Recipients Of Spleen Cells Research Articles

Reduction of lethal graft-versus-host disease: transplantation of cultured murine bone marrow across minor histocompatibility differences

The murine bone marrow culture technique was used to prepare donor marrow for bone marrow transplantation across minor histocompatibility complex differences. Previous studies have shown that theta-positive cells are rapidly lost from such cultures and that transplantation of cultured marrow across major histocompatibility complex differences results in a delay in the development of lethal graft-v-host disease (GVHD). In this study, a total of 1 to 2 X 10(7) nonadherent cells (740 to 1560 CFUs [colony-forming units]) from three-day-old cultures were used as a source of donor marrow. Three strain combinations were evaluated; LP/J into C57BL/6; BIO.BR into CBA/J; and C57BL/6 into LP/J. Donor mice were immunized with recipient spleen cells prior to culture in order to increase the graft-v-host response. For LP/J marrow into C57BL/6 mice, 5 X 10(7) donor spleen cells transplanted along with the marrow were needed to induce lethal GVHD. However, lethal GVHD was seen without the addition of spleen cells for BIO.BR into CBA/J and C57BL/6 into LP/J strain combinations. Most animals receiving fresh marrow were dead of GVHD five weeks after transplantation. With the use of cultured marrow the three-month survival was 80%, 51%, and 93%, respectively, for LP/J into C57BL/6, BIO.BR into CBA/J, and C57BL/6 into LP/J strain combinations. Long-term donor engraftment in all recipient animals receiving cultured marrow was confirmed by analyzing hemoglobin polymorphisms between the strain combinations. These results demonstrate that in contrast to transplantation across major histocompatibility complex differences, the use of cultured cells for bone marrow transplantation across minor histocompatibility complex differences allows for engraftment while reducing the risk of lethal GVHD.

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. I. Impairment of mitogen responses and suppressor phenomena.

In our laboratory, we have developed a murine model to examine GVHD across minor histocompatibility antigens. In our model, GVHD is induced by injecting B10.D2 spleen cells into irradiated BALB/c recipients. Seven to 10 days after irradiation and injection of cells, there are significant changes in cell function in the recipient spleens. In the B10.D2----BALB/c (600 rad) model, recipient spleen cells are profoundly unresponsive to Con A and LPS stimulation but show increased B cell activity measured by Staphylococcus aureus protein A plaque-forming activity. Spleen cells from such GVH mice profoundly suppress the mitogenic responses of normal BALB/c or B10.D2 spleen cells to Con A and LPS. The degree of impairment of the mitogenic response and the ability to suppress normal cells is proportional to the dose of cells used to induce GVH reactions. Both the inability to respond to mitogens and the capacity to suppress are also related to the dose of irradiation given to the recipients. In addition, immunosuppression across minor histocompatibility antigens shows an unevenhandedness. If we inject parental B10.D2 or BALB/c cells into F1 recipients (P----F1), there is greater inhibition of mitogenic responses when B10.D2 parental cells are given than when BALB/c cells are given to the irradiated F1 recipients. These experiments show that significant immunosuppression occurs during GVH reactions across minor histocompatibility barriers. The degree of suppression varies according to the dose of cells used to induce GVH, the dose of irradiation to the recipient and the "strength" of the GVH recognition system. Such experiments provide models for GVH disease seen in humans who receive treatment for leukemia or other diseases that involves recipient irradiation and infusion of HLA-identical bone marrow.

Helper effects required during in vivo priming for a cytolytic response to the H-Y antigen in nonresponder mice.

Induction of H-Y-specific cytotoxic T lymphocyte (CTL) responses in nonresponder female mice was attempted by i.v. injection of allogeneic male cells, followed by in vitro restimulation of recipient spleen cells with syngeneic male cells. Responses were obtained only in two strain combinations in which the recipients, although phenotypically nonresponders, carried responder alleles at class I major histocompatibility complex (MHC) loci, and the immunizing cells differed from the recipients at class II MHC loci. The two positive strain combinations were B10.A(2R) anti-B10.A(4R), and B10.GD anti-B10.D2(R101). In the first combination, both recipient and donor are nonresponders to H-Y, and the CTL are induced via a bystander effect of another CTL response to a previously undetected minor histocompatibility (H) antigen. This "carrier" antigen can only induce CTL against H-Y and itself when the immunizing cells express class II MHC molecules. Furthermore, the presence of H-Y and the carrier antigen on the same cell is a prerequisite for the generation of H-Y-specific CTL. In the second combination, the recipient is a nonresponder, whereas the donor is a responder. The two strains differ at only E alpha and E beta class II MHC loci. For the induction of CTL, H-Y and the foreign E molecule must be expressed on the same cells. Thus, the B10.D2(R101) cells that express E molecules on their surface probably provide the E-nonexpressor B10.GD recipients with a stimulus for the generation of H-Y-specific T helper cells. The data are consistent with the notion that antigen-specific class II MHC-restricted T helper cells are involved in the initiation of CTL responses to minor H antigens.

Cellular immunity to heterotopic corneal allografts in the rat.

Although the immune nature of corneal allograft rejection has been recognized for over thirty years, the specific mechanisms involved in such reactions remain obscure. We investigated the cellular immune responses of PVG (RT1c) rats that were grafted with fully allogeneic ACI (RT1a) skin, ACI cornea, or PVG cornea to the chest wall or given sham grafts. Cell-mediated lymphocytotoxicity (CML) was tested at 10 days posttransplant by placing recipient spleen cells in culture with irradiated ACI stimulator cells, and six days later measuring specific lysis of 51Cr-labeled target lymphoblasts at several effector-to-target ratios. Effector cells from animals receiving allogeneic skin or cornea grafts lysed targets from the donor (ACI) strain at levels significantly (P less than 0.01) above those obtained using effectors from control (sham grafted or syngeneic corneal graft recipient) animals. Significant lysis was also seen using target cells from PVG.1A (RT1a) or PVG.R1 (RT1r1) congenic rats, which differ from recipients only at the RT1 complex and the RT1.A (class I antigen) region, respectively. Stimulator cells from PVG.1A and PVG.R1 animals also permitted detection of specific responses in secondary CML, but syngeneic PVG stimulators did not, indicating that in vitro restimulation of effector cells can be met by using stimulator cells bearing only allogeneic class I major histocompatibility complex (MHC) antigens. These results indicate that corneal allografts evoke specific cellular immune responses in the rat, and that class I MHC antigens act as effective targets for these responses.

Graft-versus-host disease in murine bone marrow transplantation. I. Modification of GVHD by preimmunization of recipients with spleen cells of primary recipients undergoing GVHD

Lethally irradiated (1000 rad) CBA/J mice were transplanted with anti-Thy 1 treated BALB/c bone marrow. Under these conditions, we uniformly observed the development of pathology suggestive of acute graft-vs.-host disease (GVHD), i.e. weight loss, diarrhoea, hypogammaglobulinemia and thymic hypoplasia. If, 2 wk before irradiation, the recipients were preimmunized with spleen cells taken from mice undergoing acute GVHD, these symptoms were avoided. Instead, such animals seemed to show long term survival either with or without signs of chronic GVHD (hypergammaglobulinemia, splenomegaly, lymphoid hyperplasia). The ability to show long term survival with bone marrow allografts was dependent upon successful immunization of the recipient mice. Long term survivors contain a splenic population not bearing detectable host MHC antigens, which can elicit a memory anti-host cytotoxic response from a population of quiescent donor lymphocytes previously immunized in vitro against host MHC antigens.

Tumor-specific immunity induced by somatic hybrids: IV. Relationship between immunogenicity and expression of surface tumor-associated antigens

Semi-allogeneic hybrid clones were derived by fusion of the TEPC-15 plasmacytoma (H-2 d) and mouse L cells of C3H (H-2 k) origin. Three representative clones were chosen to study the relationship between the expression of different membrane antigens and their immunogenicities leading to protection of recipient mice from the parent TEPC-15 plasmacytoma. The level of surface tumor-specific transplantation antigens (TSTA) was measured by radioimmunoprecipitation with syngeneic anti-TSTA and by inhibition of anti-TSTA binding to the TEPC-15 tumor cells. The most immunogenic hybrid clone (LTC-1) expressed the highest level of TSTA and the weakly immunogenic (LTC-2) and nonimmunogenic (LTC-4) hybrid clones exhibited relatively low levels of TSTA on the surface. Moreover, the strongly immunogenic LTC-1 hybrid cells, but not the parent tumor cells, were effective in priming recipient spleen cells to generate TEPC-15 tumor-specific cytotoxic cells upon subsequent in vitro exposure to the TSTA-bearing cells. Therefore, the level of TSTA on the semi-allogeneic hybrid clones may play an important role in enhancing the immunogenicity of TSTA.

Graft-Versus-Host Reactions in F 1 Hybrid Mice: MHC-Restriction-Independent Generalized Depression of Virus-Specific Cytotoxic T Cell Response

A graft-versus-host reaction (GVHR) was induced in F 1 hybrid mice by intravenous injection of parental spleen cells. A possible effect generated by recipient F 1 mice of a GVHR on the restriction specificities of anti-viral cytotoxic T cells was investigated. (A/J × C57BL/6) F 1 recipients were injected with 1 × 10 8 or 2 × 10 7 spleen cells from either parent. Zero to two weeks later during an acute GVHR or 12 to 26 weeks later during a chronic GVHR, spleen cells from these F1 recipients were assayed for their capacity to generate vaccinia (or lymphocytic choriomeningitis) virus-specific cytotoxic T cells. No effect was seen when parental cells were injected with virus on the same day. During the acute phase of the GVHR, recipients of spleen cells of both doses or from either parent injected 7 or 14 days previously generated markedly fewer cytotoxic T cells with respect to both parental restriction specificities. In the more chronic situation only, recipients of 1 × 10 8 parental spleen cells showed depressed virus-specific cytotoxic responses, again both restriction specificities were affected comparably. Therefore, a GVHR depresses generation of virus-specific cytotoxic T cells in a dose- andtime-dependent way, but does not measurably disturb the balance of parental 1 versus parental 2 restriction specificity in an F 1 recipient.

Cellular immune response to sheep erythrocytes interrelationship between proliferation of popliteal lymph node cells and footpad swelling

Mice were sensitized with graded doses of sheep erythrocytes by the intravenous or subcutaneous route and challenged for delayed-type hypersensitivity (DTH) at different times thereafter. The DTH response as assessed by footpad swelling (FPS) was compared to the spontaneous proliferative response of the popliteal lymph node cells (PLNC). Proliferation of PLNC was optimal after sensitization regimens resulting in optimal FPS. The same was true for mice sensitized under cyclophosphamide modulation. Proliferation of PLNC induced by SRBC was antigen-specific, although some crossreactivity with horse red blood cells was observed. Proliferation of PLNC could be abrogated by treatment with anti-Thy-1.2 antiserum plus complement demonstrating the T cell nature of proliferating cells. In accordance with published data, FPS of mice presensitized with a high dose of SRBC as well as FPS of recipients of spleen cells from high-dose-sensitized donors was suppressed. In marked contrast, PLNC proliferation was not diminished in these mice. Although proliferation of PLNC did not parallel FPS under all circumstances, it seems to be a correlate of the cellular immune response to SRBC.

Skin allografts generate an enhanced production of histamine and histamine-producing cell-stimulating factor (HCSF) by spleen cells in response to T cell mitogens.

In response to T cell mitogens, spleen cells produce a large amount of histamine, whereas no or a slight increase is observed after B cell mitogen stimulation. This increased histamine production results from the effect of a factor having all the characteristics of HCSF (histamine-producing cell-stimulating factor) already described in secondary MLC supernatant. This factor is produced by Thy-1, 2, Lyt-1, 2-positive cells. Spleen cell cultures derived from skin-allografted mice during rejection produce more histamine in response to T cell mitogens than do spleen cells from normal or syngeneic grafted mice. Such a phenomenon is not observed in response to B cell mitogens. A striking association is found between enhanced histamine synthesis and skin allograft rejection. This phenomenon results from a) a five to 10-fold increase in HCSF production by allograft recipient spleen cells in response to T cell mitogens, and b) an increase in HCSF sensitivity of these spleen cells.

Evidence for a lymphokine enhancing arginase activity during allograft rejection.

The production of urea and ornithine is increased greatly in spleen cell cultures of an allograft recipient in the presence of donor cells (secondary MLC) in comparison to that of primary MLC (without previous allograft). This phenomenon appears after 24 hr of culture and reaches its maximum at 48 hr. The greatest increase in urea production is observed when the recipient spleen cells are collected at the time of allograft rejection. To obtain this extra production of urea, the stimulating cells in MLC should specifically be of the donor type or at least bear one homology with donor cells at the K or D locus. The increased production of urea and ornithine during MLC results from the action of a lymphokine released by recipient cells in the presence of donor cells. This factor acts upon cells present in bone marrow, spleen, and elicited peritoneal cells but is absent or is present in smaller quantities in thymus and lymph node cells. Target cells of this factor possess numerous macrophage features and could be immature cells of the macrophage line. The lymphokine responsible for this phenomenon is heat-stable, destroyed by trypsin, chymotrypsin, and neuraminidase, and has a m.w. around 32,000. It acts upon its target cells by increasing arginase activity, which results in the production of a large amount of ornithine, an important precursor of polyamine biosynthesis.

Effect of fractional cyclophosphamide dosage on sheep red blood cell-delayed-type hypersensitivity response in mice.

The CY-enhancing effect on DTH response of mice against SRBC was studied by administering to sensitized animals graded amounts of drug at various times during the immune response. The use of a staggered schedule for CY administration made it clear that this enhancing effect could be augmented even further by lowering the standard 200 mg/kg CY dose. Animals immunized on day 0 with 1 X 10(8) SRBC receiving 50 mg/kg doses on days -1, +1, and +4 showed higher DTH responses on day +7 than those similarly sensitized 1 day after the administration of 200 mg/kg body weight. In addition, we wanted to demonstrate that the TDTH effector cell is sensitive to CY in vivo, because a single 50 mg/kg dose inoculated on day +6 can lower by 50% an already established DTH response. This effect was not due to an effect of CY treatment on bone marrow-derived cells recruited to DTH responses; inhibition of DTH responses were transferred with spleen cells of CY-treated recipients. The action of CY is not dose-dependent; the administration on day +6 of a single dose of 200 mg/kg results in no further depression of the DTH reaction. We conclude that CY affects not only T supp cells, but also cell type(s) involved in the cell-mediated response of mice against SRBC, and that the DTH-enhancing effect of the drug is a blend of its action upon all these cells.

Demonstration of a concanavalin A-induced lymphokine enhancing arginase activity. Effect of presensitization by a skin allograft.

In response to concanavalin A (Con A) mouse spleen cells produce increased amounts of urea. This increase results from arginase activity degrading arginine into urea and ornithine. It is mediated by a soluble protein factor, characterized by its heat stability (10 min at 80 degrees C) and a molecular weight of approximately 32 000. The factor is produced by T cells and acts on a cell population mainly present in bone marrow and spleen. Splenocytes collected from skin allograft recipients during rejection produce more urea in response to Con A than those obtained from normal or syngeneically grafted mice. The maximum urea increase is observed just before and maintained during rejection, declining rapidly thereafter. This phenomenon is explained (a) by a 10-15-fold increase in arginase-enhancing factor production by Con A-stimulated allograft recipient spleen cells and (b) by an increased responsiveness of these splenocytes to the lymphokine.

Adoptive transfer of resistance to Plasmodium berghei with spleen cells and serum from Fansidar-cured mice.

The ability of splenic leukocytes or serum to transfer immunity to Plasmodium berghei was studied in C57BL/6 mice. Splenic leukocytes or serum was removed from mice which had been inoculated previously 1 to 4 or 5 times with P. berghei and cured 1 to 4 or 5 times with Fansidar (pyrimethamine plus sulfadoxine) and transferred to syngeneic recipients 1 or 2 days before parasite challenge. Partial protection was observed in recipients of immune serum or unfractionated splenic leukocytes. Significantly more protection was observed in recipients of preparations from immune mice enriched for immunoglobulin-positive (B-lymphocyte) cells or B-lymphocyte plus immunoglobulin-negative (T-lymphocyte) cells than in recipients of preparations enriched for T-lymphocytes. Recipients of B- or B+T-lymphocyte-enriched spleen cells from immune mice had significantly longer survival times than did recipients of T-lymphocyte-enriched spleen cells and recipients of spleen cells from normal mice. Transferred immunity lasted less than 2 months and did not protect recipients against death induced by the malarial parasite. The ability of splenic leukocytes to transfer resistance to recipients was independent of the number of times donors were inoculated with P. berghei and cured with Fansidar.

Induction of IgG memory responses with polyvinylpyrrolidone (PVP) is antigen dose dependent.

Irradiated recipients of spleen cells from mice primed with a very low dose (0.0025 microgram) of the thymus-independent (TI) antigen polyvinylpyrrolidone (PVP) produced PVP-specific IgG memory responses after secondary challenge with a T-dependent (TD) form of PVP, PVP-HRBC. The IgG memory responses induced by low doses of PVP were similar in magnitude to those induced by the TD antigen PVP-HRBC. The induction of IgG memory by the TI form of antigen was markedly dependent on the dose of PVP used to prime donor mice. Spleen cells from mice primed with an amount of PVP (0.25 microgram) that induces an optimal primary IgM response did not produce significant IgG antibody after challenge with PVP-HRBC. The inability of higher doses of PVP to induce IgG memory may be due, at least in part, to the fact that such doses of PVP were found to induce tolerance in PVP-specific B cells and could suppress the induction of memory induced by PVP-HRBC. Low doses of PVP did not interfere with the induction of memory by PVP-HRBC. Expression of IgG memory responses in recipients of PVP-HRBC or low-dose PVP-primed cells was found to be T cell dependent. Moreover, only primed T cells could reconstitute the response of recipients of primed B cells, suggesting that the ability of PVP to induce IgG memory may be related to its ability to prime T helper cells. Expression of the IgG memory response in recipient mice also required the use of a TD antigen for secondary challenge, i.e., mice challenged with PVP did not develop IgG. The inability of PVP to elicit IgG antibody was due in part to the fact that 0.25 microgram PVP was tolerogenic, i.e., it could interfere with the expression of memory in mice simultaneously challenged with both PVP and PVP-HRBC.

Histamine production during the anti-allograft response. Demonstration of a new lymphokine enhancing histamine synthesis.

Histamine production is greatly increased during culture of allograft recipient spleen cells in the presence of immunizing cells (secondary mixed leukocyte cultures [MLC]) as compared to that found in primary MLC (i.e., without previous allograft). This phenomenon appears after 24 h of culture and reaches its maximum at 48 h. Optimal increased histamine production is observed when MLC is performed with spleen cells removed from mice during rejection. This increased production of histamine during secondary MLC results from the action of a lymphokine: the histamine-producing cell stimulating factor (HCSF). This factor is released by T lymphocytes. Its production requires specific stimulation of the recipient lymphocytes because increase in histamine production during secondary MLC can be only observed when recipient cells are cultured with stimulating cells bearing at least one homology at K or D loci with immunizing cells. HCSF acts on a cell which is present in bone marrow, spleen, blood, and peritoneal cells but absent in thymus or lymph node cells. This target cell is found in the less-dense layer of a discontinuous Ficoll-gradient of bone marrow cells. HCSF is heat stable, destroyed by trypsin treatment, and has a molecular weight between 50,000 and 100,000. It acts on its target cells by increasing histidine decarboxylase activity.

Prostaglandin (PG) release in the mixed lymphocyte culture; effect of presensitization by a skin allograft; nature of the PG-producing cell.

AbstractA release of prostaglandin (PG) has been demonstrated in lymphocyte culture supernatants. The amount of PG produced is strikingly increased in mixed cultures between allograft donor and recipient. This phenomenon is not yet detectable after 6 h of culture, appears at 24 h and reaches its maximum at 48 h. Little or no increase in PG production is found in the supernatant of primary mixed lymphocyte cultures (i.e. without previous allograft) at least in the first two days of culture. The difference between allografted and control animals begins at day 6 after grafting, reaches a maximum at day 12, remains high until day 20 and decreases thereafter.This increased amount of PG in mixed cultures between donor and recipient spleen cells is probably, at least to a large extent, produced by macrophages. Evidence for this interpretation includes the following observations: (a) cells responsible for the increase are adherent cells and (b) the phenomenon is no longer found following a treatment of spleen cells with silica.When mouse peritoneal macrophages (or adherent spleen cells) are incubated in vitro with supernatants of mixed cultures between allograft donor and recipient, their PG production is considerably increased. Hence, it is suggested that the above‐described phenomenon could result from the stimulation of macrophages by lymphokines released in mixed cultures following allografts.If such a release of PG takes place within the graft, it may play a regulatory role in the control of the magnitude of the anti‐allograft immune response.

Failure of allogeneic thymocytes to survive in nude mice.

Athymic nude mice or thymectomized mice, irradiated and reconstituted with T-depleted bone marrow cells ("B mice"), were injected with allogeneic or syngeneic thymocytes bearing caryotypically distinct chromosomes. The fate of the thymocytes was investigated after various periods of time using two methods: (a) frequency of the cells bearing the Thy-1 antigen as detected by immunofluorescence with a heteroantiserum among the recipient spleen cells, (b) presence of chromosomally detectable donor cells in phytohemagglutinin-stimulated spleen cell cultures. These two methods indicate that allogeneic thymocytes disappear after about 7 days, semiallogeneic thymocytes after about 20 days, while syngeneic thymocytes, even when injected in 50 times lower number (10(6) cells), are detectable for months, thanks to the sensitivity of the caryotypic method. Allogeneic thymocytes induce the production of high titers of alloantibodies, which were shown to react specifically against their own H-2, a phenomenon interpreted as a "suicidal" allogeneic collaboration. These experiments demonstrate that the failure of allogeneic thymocytes, in contrast to syngeneic thymocytes, to achieve long-term restoration of the immune responsiveness of T-depleted mice is due to a rejection of the foreign thymocytes, and not to a failure of T and B cells to collaborate across the histocompatibility barrier.

An immunoregulatory factor associated with spleen cells from tumor‐bearing animals. II. Characterization of the cell populations involved in its production and release

AbstractCulture supernatants of spleen cells from tumor‐bearing mice (TBM) have been found to enhance tumor growth, when injected together with tumor cells, and to augment the generation of antibody‐producing cells (APC), when injected into mice together with sheep erythrocytes (SRBC). In an attempt to characterize the cellular entity in the spleen‐cell population which is involved in release of the enhancing factor (EF), “B”, adult‐thymectomized, and nude mice were inoculated with 3LL tumor cells, and their spleen‐cell supernatants were tested. We found that spleen‐cell supernatants from the three different categories of 3LL tumor‐bearing mice were highly effective in augmenting anti‐SRBC APC production and in accelerating tumor growth. In a second line of experiments, T or B lymphocytes were eliminated from spleens of TBM before the cells were incubated in vitro. Culture supernatants of TBM spleen cells adherent to nylon wool colums, which were enriched mainly for B lymphocytes and macrophages, manifested higher EF activity than supernatants of eluted spleen cells which were enriched for T lymphocytes. Supernatants of TBM spleen cells, which were pretreated with anti‐Thy 1,2 serum in the presence of complement before culturing, were found to be highly effective in potentiation of APC generation and in enhancement of tumor growth. These results suggest that EF is not released by lymphoid cells that possess characteristics of mature T lymphocytes. EF was produced in culture by 3LL tumor cells; 90 min after injection of 3LL supernatant, the recipient spleen cells were capable of releasing EF in vitro. Trypsin treatment did not affect EF production by tumor cells but stopped its release from spleen cells of TBM. It therefore appears that EF is produced by the tumor cells and is then adsorbed onto spleen cells.

In vitro immune blastogenesis during contact sensitivity in tumor-bearing mice : II. Mechanism of inhibition

The addition of optimal concentrations of normal or tumor-bearing host (TBH) splenic macrophages to 2,4-dinitro-1,5-difluorobenzene (DN2FB) immune spleen cells augmented the responsiveness of these cells during in vitro antigen stimulation. At supraoptimal in vitro and in vivo concentrations, however, macrophages were highly inhibitory of immune blastogenesis. Suppressor T-cells were detected in the spleens and thymuses of fibrosarcoma-bearing BALB/c mice. These cells produced a soluble factor which impaired in vitro responsiveness to the sensitizing antigen, and did not absorb to macrophages. Both, adoptively transferred, macrophages and suppressor T-cells were capable of suppressing antigen responsiveness of normal immune recipient spleen cells. Suppressor T-cell inhibition was apparently expressed at the molecular level of DNA polymerase enzymes.

Experimental models for prevention of graft-versus-host reaction in bone marrow transfusion. II. Inability to prevent graft-versus-host reaction in an H-2 identical combination.

Splenomegaly was strong in the degree and continued for a long period of time in adult F1 hybrids between AKR (H-2k) and C3H/He (H-2k) mice after transfer of spleen cells from normal C3H/He mice. In spleen cells of such F1 recipients, cytotoxicity was detected by an in vivo neutralization test using methylcholanthrene-induced sarcoma or AKR origin as target cells. All of newborn F1 recipients died within 17 days after cell transfer. Induction of splenomegaly and cytotoxicity was not prevented by repeated pretreatments of donors with sonicated AKR spleen cells in saline, which suppressed completely such phenomena of graft-versus-host reaction in an H-2 nonidentical combination. Induction of cytotoxicity in the spleen of F1 recipients was not prevented by a pretreatment of donors with AKR spleen cells in complete Freund's adjuvant, which suppressed the induction of cytotoxicity in an H-2 nonidentical combination. Graft-versus-host reaction appears to be stronger in a combination between parental strains of which major histocompatibility antigens were identical.