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Response to letter by Down and Mauch
219
Correspondence acute animal lethality. Subsequent studies using another iso-zyme marker, glucose phosphate isomerase (Gpi-I), have yielded very comparable results at times beyond 3 months and are consistent with stable engraftment arising from high self-renewing stem cells that are both more primitive and more radioresistant than CFU-S (3, 7, 9, 10). The paper by Storb et al. (8) shows little if any effect of fractionated TBI (at total doses of 3 and 4 Gy) on acute hematological toxicity in their canine study. Taking into account the lower tolerance of dogs to target cell depletion (4) this finding does not conflict with the rodent data on CFU-S and acute lethality. We were therefore surprised by the comment of Storb et al. that their data is in apparent contrast with our data on long-term chimerism: we are clearly using different endpoints resulting from radiation killing of different cell types. The authors then suggest that red blood cells at 3 months still represent the progeny of “rather mature” cells and not primitive stem cells. We would contend that it is the mature cells that are involved in the acute myelodepression as measured in dogs by Storb et al. while steady-state blood chimerism in mice at 3 months and beyond reflects repopulation originating from primitive stem cells. More relevant to the topic of erythrocyte repopulation kinetics and the phenomenon of stem cell heterogeneity is a recent chimerism study that incorporated manipulation of donor marrow on the basis of lectin affinity and the treatment of host with different cytotoxic agents (1). By following the development of erythroid chimerism with time after transplant we were able to relate the timing of engraftment to the frequency of hemopoietic cell subsets, either in donor or host marrow. This approach demonstrated that progenitors and CFU-S equivalent cells provide only transient support along the erythroid lineage that largely disappears by 3 months. It is reasonable to assume that a similar phenomenon could exist in larger species such as humans or dogs but slower blood cell renewal rates would require much longer assessment times before detection of progeny arising from the long-term repopulating stem cell subset. It is noteworthy to add that these elusive “true” stem cells are now becoming central to the problems of gene insertion therapy in autologous BMT aimed at permanent correction of various hematological disorders. While we do not wish to belittle the importance of progenitor and CFU-S-like populations for their role in avoiding acute marrow failure after irradiation (5, 6), broadening our attention to include longer repopulating stem cells, as well as cell populations involved in transplant immunity, would provide a better overall perspective of how fractionated TBI can impact on hemopoietic engraftment in clinical BMT. JULIAN D. DOWN, PH.D.
Department of Radiobiology University of Groningen Bloemsingel I 97 I3 BZ Groningen The Netherlands PETER M. MAUCH, M.D. Joint Center for Radiation Therapy Harvard Medical School 50 Binney St. Boston, MA 02 115 1. Down, J. D.; Ploemacher, R. E. Transient and permanent engraftment potential of murine hematopoietic stem cell subsets: Differential effects of host conditioning with gamma radiation and cytotoxic drugs. Exp. Hematol. 21:913-921; 1993. 2. Down, J. D.; Tarbell, N. J.; Thames, H. D.; Mauch, P. M. Syngeneic and allogeneic bone marrow engraftment after total body irradiation: Dependence on dose, dose rate and fractionation. Blood 77:661669; 1991. 3. Down, J. D.; van OS, R. Donor bone marrow and host irradiation dose-response relationships for long-term hemopoietic repopulation (Abstr.). Int. J. Radiat. Biol. 63:795; 1993. 4. Hendry, J. H.; Roberts, S. A. Analysis of dose-incidence relationships for marrow failure in different species in terms of radiosensitivity of tissue-rescuing units. Radiat. Res. 122: 155- 160; 1990. 5. Jones, R. J.; Wagner, J. E.; Celano. P.; Zicha, M. S.; Sharkis, S. J. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 347: 188- 189; 1990. 6. Ploemacher, R. E.; Brons, N. H. C. Separation of CFU-S from primitive cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: Evidence for a pre-CFU-S cell. Exp. Hematol. 17:263-266; 1989.
7. Ploemacher, R. E.; van OS, R.; van Beurden, C. A. J.; Down, J. D. Murine hemopoietic stem cells with long term engraftment and marrow repopulating ability are less radiosensitive to gamma radiation than are spleen colony forming cells. Int. J. Radiat. Biol. 61: 489-499: 1992. 8. Storb, R.; Raff, R. F.; Graham, T.; Appelbaum, F. R.; Deeg, H. J.; Schuening. F. G.; Schulman, H.; Pepe, M. Marrow toxicity of fractionated vs. single dose total body irradiation is identical in a canine model. Int. J. Radiat. Oncol. Biol. Phys. 26:275-283; 1993. 9. van OS, R.; Down, J. D. Radiation dose fractionation and dose rate relationships for long term bone marrow repopulation (Abstr.). Int. J. Radiat. Biol. 62:361; 1992. 10. van OS, R.; Konings, A. W. T.; Down. J. D. Radiation dose as a factor in host preparation for bone marrow transplantation across different genetic barriers. Int. J. Radiat. Biol. 61:501-510; 1992.
RESPONSE
TO LETTER BY DOWN AND MAUCH
To the Edifor: At the time our paper (5) was submitted, the later papers of Down and van OS, Ploemacher et al., and van OS et ai. (1, 2, 6. 7) showing data on red blood cell repopulation by donor cells beyond 3 months were not available to us. Had we known them, we still would have expressed surprise about the apparent discrepancy of their and our results on the myelotoxicity of fractionated vs. single-dose TBI. However, we would not have put forth the argument that they needed to observe their mice for more than 3 months to address the issue of sublethal repair in more primitive stem cells. In the meantime, we also would no longer express any surprise. Down et al. used a TBI dose rate of 1 Gy/min, while we used I Gy/min in our original study. We have now repeated our study at a dose rate of .6 Gy/min (unpublished). Current results conform to those of Down et al. and show that single-dose TBI is significantly more myelotoxic than the same overall dose of fractionated TBI. This is consistent with DNA repair in early hematopoietic cells during interfraction intervals when TBI is administered at a relatively high dose rate. Our 1993 paper did not address issues of gene transfer into hematopoietic stem cells or of immunosuppressive qualities of fractionated vs. single-dose TBI, as elaborated on by Down et al. Those issues were addressed in earlier papers (3, 4). RAINERSTORB, M.D. Fred Hutchinson Cancer Research Center 1124Columbia St., M3 18 Seattle, WA 98 104
1. Down, J. D.; van OS, R. Donor bone marrow and host irradiation dose-response relationships for long-term hemopoietic repopulation (Abstr.). lnt. J. Radiat. Biol. 63:795; 1993. 2. Ploemacher, R. E.: van OS, R.; van Beurden, C. A. J.; Down, J. D. Murine hemopoietic stem cells with long term engrafiment and marrow repopulating ability are less radiosensitive to gamma radiation than are spleen colony forming cells. Int. J. Radiat. Biol. 61: 489-499: 1992. 3. Schuening, F. G.; Kawahara, K.; Miller, D. A.; To. R.: Goehle, S.; Stewart, D.; Mullally, K.; Fisher, L.; Graham, T. C.; Appelbaum, F. R.: Hackman, R.; Osborne, W. R. A.: Storb, R. Retrovirus-mediated gene transduction into long-term repopulating marrow cells ofdogs. Blood 78:2568-2576; 1991. 4. Storb, R.; Raff, R. F.; Appelbaum, F. R.; Graham, T. C.: Schuening, F. G.; Sale, G.; Pepe, M. Comparison of fractionated to single-dose total body irradiation in conditioning canine littermates for DLAidentical marrow grafts. Blood 74: 1139-l 143; 1989. 5. Storb, R.; Raff, R. F.; Graham, T.; Appelbaum, F. R.; Deeg, H. J.; Schuening, F. G.; Shulman, H.; Pepe, M. Marrow toxicity of fractionated versus single dose total body irradiation is identical in a canine model. Int. J. Radiat. Oncol. Biol. Phys. 26:275-283; 1993. 6. van OS. R.; Down, .I. D. Radiation dose fractionation and dose rate relationships for long term bone marrow repopulation (Abstr.). Int. J. Radiat. Biol. 62:361; 1992. 7. van OS, R.; Konings, A. W. T.; Down, J. D. Radiation dose as a factor in host preparation for bone marrow transplantation across different genetic barriers. Int. J. Radiat. Biol. 61:501-510: 1992.
Correspondence acute animal lethality. Subsequent studies using another iso-zyme marker, glucose phosphate isomerase (Gpi-I), have yielded very comparable results at times beyond 3 months and are consistent with stable engraftment arising from high self-renewing stem cells that are both more primitive and more radioresistant than CFU-S (3, 7, 9, 10). The paper by Storb et al. (8) shows little if any effect of fractionated TBI (at total doses of 3 and 4 Gy) on acute hematological toxicity in their canine study. Taking into account the lower tolerance of dogs to target cell depletion (4) this finding does not conflict with the rodent data on CFU-S and acute lethality. We were therefore surprised by the comment of Storb et al. that their data is in apparent contrast with our data on long-term chimerism: we are clearly using different endpoints resulting from radiation killing of different cell types. The authors then suggest that red blood cells at 3 months still represent the progeny of “rather mature” cells and not primitive stem cells. We would contend that it is the mature cells that are involved in the acute myelodepression as measured in dogs by Storb et al. while steady-state blood chimerism in mice at 3 months and beyond reflects repopulation originating from primitive stem cells. More relevant to the topic of erythrocyte repopulation kinetics and the phenomenon of stem cell heterogeneity is a recent chimerism study that incorporated manipulation of donor marrow on the basis of lectin affinity and the treatment of host with different cytotoxic agents (1). By following the development of erythroid chimerism with time after transplant we were able to relate the timing of engraftment to the frequency of hemopoietic cell subsets, either in donor or host marrow. This approach demonstrated that progenitors and CFU-S equivalent cells provide only transient support along the erythroid lineage that largely disappears by 3 months. It is reasonable to assume that a similar phenomenon could exist in larger species such as humans or dogs but slower blood cell renewal rates would require much longer assessment times before detection of progeny arising from the long-term repopulating stem cell subset. It is noteworthy to add that these elusive “true” stem cells are now becoming central to the problems of gene insertion therapy in autologous BMT aimed at permanent correction of various hematological disorders. While we do not wish to belittle the importance of progenitor and CFU-S-like populations for their role in avoiding acute marrow failure after irradiation (5, 6), broadening our attention to include longer repopulating stem cells, as well as cell populations involved in transplant immunity, would provide a better overall perspective of how fractionated TBI can impact on hemopoietic engraftment in clinical BMT. JULIAN D. DOWN, PH.D.
Department of Radiobiology University of Groningen Bloemsingel I 97 I3 BZ Groningen The Netherlands PETER M. MAUCH, M.D. Joint Center for Radiation Therapy Harvard Medical School 50 Binney St. Boston, MA 02 115 1. Down, J. D.; Ploemacher, R. E. Transient and permanent engraftment potential of murine hematopoietic stem cell subsets: Differential effects of host conditioning with gamma radiation and cytotoxic drugs. Exp. Hematol. 21:913-921; 1993. 2. Down, J. D.; Tarbell, N. J.; Thames, H. D.; Mauch, P. M. Syngeneic and allogeneic bone marrow engraftment after total body irradiation: Dependence on dose, dose rate and fractionation. Blood 77:661669; 1991. 3. Down, J. D.; van OS, R. Donor bone marrow and host irradiation dose-response relationships for long-term hemopoietic repopulation (Abstr.). Int. J. Radiat. Biol. 63:795; 1993. 4. Hendry, J. H.; Roberts, S. A. Analysis of dose-incidence relationships for marrow failure in different species in terms of radiosensitivity of tissue-rescuing units. Radiat. Res. 122: 155- 160; 1990. 5. Jones, R. J.; Wagner, J. E.; Celano. P.; Zicha, M. S.; Sharkis, S. J. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 347: 188- 189; 1990. 6. Ploemacher, R. E.; Brons, N. H. C. Separation of CFU-S from primitive cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: Evidence for a pre-CFU-S cell. Exp. Hematol. 17:263-266; 1989.
7. Ploemacher, R. E.; van OS, R.; van Beurden, C. A. J.; Down, J. D. Murine hemopoietic stem cells with long term engraftment and marrow repopulating ability are less radiosensitive to gamma radiation than are spleen colony forming cells. Int. J. Radiat. Biol. 61: 489-499: 1992. 8. Storb, R.; Raff, R. F.; Graham, T.; Appelbaum, F. R.; Deeg, H. J.; Schuening. F. G.; Schulman, H.; Pepe, M. Marrow toxicity of fractionated vs. single dose total body irradiation is identical in a canine model. Int. J. Radiat. Oncol. Biol. Phys. 26:275-283; 1993. 9. van OS, R.; Down, J. D. Radiation dose fractionation and dose rate relationships for long term bone marrow repopulation (Abstr.). Int. J. Radiat. Biol. 62:361; 1992. 10. van OS, R.; Konings, A. W. T.; Down. J. D. Radiation dose as a factor in host preparation for bone marrow transplantation across different genetic barriers. Int. J. Radiat. Biol. 61:501-510; 1992.
RESPONSE
TO LETTER BY DOWN AND MAUCH
To the Edifor: At the time our paper (5) was submitted, the later papers of Down and van OS, Ploemacher et al., and van OS et ai. (1, 2, 6. 7) showing data on red blood cell repopulation by donor cells beyond 3 months were not available to us. Had we known them, we still would have expressed surprise about the apparent discrepancy of their and our results on the myelotoxicity of fractionated vs. single-dose TBI. However, we would not have put forth the argument that they needed to observe their mice for more than 3 months to address the issue of sublethal repair in more primitive stem cells. In the meantime, we also would no longer express any surprise. Down et al. used a TBI dose rate of 1 Gy/min, while we used I Gy/min in our original study. We have now repeated our study at a dose rate of .6 Gy/min (unpublished). Current results conform to those of Down et al. and show that single-dose TBI is significantly more myelotoxic than the same overall dose of fractionated TBI. This is consistent with DNA repair in early hematopoietic cells during interfraction intervals when TBI is administered at a relatively high dose rate. Our 1993 paper did not address issues of gene transfer into hematopoietic stem cells or of immunosuppressive qualities of fractionated vs. single-dose TBI, as elaborated on by Down et al. Those issues were addressed in earlier papers (3, 4). RAINERSTORB, M.D. Fred Hutchinson Cancer Research Center 1124Columbia St., M3 18 Seattle, WA 98 104
1. Down, J. D.; van OS, R. Donor bone marrow and host irradiation dose-response relationships for long-term hemopoietic repopulation (Abstr.). lnt. J. Radiat. Biol. 63:795; 1993. 2. Ploemacher, R. E.: van OS, R.; van Beurden, C. A. J.; Down, J. D. Murine hemopoietic stem cells with long term engrafiment and marrow repopulating ability are less radiosensitive to gamma radiation than are spleen colony forming cells. Int. J. Radiat. Biol. 61: 489-499: 1992. 3. Schuening, F. G.; Kawahara, K.; Miller, D. A.; To. R.: Goehle, S.; Stewart, D.; Mullally, K.; Fisher, L.; Graham, T. C.; Appelbaum, F. R.: Hackman, R.; Osborne, W. R. A.: Storb, R. Retrovirus-mediated gene transduction into long-term repopulating marrow cells ofdogs. Blood 78:2568-2576; 1991. 4. Storb, R.; Raff, R. F.; Appelbaum, F. R.; Graham, T. C.: Schuening, F. G.; Sale, G.; Pepe, M. Comparison of fractionated to single-dose total body irradiation in conditioning canine littermates for DLAidentical marrow grafts. Blood 74: 1139-l 143; 1989. 5. Storb, R.; Raff, R. F.; Graham, T.; Appelbaum, F. R.; Deeg, H. J.; Schuening, F. G.; Shulman, H.; Pepe, M. Marrow toxicity of fractionated versus single dose total body irradiation is identical in a canine model. Int. J. Radiat. Oncol. Biol. Phys. 26:275-283; 1993. 6. van OS. R.; Down, .I. D. Radiation dose fractionation and dose rate relationships for long term bone marrow repopulation (Abstr.). Int. J. Radiat. Biol. 62:361; 1992. 7. van OS, R.; Konings, A. W. T.; Down, J. D. Radiation dose as a factor in host preparation for bone marrow transplantation across different genetic barriers. Int. J. Radiat. Biol. 61:501-510: 1992.