BRIEF COMMUNICATION Increased rejection of murine ... - Nature

Leukemia (1997) 11, 1045–1048  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

BRIEF COMMUNICATION Increased rejection of murine allogeneic bone marrow in presensitized recipients R van Os1,2, T de Witte3, JH Dillingh1, PM Mauch2 and JD Down1 Department of Radiobiology, University of Groningen, Groningen, The Netherlands; 2Joint Center for Radiation Therapy, Harvard Medical School, Boston, MA, USA; and 3Division of Hematology, University of Nijmegen, Nijmegen, The Netherlands

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The role of presensitizing murine recipients with donor spleen cells prior to T cell-depleted or -repleted H-2 compatible allogeneic bone marrow transplantation (BMT) was investigated at two different doses of total body irradiation (TBI). Recipients that were presensitized with 2 3 107 irradiated donor spleen cells at 1 week before a sublethal dose of 6 Gy TBI and BMT showed no evidence of donor blood chimerism while unsensitized recipients showed about 80% donor engraftment as determined by blood Gpi phenotyping. After raising the TBI dose to 9.5 Gy an increase in mortality from marrow failure was observed in presensitized animals. No significant engraftmentpromoting effect of up to 2 3 106 T cells (20% of total marrow dose) was seen either in presensitized or unsensitized mice. It can be concluded that presensitized recipients are more susceptible to acute marrow rejection and that T cells added to the bone marrow did not influence the level of donor engraftment in these recipients. Keywords: presensitization; T cell depletion; allogeneic; bone marrow transplantation; total body irradiation

Graft-versus-host disease (GVHD) still remains a major complication after allogeneic bone marrow transplantation (BMT).1 Methods of depleting T lymphocytes from the donor bone marrow graft have successfully decreased the incidence of GVHD but these are associated with an increasing number of engraftment failures as well as leukemic relapses.1,2 These problems have prompted a number of investigators to study the role of donor T lymphocytes on murine bone marrow engraftment.3,4 The most important factors for engraftment appear to be the number of donor cells in the graft, the extent of host preparation and the degree of genetic difference between donor and recipient. In addition, BMT patients often receive blood transfusions prior to transplantation which may immunize the patient against allogeneic cells. This preimmunization induced a high incidence of bone marrow graft rejection, especially in aplastic anemia patients who are prepared with less intense conditioning regimens.5 The relative role of pre-immunization in T cell-depleted BMT is not known. We have investigated the roles of donor T lymphocytes and host presensitization in an H-2-matched murine BMT model at two different doses of total body irradiation (TBI). These variables were compared in one experimental system where survival as well as donor chimerism were principle endpoints. C57BL/6JIco (B6 (H-2b, Gpi-1b)) recipient male mice (Iffa Credo, L’Arbresle, France) and BALB.B10LiLa (H-2b, Gpi-1a) male donor mice (bred at the Department of Radiobiology, University of Groningen) were used at 3–5 months of age. The donor (BALB.B) and host (B6) mice used in this H-2 compatible allogeneic combination are both H-2b but are misCorrespondence: R van Os, Joint Center for Radiation Therapy, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA Received 27 February 1997; accepted 18 March 1997

matched at multiple minor histocompatibility loci. These donor–host combinations differ with respect to the Gpi-1 phenotype which allows determination of the level of donor blood chimerism as described previously.6 Recipient mice were irradiated using a 60Co source in a Gammatron-3 Unit (Siemens, Germany) at a high-dose rate between 40 and 50 cGy/min. Presensitization was performed by intravenous injection of 2 × 107 lethally irradiated (20 Gy) donor spleen cells 1 week prior to irradiation and BMT. Bone marrow was depleted of T cells by monoclonal antibody (anti-Thy1.2) and complement treatment according to a method previously described.7 This procedure reduced T cell frequencies from 1.45 ± 0.27% (s.e.m.) in normal marrow to 0.18 ± 0.05% as determined using FITC-conjugated anti-Thy1 labeling and immunofluorescent microscopy. For all experiments, a transplanted bone marrow cell dose of 107 nucleated cells was used. Spleen cells were collected from the donors and enriched for T cells by passing the cells over a nylonwool column according to a method described by Julius et al.8 T cell-enriched spleen cell suspensions were added to the bone marrow so that each mouse received 2 × 106 T cells (20% of the total marrow dose) or none at all. The different cell suspensions were injected intravenously at 4–6 h after the end of irradiation. In mixed lymphocyte cultures (MLC), normal B6 spleen responder cells were not reactive against H-2 compatible BALB.B cells. However, B6 spleen cells that were presensitized in vivo against BALB.B cells demonstrated a significant immune response (data not shown). The frequency of proliferating T lymphocytes (pPTL) in B6 spleen cells reactive against H-2 compatible BALB.B cells was determined in a 7-day limiting dilution culture of B6 responder cells in the presence of irradiated (20 Gy) donor (BALB.B) stimulator cells as described.9 This frequency was increased by about 100-fold at 1 week after presensitization with BALB.B cells when compared with unsensitized animals (Figure 1). Total body irradiation with 6 or 9.5 Gy reduced the numbers of pPTL per spleen but the residual number of lymphocyte precursors in presensitized animals was significantly higher than in unsensitized mice. Transplantation of H-2-matched BM into B6 mice that were conditioned with a sublethal dose of 6 Gy did not reach a stable level of erythroid donor engraftment until 12 weeks after BMT due to the long half-life of host erythrocytes (Figure 2a). Adding 2 × 106 T cells showed only a slight gain in the level of long-term engraftment. In mice that were presensitized with 2 × 107 irradiated donor cells prior to TBI and BMT, no donor cells could be detected throughout the post-transplant period and these mice survived with autologous hemopoietic recovery. Adding T cells did not improve long-term donor engraftment in presensitized mice. No significant differences in survival or in blood cell reconstitution were observed between T cell-depleted and -repleted BMT (data not shown).

Brief communication R van Os et al

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Figure 1 Immune reactivity in B6 mice as measured by pPTL frequencies in response to H-2 compatible (BALB.B) cells. Host and donor spleen cells were cultured as responders and irradiated stimulator cells respectively and 3 H-Tdr uptake was determined after 7 days. Spleen cells were collected from irradiated hosts at 24 h after TBI (without a BMT). Frequencies with 95% confidence intervals were calculated and are shown as error bars. Presensitized mice were injected with 2 × 107 irradiated donor cells on day −7.

A TBI dose of 9.5 Gy is potentially lethal and requires at least transient engraftment of transplanted bone marrow to survive the acute effects of radiation. Unsensitized recipients prepared with 9.5 Gy TBI demonstrated good survival and achieved full donor engraftment as early as 8 weeks postBMT, whereas presensitized recipients all died early from acute marrow failure with no evidence of donor type chimerism (Figure 2b). The recovery of peripheral leukocyte and erythrocyte counts after 6 Gy TBI and BMT was delayed in presensitized recipients while this was not altered by T cell repletion (data not shown). Comparison between erythroid engraftment and engraftment among the lymphohemopoietic tissues on termination of the experiment at 20 weeks postBMT showed that only in spleen and thymus were higher engraftment levels found in unsensitized mice (6 Gy TBI) that received extra T cells. No significant differences were found in erythrocytes and bone marrow cells (Table 1). Using an H-2 compatible BMT model (B10.BR → CBA), Down et al4 previously showed that low doses of T cell repletion enhanced GVHD, but had no effect on the level of donor chimerism. Transplantation of B10.BR marrow into CBA represents one of the few murine H-2 compatible BMT models that result in the induction of lethal GVHD without the addition of extra T lymphocytes to the bone marrow inoculum.10 In that model, BMT led to GVHD when low doses of T cells were added back to T-depleted marrow4,10 whereas in the current study a relatively high number of T cells neither improved engraftment nor significantly led to GVHD severe enough to induce lethality. In contrast to the lack of a T cell effect on engraftment in the two BMT models mentioned above, it was recently shown that CD8-positive lymphocytes were able to facilitate engraftment of class II MHC-disparate marrow and of marrow heterozygous for class I and II MHC.11 It is therefore likely that the actual type of genetic disparity between donor and host mice as well as the number of T cells transplanted determines whether or not donor T cells can have a positive effect on engraftment. A similar phenomenon may exist among BMT patients were there is an association between graft failure and T cell depletion of the graft.

Figure 2 Development of engraftment in B6-mice transplanted with H-2 compatible (BALB.B) T cell-depleted or -repleted bone marrow after 6 Gy (a) or 9.5 Gy (b) TBI. The level of donor chimerism at different time intervals after BMT was determined from blood Gpi phenotyping. H-2-matched BMT (BALB.B → B6) was performed on day 0 with 107 T cell-depleted bone marrow cells with (p) or without 2 × 106 T cells (P). Presensitized mice were injected with 2 × 107 irradiated donor cells on day −7 and transplanted with T-depleted bone marrow (no T cells, H) or T-repleted bone marrow (2 × 106, h). Results are shown from one experiment with five mice per group (mean ± 1 s.e.m.).

The results presented in this paper show that an enhanced alloreactivity following recipient presensitization leads to a very marked rejection of allogeneic marrow. This was predicted from measurements obtained from the MLC and pPTL assays. The results also suggest that radiation dose is critical for allogeneic engraftment and survival in presensitized animals. TBI doses of 6 Gy and above were enough to induce donor chimerism in unsensitized recipients of H-2 matched BMT.4,7,9,12 In presensitized animals bone marrow was promptly rejected but the low TBI dose allowed survival through recovery of host hemopoiesis. Raising the TBI dose to 9.5 Gy did not appear to be adequate enough to prevent rejection of allogeneic bone marrow in presensitized mice. This led to death from acute bone marrow aplasia. Under conditions of


Table 1

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Comparison of donor engraftment in erythrocytes, bone marrow, spleen and thymus at 20 weeks after allogeneic BMT

TBI dose

T cell dose

% Donor engraftment a

Survival Erythrocytes

BALB.B → B6 – unsensitized 6 Gy 0 2 × 106 9.5 Gy 0 2 × 106

24/25 5/5 5/5 4/5

82 ± 6 85 ± 1 99 ± 1 100

BALB.B → B6 – presensitized b 6 Gy 0 2 × 106 9.5 Gy 0 2 × 106

10/10 5/5 0/5 0/5

0.3 ± 0.3 1.4 ± 1.3 ND ND

Bone marrow

70 82 96 98

± ± ± ±

9 8 2 2

1±1 4±2 ND ND

Spleen

70 93 96 98

± ± ± ±

8 3* 2 2

1±1 2±2 ND ND

Thymus

70 99 87 83

± ± ± ±

9 1* 5 9

2±1 2±2 ND ND

Percentage of donor chimerism as determined from Gpi phenotyping is expressed as the mean 6 the standard error of the mean. Presensitized recipients received 2 × 107 irradiated (20 Gy) donor spleen cells 7 days prior to TBI and BMT. *Significant difference (P , 0.05) between no T cells and 2 × 106 T cells.

a

b

presensitization, adding as much as 2 × 106 T lymphocytes (about 20% of the bone marrow inoculum) to the bone marrow did not alleviate this effect. The use of alternative irradiation protocols, such as fractionation or low-dose rate TBI were shown to decrease the incidence of allogeneic donor chimerism in experimental9,12 and clinical BMT.13 Therefore, further studies will be needed to elucidate experimentally the role of T cell depletion under conditions of altered TBI. In clinical BMT, additional immune suppression may be required to prevent rejection of T celldepleted marrow grafts, especially in patients who are already presensitized from previous blood transfusions. This could be achieved by using anti-thymocyte globulin or monoclonal antibodies directed against host immune effector cells or by irradiation of lymphoid organs. In the latter case, total lymphoid irradiation (TLI) has been shown to improve engraftment of T cell-depleted allogeneic marrow.14 Furthermore, in aplastic anemia, where minimal host conditioning is used, TLI has been shown to reduce graft rejection for previously heavily transfused recipients.15 These particular recipients appear at more risk for graft rejection than non-transfused recipients. In our transplantation models, we observed enhanced allograft rejection in presensitized recipients. The addition of T cells to the donor marrow inoculum had no effect on engraftment in these recipients. The role of residual immunity from previous transfusions in BMT recipients at increased risk for graft rejection needs further evaluation in the context of designing safer but more effective conditioning therapy. Acknowledgements We wish to thank Dr J Ferarra (Dana Farber Cancer Institute, Boston) for helpful discussions. This study was supported by a grant from the Dutch Cancer Society (GUKC 92-16) and NIH RO1 Grant-CA 10941.

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