushu University, Fukuoka
Introduction The occurrence of the graft-versus-host (GVH) reaction interferes with a wide ap plication of bone marrow transfusion in clinical fields. In model systems with inbred mice, GVH reaction in FI recipients occurs not only in an H-2 nonidentical but also in an H-2 identical combination of which par ental strains have different antigens in mi nor histocompatibility and M locus systems [10, 11]. In an H-2 nonidentical system, a pretreatment of donors with sonicated lymphoid cells of a parental partner strain suppressed completely cytotoxicity and splenomegaly in FI recipients which are the most prominent features of GVH reaction [11]. Pretreatment of such donors with lymphoid cells of a partner strain in
Freund’s complete adjuvant (FCA) sup pressed cytotoxicity but augmented splen omegaly in FI recipients. In an H-2 identi cal combination of which donors and recipi ents were different in minor histocompati bility and M locus systems, GVH reaction continued for a longer period of time and a lethal effect of runt disease was more pro nounced than in an H-2 nonidentical combi nation [12]. In such an H-2 identical combi nation, the pretreatments of donors scarcely affected the occurrence of cytotoxicity and splenomegaly in contrast to an H-2 non identical combination. Thus, the prevention of GVH reaction by such pretreatments of donors in H-2 nonidentical combinations may be worthwhile to be studied in detail for a wide application of bone marrow transfusion. In the present experiments, re
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 6:15:10 AM
Abstract. When lymphoid cells were obtained from AKR donors 12 h after a treatment with C57BL/6 cells in complete Freund’s adjuvant and transferred to (AKR X C57BL/6) F I mice, splenomegaly in FI recipients was augmented but cytotoxicity was suppressed. The suppression of cytotoxicity was antigen-specific. When cell transfer was carried out at stages as early as 3 or 6 h after the treatment of donors, cytotoxicity was enhanced but splenomegaly was suppressed. Irreversible deviation of immune response from the genera tion of cytotoxicity to the development of splenomegaly appears to occur within 12 h after such a treatment of donors.
58
versible and irreversible deviation of im mune response from the generation of cyto toxicity to the development of splenomegaly by a treatment of donors with parental lymphoid cells in FCA was studied especial ly in terms of time-course of a treatment of donors.
Materials and Methods Animals. Female and male mice of inbred AKR and C57BL/6 strains were obtained from the Breeding Unit of Kyushu University. FI hybrids between AKR and C57BL/6 mice were raised in our laboratory. 8-week-old female mice of AKR strain were used as donors and 6-week-old mice of FI hybrid were used as recipients of which each group included approximately an equal number of female and male mice. In experiments for study ing on a lethal effect of runt disease, 1- to 5-dayold FI hybrids were used as recipients. Preparation of cell suspensions. Mice were bled by cutting the femoral artery under anesthes ia with ether. Spleens and cervical, axillary, in guinal and mesenteric lymph nodes were removed and squeezed with two slide glasses in Hanks’ bal anced salt solution (HBSS) in Petri dishes. The cell suspension was passed through a gauze filter to remove large residual fragments. This cell sus pension was designated as lymphoid cell suspen sion. The femurs were removed and both ends were cut by scissors. The marrow was flushed out with HBSS using a syringe equipped with a Mantoux needle. The fragments of the bone marrow were pipetted gently in tubes to prepare a single cell suspension. This cell suspension was designat ed as bone marrow cell suspension. Cell suspen sion of methylcholanthrene (MCA)-induced sar coma of C57BL/6 origin was prepared by trypsinization of small fragments according to the con ventional method. Viable cells were counted by the trypan blue exclusion method and adjusted to the desired concentrations. Preparation of anti-C57BL/6 serum and antiQ-AKR serum. AKR mice were subcutaneously injected with 3 X 107 lymphoid cells of C57BL/6 mice in FCA. Intravenous injections with 1 X 107
lymphoid cells in saline were carried out six times every 4 days with an interval of 2 weeks from the primary immunization. The blood was harvested 5 days after the last immunization. Serum was pooled from 20 animals and inactivated at 56 °C for 30 min. The serum was divided into small tubes and stocked at -20 °C until use. A treatment with 1/4-diluted serum and rabbit complement killed over 90°/o of the lymphoid cells of C57BL/6 mice. Anti-0 serum was prepared according to Reif and Allen [18]. Conditioning of donors. AKR mice were in jected subcutaneously with 2 X 106 lymphoid cells of C57BL/6 mice in FCA into both sides of the flank and used as donors 2, 6,12, 24 and 72 h later. Conditioning of recipients. In experiments de signed to observe cytotoxicity and splenomegaly, adult ABF1 mice were used as recipients without any treatment. In experiments designed to observe a lethal effect of GVH reaction, newborn ABF1 mice were exposed to 400 rad of whole body irra diation and inoculated with donor cells 1-3 h later. Splenomegaly. The spleen index represented the ratio of the spleen weight (relative to body weight) of the treated animals to the spleen weight (relative to body weight) of the controls treated with an equivalent number of syngeneic FI lymphoid cells [17]. Cytotoxicity test. Spleens were removed from the recipients 5 or 10 days after the injection of donor cells. Cell suspensions were prepared as de scribed above. Spleen cell suspensions were treat ed with 1/4-diluted anti-C57BL/6 serum and rab bit complement at 37 °C for 30 min to eliminate the recipient cells carrying H-2b antigens. After washing, 1.25 X 107 viable spleen cells were add ed to 5 X 10s MCA tumor cells of C57BL/6 ori gin and the mixtures were subcutaneously injected into AKR mice exposed 500 rad of whole body ir radiation. Tumor growth was measured 10 days after the injection and tumor size was recorded as the area (mm2) of the largest diameter times the smallest diameter. Counting of macrophages in the spleens of re cipients. Spleen cell suspension was incubated with lymphocyte separator reagent (Technicon Instru ments Corp., N.Y.) for 20 min and smears of these cells were stained with Giemsa’s solution. Cells engulfing iron particles were counted as ma crophages under a microscope.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 6:15:10 AM
Miyazaki/Nomoto/Kuroiwa/Goya/Takeya
59
Depressed Cytotoxicity in GVH Reaction
Results Effects of a Pretreatment of Donors with Parental Antigens in FCA Donor cells including 1.5 X 107 lymph oid cells and 1.0 X 107 bone marrow cells were injected intravenously into adult FI recipients. Splenomegaly and cytotoxicity were observed 5 and 10 days after the cell transfer. When donor cells were obtained from normal AKR mice, splenomegaly was detected at 5 and 10 days and cytotoxicity was detected at 10 days (table I). Cytotoxic ity was abolished by a treatment of FI cells with anti-0 serum and complement. When donor cells were obtained 3 or 6 h after the treatment with C57BL/6 lymphoid cells in FCA, the generation of cytotoxicity was fa cilitated but splenomegaly was suppressed. When donor cells were obtained 12 h after such a treatment or later, cytotoxicity was
not raised, but splenomegaly was scarcely affected or slightly augmented. When AKR donors were pretreated with AKR cells in FCA for 24 h, the generation of cytotoxicity in F I recipients was not suppressed 10 days after cell transfusion (table II). Lethal Effect of GVH Reaction in Irradiated Neonatal Recipients F I recipients of 1-5 days of age were ex posed to 400 rad and injected intraperitoneally with 1 X 107 donor lymphoid cells within 3 h. The number of FI recipients in each group was 20-25 animals. Survival rates of recipients were observed for 50 days. A few of the irradiated mice died within 7 days if they were not given donor cells (fig. 1). When irradiated FI recipients were injected with normal AKR cells, 80% of them died within 35 days but the remain ing 20% survived beyond 50 days. When
Table I. Augmentation and suppression of cytotoxicity and splenomegaly in FI recipients by a treatment of donors with antigen in FCA at various times before cell transfer
3 6 12 24 72 Normal AKR
Spleen index 5 daysi
1.13 1.17 ± 1.47 ± 1.61 ± 1.59 ± 1.42 ±
0.122 0.15 0.26 0.28 0.29 0.27
Tumor growth in neutralization tests io days
1.15 _L0.14 1.14 ± 0.13 1.32 ± 0.18 1.47 ± 0.25 1.60 ± 0.31 1.49 ± 0.25
in the presence of spleen cells in FI recipients 5 days
10 days
12.2 X 6.1 ( I )3* 17.4 ± 5.2(+)* 38.8 ± 12.5( —) 45.5 ± 13.5 ( ) 39.2 ± 10.8 ( ) 38.4 ± 26.6 ( —)
14.5 X 6.5 ( + ‘) 15.9 ± 5.3 ( + )* 42.5 ± 13.9 ( —) 38.1 ± 11.5(—) 45.8 ± 12.0 ( ) 12.0 ± 5.1( + )*
controls inoculated with tumor cell alone 38.0 X 10.2 30.0 ± 6.9 36.5 ± 8.9 43.9 ± 12.6 42.0 ± 15.5 42.0 ± 22.1
1 Assays of splenomegaly and cytotoxicity were carried out 5 or 10 days after cell transfer. 2 Average value of 5-10 animals ± SE. 3 Average value of 5-10 animals ± SE. Plus or minus in the parentheses indicates the presence or absence of cytotoxicity. Growth of indicator tumors was expressed as areas of the largest diameter times the smallest diameter (mm2). *p
Experimental Models for Prevention of Graft-versus-Host Reaction in Bone Marrow Transfusion III. Reversible and Irreversible Differentiation of Lymphocytes Destined for Cytotoxicity to Effector Cells for Splenomegaly Sumio Miyazaki, Kikuo Nomoto, Ataru Kuroiwa, Nagahide Goya and Kenji Takeya Departments of Pediatrics and Microbiology, Faculty of Medicine, Kyushu University, Fukuoka
Introduction The occurrence of the graft-versus-host (GVH) reaction interferes with a wide ap plication of bone marrow transfusion in clinical fields. In model systems with inbred mice, GVH reaction in FI recipients occurs not only in an H-2 nonidentical but also in an H-2 identical combination of which par ental strains have different antigens in mi nor histocompatibility and M locus systems [10, 11]. In an H-2 nonidentical system, a pretreatment of donors with sonicated lymphoid cells of a parental partner strain suppressed completely cytotoxicity and splenomegaly in FI recipients which are the most prominent features of GVH reaction [11]. Pretreatment of such donors with lymphoid cells of a partner strain in
Freund’s complete adjuvant (FCA) sup pressed cytotoxicity but augmented splen omegaly in FI recipients. In an H-2 identi cal combination of which donors and recipi ents were different in minor histocompati bility and M locus systems, GVH reaction continued for a longer period of time and a lethal effect of runt disease was more pro nounced than in an H-2 nonidentical combi nation [12]. In such an H-2 identical combi nation, the pretreatments of donors scarcely affected the occurrence of cytotoxicity and splenomegaly in contrast to an H-2 non identical combination. Thus, the prevention of GVH reaction by such pretreatments of donors in H-2 nonidentical combinations may be worthwhile to be studied in detail for a wide application of bone marrow transfusion. In the present experiments, re
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 6:15:10 AM
Abstract. When lymphoid cells were obtained from AKR donors 12 h after a treatment with C57BL/6 cells in complete Freund’s adjuvant and transferred to (AKR X C57BL/6) F I mice, splenomegaly in FI recipients was augmented but cytotoxicity was suppressed. The suppression of cytotoxicity was antigen-specific. When cell transfer was carried out at stages as early as 3 or 6 h after the treatment of donors, cytotoxicity was enhanced but splenomegaly was suppressed. Irreversible deviation of immune response from the genera tion of cytotoxicity to the development of splenomegaly appears to occur within 12 h after such a treatment of donors.
58
versible and irreversible deviation of im mune response from the generation of cyto toxicity to the development of splenomegaly by a treatment of donors with parental lymphoid cells in FCA was studied especial ly in terms of time-course of a treatment of donors.
Materials and Methods Animals. Female and male mice of inbred AKR and C57BL/6 strains were obtained from the Breeding Unit of Kyushu University. FI hybrids between AKR and C57BL/6 mice were raised in our laboratory. 8-week-old female mice of AKR strain were used as donors and 6-week-old mice of FI hybrid were used as recipients of which each group included approximately an equal number of female and male mice. In experiments for study ing on a lethal effect of runt disease, 1- to 5-dayold FI hybrids were used as recipients. Preparation of cell suspensions. Mice were bled by cutting the femoral artery under anesthes ia with ether. Spleens and cervical, axillary, in guinal and mesenteric lymph nodes were removed and squeezed with two slide glasses in Hanks’ bal anced salt solution (HBSS) in Petri dishes. The cell suspension was passed through a gauze filter to remove large residual fragments. This cell sus pension was designated as lymphoid cell suspen sion. The femurs were removed and both ends were cut by scissors. The marrow was flushed out with HBSS using a syringe equipped with a Mantoux needle. The fragments of the bone marrow were pipetted gently in tubes to prepare a single cell suspension. This cell suspension was designat ed as bone marrow cell suspension. Cell suspen sion of methylcholanthrene (MCA)-induced sar coma of C57BL/6 origin was prepared by trypsinization of small fragments according to the con ventional method. Viable cells were counted by the trypan blue exclusion method and adjusted to the desired concentrations. Preparation of anti-C57BL/6 serum and antiQ-AKR serum. AKR mice were subcutaneously injected with 3 X 107 lymphoid cells of C57BL/6 mice in FCA. Intravenous injections with 1 X 107
lymphoid cells in saline were carried out six times every 4 days with an interval of 2 weeks from the primary immunization. The blood was harvested 5 days after the last immunization. Serum was pooled from 20 animals and inactivated at 56 °C for 30 min. The serum was divided into small tubes and stocked at -20 °C until use. A treatment with 1/4-diluted serum and rabbit complement killed over 90°/o of the lymphoid cells of C57BL/6 mice. Anti-0 serum was prepared according to Reif and Allen [18]. Conditioning of donors. AKR mice were in jected subcutaneously with 2 X 106 lymphoid cells of C57BL/6 mice in FCA into both sides of the flank and used as donors 2, 6,12, 24 and 72 h later. Conditioning of recipients. In experiments de signed to observe cytotoxicity and splenomegaly, adult ABF1 mice were used as recipients without any treatment. In experiments designed to observe a lethal effect of GVH reaction, newborn ABF1 mice were exposed to 400 rad of whole body irra diation and inoculated with donor cells 1-3 h later. Splenomegaly. The spleen index represented the ratio of the spleen weight (relative to body weight) of the treated animals to the spleen weight (relative to body weight) of the controls treated with an equivalent number of syngeneic FI lymphoid cells [17]. Cytotoxicity test. Spleens were removed from the recipients 5 or 10 days after the injection of donor cells. Cell suspensions were prepared as de scribed above. Spleen cell suspensions were treat ed with 1/4-diluted anti-C57BL/6 serum and rab bit complement at 37 °C for 30 min to eliminate the recipient cells carrying H-2b antigens. After washing, 1.25 X 107 viable spleen cells were add ed to 5 X 10s MCA tumor cells of C57BL/6 ori gin and the mixtures were subcutaneously injected into AKR mice exposed 500 rad of whole body ir radiation. Tumor growth was measured 10 days after the injection and tumor size was recorded as the area (mm2) of the largest diameter times the smallest diameter. Counting of macrophages in the spleens of re cipients. Spleen cell suspension was incubated with lymphocyte separator reagent (Technicon Instru ments Corp., N.Y.) for 20 min and smears of these cells were stained with Giemsa’s solution. Cells engulfing iron particles were counted as ma crophages under a microscope.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 6:15:10 AM
Miyazaki/Nomoto/Kuroiwa/Goya/Takeya
59
Depressed Cytotoxicity in GVH Reaction
Results Effects of a Pretreatment of Donors with Parental Antigens in FCA Donor cells including 1.5 X 107 lymph oid cells and 1.0 X 107 bone marrow cells were injected intravenously into adult FI recipients. Splenomegaly and cytotoxicity were observed 5 and 10 days after the cell transfer. When donor cells were obtained from normal AKR mice, splenomegaly was detected at 5 and 10 days and cytotoxicity was detected at 10 days (table I). Cytotoxic ity was abolished by a treatment of FI cells with anti-0 serum and complement. When donor cells were obtained 3 or 6 h after the treatment with C57BL/6 lymphoid cells in FCA, the generation of cytotoxicity was fa cilitated but splenomegaly was suppressed. When donor cells were obtained 12 h after such a treatment or later, cytotoxicity was
not raised, but splenomegaly was scarcely affected or slightly augmented. When AKR donors were pretreated with AKR cells in FCA for 24 h, the generation of cytotoxicity in F I recipients was not suppressed 10 days after cell transfusion (table II). Lethal Effect of GVH Reaction in Irradiated Neonatal Recipients F I recipients of 1-5 days of age were ex posed to 400 rad and injected intraperitoneally with 1 X 107 donor lymphoid cells within 3 h. The number of FI recipients in each group was 20-25 animals. Survival rates of recipients were observed for 50 days. A few of the irradiated mice died within 7 days if they were not given donor cells (fig. 1). When irradiated FI recipients were injected with normal AKR cells, 80% of them died within 35 days but the remain ing 20% survived beyond 50 days. When
Table I. Augmentation and suppression of cytotoxicity and splenomegaly in FI recipients by a treatment of donors with antigen in FCA at various times before cell transfer
3 6 12 24 72 Normal AKR
Spleen index 5 daysi
1.13 1.17 ± 1.47 ± 1.61 ± 1.59 ± 1.42 ±
0.122 0.15 0.26 0.28 0.29 0.27
Tumor growth in neutralization tests io days
1.15 _L0.14 1.14 ± 0.13 1.32 ± 0.18 1.47 ± 0.25 1.60 ± 0.31 1.49 ± 0.25
in the presence of spleen cells in FI recipients 5 days
10 days
12.2 X 6.1 ( I )3* 17.4 ± 5.2(+)* 38.8 ± 12.5( —) 45.5 ± 13.5 ( ) 39.2 ± 10.8 ( ) 38.4 ± 26.6 ( —)
14.5 X 6.5 ( + ‘) 15.9 ± 5.3 ( + )* 42.5 ± 13.9 ( —) 38.1 ± 11.5(—) 45.8 ± 12.0 ( ) 12.0 ± 5.1( + )*
controls inoculated with tumor cell alone 38.0 X 10.2 30.0 ± 6.9 36.5 ± 8.9 43.9 ± 12.6 42.0 ± 15.5 42.0 ± 22.1
1 Assays of splenomegaly and cytotoxicity were carried out 5 or 10 days after cell transfer. 2 Average value of 5-10 animals ± SE. 3 Average value of 5-10 animals ± SE. Plus or minus in the parentheses indicates the presence or absence of cytotoxicity. Growth of indicator tumors was expressed as areas of the largest diameter times the smallest diameter (mm2). *p
