Induction of mixed allogeneic chimerism for leukemia

Leukemia

Pergamon

Research Vol. 21, No. IO, pp. 907-909, 1997. Q 1997 Elsewer Science Ltd. All rights reserved tinted in Great Britam 0145S2126/97 $17.00 + 0.00

PII: SO145-2126(97)00011*

INDUCTION

OF MIXED

ALLOGENEIC

CHIMERISM

FOR LEUKEMIA

Victor I. Seledtsov, Galina V. Seledtsova, Elias V. Avdeev, Denis M. Samarin and Vladimir A. Kozlov Department of Experimental Immunology, Institute of Clinical Immunology, 630099 Novosibirsk, Russia (Received 9 December 1996.Accepted 28 December 1996) Abstract-Hybrid (C56BL/6xDBA) (BDFI; H-2b/H-2d) mice bearing the P815 leukemia (H-2d) were grafted with a (CBAxC57BlJ6)Fl (CBFI; H-2k/H-2b) cell suspension, comprising bone marrow cells (BMC; 25 x 106/mouse) and spleen cells (SC; 55 x 106/mouse) on day -4, then treated with cyclophosphamide (200 mg/kg) on day -2 and finally grafted once more with CBFI cells (25 x IO6 BMC+7 x IO6 SC) on day 0. Allogeneic cell graftings performed in this way induced durable mixed hematopoietic chimerism and significantly prolonged the survival of recipients, compared with that of leukemia-bearers grafted with syngeneic cells. The results obtained raise the possibility of using allogeneic hematopoietic tissue transplantation in combination with non-lethal cytoreductive therapy to induce a long-lasting graft-vs-leukemia effect. c 1997 Elsevier Science Ltd

Key words: graft-vs-leukemia

effect, mixed

allogeneic

hematopoietic

chimerism.

lympho-hematopoiesis in a leukemia-bearer might be clinically useful to suppress leukemia development. The data presented here indicate that the mixed chimerism induced in BDFl mice bearing P81.5 leukemia by a combination of the CBFl (bone marrow+spleen) cell graftings with cyclophosphamide (CP) treatment may be associated with a significant GVL effect. Importantly, the approach outlined in this study does not include total myeloablation of a recipient before cell transplantations, thereby, predicting rapid formation of GVHD-controlling barrier from surviving host hematopoietic cells.

Introduction It is firmly established that allogeneic marrow recipients have a lower relapse rate for leukemia compared with autologous marrow recipients. Allogeneic donor T cells have been found to be responsible for most of the immune-mediated antileukemia effects associated with transplantation. On the other hand, donor T-lymphocytes are also capable of causing graft-vs-host disease (GVHD). Fatal complications of GVHD limit the survival of the allogeneic marrow recipients and diminish the overall benefit of the graft-vs-leukemia (GVL) effect (reviewed in Ref. [l]). An improved survival might be achieved by using approaches that would provide GVL effect, while controlling GVHD. One such approach has been previously reported in a murine model [2] based on the infusion of T-celldepleted syngeneic marrow prior to allogeneic bone marrow transplantation in lethally irradiated mice. In this case, the GVL effect is mediated by the allogeneic donor cells, while GVHD is controlled by syngeneic cells identified, at least in part, as natural suppressor cells [3]. In the light of these results it seems reasonable to think that the induction of durable mixed (allo+auto)

Materials and Methods Animals (C57BL/6xDBA)Fl (BDFI; H-2b/H-2d) and (C57BL/6xCBA)Fl (CBFl; H-2b/H-2k) mice were bred in our own facilities. Both males and females of ages ranging between 4-7 months were used. Autoclaved food and HCl-acidified (pH 2.8) drinking water with antibiotics were provided. Tumor cell line P815 mastocytoma cells of DBA (H-2d) origin were obtained from Moscow Oncologic Scientific Center of the Russian Academy of Medical Sciences and maintained in BDFl mice in ascites form.

Abbreviations: BMC, bone marrow cells; CP, cyclophosphamide; GVHD, graft-vs-host disease;GVL, graft-vs-leukemia; MHC, major histocompatibility complex; SC, spleen cells. Correspondence to: Victor I. Seledtsov, Department of Experimental Immunology, Institute of Clinical Immunology, 630091, Novosibirsk, Russia.

Cell transplantation procedures, survival registration and chimerism analysis BDFl male mice were injected with 7 x lo5 P815 907

908

V. I. Seledtsov et al.

cells intraveneously (i.v.) on day -6, grafted with a CBFl cell suspension, comprising bone marrow cells (BMC; 25 x 106/mouse) and spleen cells (SC; 55 x 106/ mouse), i.v. on day -4, treated with 200 mg/kg CP (Biochimic, Saransk, Russia) on day -2 and grafted once more with CBFl cells (25 x lo6 BMC plus 7 x lo6 SC) on day 0. The control mice bearing the P8 15 leukemia underwent CP treatment alone or in combination with syngeneic cell transplantations. Control for GVHD included the mice that did not receive an injection of P815 cells prior to the cell transplantation procedures and CP treatment. Each group consisted of 10-14 mice. Mice were observed daily for survival and dead animals were autopsied. Evidence of tumors was grossly detected with massively enlarged spleens and livers. When necessary, microscopic observation of sections from spleens and livers was performed to confirm the presence or absence of tumors. Mice with significantly decreased body weight and other apparent signs of GVHD development were killed. The percentage of donor cells in bone marrow were assessed by a cytotoxicity assay using allospecific mouse antisera and Low-Tox-M rabbit complement (Cedarlane) in the standard way. BDFl anti-CBA and CBFl anti-DBA sera, reacting with donor and host cells, respectively, was prepared by weekly alloimmunizations of mice as described previously [4]. Levels of donor chimerism were calculated as: % donor chimerism = (donor cells/host + donor cells)x 100. Statistical analysis The statistical significance of the data was determined using the Mann-Whitney U-test. A P-value of <0.05 was considered to be statistically significant. Results In the initial experiments for inducing mixed (BDFl+CBFl) chimerism in P815 leukemia-bearing BDFl mice we used a tolerance induced system, described previously in adult mice [5,6], that comprises an i.v. injection of 5 x lo7 allogeneic SC plus 3 X lo7 allogeneic BMC followed, 2 days later, by an intraperitoneal (i.p.) administration of 200 mg/kg of CP. As indicated before, this approach readily allows the induction of a long-lasting allograft tolerance in a tolerogenspecific manner in a variety of strain combinations [6]. The major mechanism for the induction of such tolerance has been demonstrated [5] to be the stimulation of effector T cells by alloantigens followed by the destruction of these cells by CP treatment. The data (not shown here) indicate that a single injection of CBFl cells into P815 leukemia-bearing BDFl mice followed by CP treatment of recipients failed to significantly prolong the survival of the recipients. These results

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Grafted with CBFl cells

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10

15

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CP control

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Postgrafting days Fig. 1. Prolongation of survival of P815 leukemia-bearing BDFl mice by CBFl cell graftings. BDFl mice were injected with 7 x lo5 P81.5 cells on day -6, grafted with CBFl cells (25 x lo6 BMC+55 x lo6 SC) on day -4, given 200 mg/kg CP on day -2, and then grafted repeatedly with CBFl cells (25 x lo6 BMC+7 x lo6 SC) on day 0. In control, BDFl mice bearing P815 leukemia underwent CP treatment alone or in combination with syngeneic cell graftings. One experiment out of two with similar pattern is shown.

prompted us to change the transplant schedule so as to elevate the level of donor lympho-hematopoiesis responsible for GVL effect in the leukemia-bearers. The changed schedule included an additional transplantation (performed 2 days after CP treatment) of CBFl cells (25 x lo7 BMC plus 7 x lo7 SC) into BDFl mice bearing P815 leukemia. As demonstrated in Fig. 1, the combination of two CBFl cell graftings with CP treatment prolonged significantly the survival of the recipients. As expected, syngeneic cell graftings performed in the same way failed to generate a GVL effect. It should be also noted that in our experimental system, when performed alone, CP treatment lacked any significant effect on the survival of the leukemia-bearers (data not shown). A cytotoxic test at 100 days postgrafting using allospecific sera and complement (see Table 1) demonstrated that 7-20% of BMC in CBFl cell transplant recipients of the control group (not bearing leukemia) were of donor origin. Thus, the findings presented here support the clinical potential of inducing mixed hematopoietic chimerism for acute leukemia.

Discussion The existing data indicate that: (1)

successful engraftment of an allogeneic hematopoietic transplant may be realized against the background of functioning host hematopoiesis [71;

909

GVL effect in a mixed allogeneic chimera

Table 1. The levels of donor chimerism in BDFl mice subjected to CBFI cell graftings in combination with CP treatment % Donor chimerism

Mean % donor chimerism + SD.

1 2 3 4

10 7 12

13+5

5

20 15

Number of mouse

15

6

BDFI mice were grafted i.v. with CBFl cells (25 x IO6 BMC+55 x IO6 SC) on day -4, given i.p. 200 mg/kg CP on day -2 and then grafted once more with CBFl cells (25 x IO6 BMC+7 x lo6 SC) on day 0. Chimerism analysis was performed on day 100.

(2)

mixed allogeneic chimerism may associate with a lessened risk of GVHD development [2, 81; and

(3)

the hematopoietic allograft present in mixed chimera may reveal a significant GVL activity ]3,81.

The above-mentioned findings have prompted us to develop an approach to generating a GVL effect. which would avoid total myeloablation of the host and the complications associated with a failure of host hematopoiesis. The results presented here indicate that a significant GVL effect may be achieved by using allogeneic hematopoietic tissue grafts in combination with a submyeloablative cytotoxic dose of CP. The donor-host (CBFl-BDFl) strain combination used in the present study modeled GVL effects in the situation that is significantly similar in genetic characteristics with those occurring in recipients of related major histocompatibility complex (MHC)-incompatible lympho-hematopoietic transplants. In our opinion, this type of transplantation might be the most available and applicable to clinical practice in generating GVL activity by means of inducing mixed hematopoietic chimerism. It should be noted, however, that in our experimental system, sharing MHC-antigens between donor and host was unlikely to play a crucial role in generating a GVL effect. Indeed, we detected a significant GVL effect in P8 15 leukemia-bearing DBA mice, which underwent C57BL6 cell grafts in the above-described way. On the other hand, in parallel experiments, both CBA (H-2k) and BALB/c (H-2d) cell transplants failed to induce a significant anti-P815 leukemia effect in DBA hosts, thereby suggesting that choice of donor may be

extremely important for generating GVL activity in mixed chimeras. Allogeneic donor T cells are firmly established to be responsible for most of the immune-mediated antileukemia effects associated with transplantation [l]. This prompts investigations to seek the boundary between beneficial GVL effects of T lymphocytes, on one hand, and their deleterious activity leading to GVHD, on the other. Murine bone marrow is known to contain low numbers of T-lymphocytes. Therefore, in the model used, spleen was the dominant source for donor Tlymphocytes. Importantly, >70% of control BDFl mice (not injected with P815 cells) subjected to CBFl cell graftings in combination with CP treatment survived during the whole of the observation period (5 months) without revealing any visible signs of GVHD (data not shown). These results, therefore, support the existing view [3, 81 that host hematopoiesis is able to control effectively GVHD development by preventing expansion of donor lymphopoiesis in the body. References 1. Butturini, A. and Gale, R. P., Graft versus leukemia. Immunology Research, 1992, 11, 24. 2. Sykes, M., Bukhary, Z. and Sachs, D. H., Graft-versusleukemia effect using mixed allogeneic bone marrow transplantation. Bone Marrow Transplant, 1989, 4, 465. 3. Sykes, M., Eisenthal, A. and Sachs, D. H., Mechanism of protection from graft-vs-host disease in murine mixed allogeneic chimeras. I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component. Journal of Immunology, 1988, 140, 2903. 4. Argyris, B. F. and Waltenbaugh, C., Role of I-J in neonatal suppression. Cellular Immunology, 1983, 80, 267. 5. Tomita, Y., Ayukawa, K., Yoshikai, Y. and Nomoto, K., Mechanisms of cyclophosphamide-induced tolerance to IE-encoded alloantigens: evidence of clonal deletion in effector cells for skin allograft rejection. Transplantation, 1992, 53, 602. 6. Tomita, Y. and Nomoto, K., Comparison of tolerance inducibility to Class I or Class II antigens between cyclophosphamide (CP)-induced tolerance and transfusion with donor cells: general effectiveness of CP-induced tolerance and difference of skin graft prolongation in each Class I antigen-disparate combination. Immunobiology, 1992, 186, 282. 7. Wu, D. D. and Keating, A., Hematopoietic stem-cells engraft in untreated transplant recipients. Experimental Hematology, 1993, 21, 25 1. 8. Bertheas, M. F., Lafage, M., Levy, P., Blaise, D., Stoppa, A. M., Viens, P., Mannoni, P. and Maraninchi, D., Influence of mixed chimerism on the results of allogeneic bone marrow transplantation for leukemia. Blood, 1991, 78, 3103.