Hematopoietic Reconstitution After Lethal Irradiation and Bone

Bone Marrow Transplantation (2000) 25, 427–433  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt Hematopoietic reconstitution after lethal irradiation and bone marrow transplantation: effects of different hematopoietic cytokines on the recovery of thymus, spleen and blood cells

D Frasca1, F Guidi1, M Arbitrio1, C Pioli1, F Poccia2, R Cicconi2 and G Doria2

1Laboratory of Immunology, AMB-PRO-TOSS, ENEA CR Casaccia, Rome; and 2Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy

Summary: ative treatments of the recipient has been used to reconsti- tute hematopoiesis. The HPC capable of long-term reconsti- Lethally irradiated mice were grafted with syngeneic tution are quiescent and slowly cycling bone marrow cells bone marrow cells or left ungrafted. Mice of each group that form multiple colonies of mixed lineages only in the were injected with different hematopoietic cytokines presence of bone marrow stromal cell-derived cytokines.6 for 5 consecutive days starting immediately after Thus, the use of hematopoietic cytokines together with irradiation or left uninjected. The recovery of lymphoid bone marrow transplantation has been shown to hasten neu- tissues induced by hematopoietic cytokines 7 days after trophil and platelet recovery, suggesting that appropriate irradiation and bone marrow cell transplantation was combinations of these factors may be used to promote full comparable to that observed at days 21–28 in reconstitution of the hematopoietic compartment.7–9 irradiated, bone marrow-grafted, but cytokine-unin- In the present work, we have compared the effects of jected mice. IL-11 or IL-6, in combination with IL-3, hematopoietic cytokines, such as IL-3, IL-11, SCF, IL-6 was able to hasten thymus, spleen and blood cell num- and the IL-6 super-agonist K-7/D-6, on the repopulation of bers and functions. SCF also displayed a detectable lymphoid organs and tissues of mice irradiated with a lethal effect when used with IL-3. Conversely, the IL-6 super- dose of X-rays and reconstituted with syngeneic bone mar- agonist K-7/D-6 was able, when injected alone, to induce row cells. IL-3 is involved in blood cell formation, as it significant recovery of thymus, spleen and blood cells. stimulates the survival and the proliferation of pluripotent Thus, K-7/D-6 appears to be a most efficient cytokine stem cells and promotes colony formation by multipotential for fast reconstitution of lymphoid tissues after progenitor cells.10,11 IL-11 is a multifunctional hemato- irradiation and bone marrow transplantation. Bone poietic cytokine capable of supporting the maintenance of Marrow Transplantation (2000) 25, 427–433. multilineage hematopoiesis in long-term bone marrow cul- Keywords: hematopoietic cytokines; lymphocytes; X- rays tures, the proliferation of an IL-6-dependent cell line, and the formation of megakaryocyte, platelet and neutrophil colonies.12–14 SCF is a potent hematopoietic growth factor acting on both early stem cells and already differentiated Hematopoiesis is a process regulated by a complex network cells, particularly mast cells, in the bone marrow.15 IL-6 of soluble factors that stimulate the growth and differen- plays a primary role in hematopoiesis, as it regulates the tiation of hematopoietic progenitor cells (HPC).1,2 HPC differentiation of B cells, megakaryocytes and platelets.16,17 have two major characteristics: self-renewal ability and the K-7/D-6 carries amino acid substitutions in two distinct capacity to differentiate into all lineages of hematopoietic regions of the wild-type IL-6 that participate in the interac- cells. The proliferation and differentiation of HPC are tion with IL-6R and shows a 70-fold increased binding influenced to a large extent by interactions among various affinity as compared to the wild-type molecule.18,19 The cell types in the hematopoietic compartment and by hema- results herein provide evidence that it is possible to acceler- topoietic cytokines produced by stromal cells and lympho- ate the repopulation of lymphoid tissues after lethal cytes. Some of these factors have been identified, charac- irradiation and bone marrow transplantation by using differ- terized and their genes cloned. Experiments performed with ent combinations of hematopoietic cytokines, such as IL-3 recombinant molecules have shown that some hematopo- and IL-11, or IL-3 and IL-6. IL-3 and SCF gave only mar- ietic cytokines act in combination with others to stimulate ginal effects. K-7/D-6, conversely, was able even when proliferative and differentiative responses of HPC, but are used alone to induce a fast recovery of lymphoid tissues 3–5 devoid of detectable activity when used alone. after irradiation and bone marrow transplantation, suggest- Bone marrow transplantation performed after myeloabl- ing that it can be considered an excellent candidate for clinical applications. Correspondence: Dr D Frasca, Laboratory of Immunology, AMB-PRO- TOSS, ENEA CR Casaccia, Via Anguillarese, 301, 00060 S Maria di Galeria, Rome, Italy Received 23 August 1999; acccepted 14 October 1999 Hematopoietic reconstitution after lethal irradiation and BMT D Frasca et al 428 Materials and methods cytokines were certified to be devoid of detectable endo- toxin. The biological activities of IL-3,10,11 IL-11,12–14 Animals SCF15 and IL-616,17 are widely described elsewhere. The doses of cytokines injected and the regimen of one injection Congenic male and female C57BL/6 Thy1.1 or Thy1.2 per day for 5 consecutive days starting immediately mice, bred and maintained in our animal facilities, were after irradiation have been determined in previous used at the age of 3 to 6 months. Normal and treated mice experiments.20,21 were distributed six per group. Cell culture Irradiation procedure Mice were individually tested. Mice were killed by decapi- C57BL/6 Thy1.1 mice were total body irradiated with 9.5 tation, their thymuses and spleens were aseptically removed Gy in a lucite chamber, 7–28 days before sacrifice. This and single cell suspensions prepared. Cell cultures were dose of X-rays is 100% lethal as it kills all mice within 30 performed in microtissue culture plates (Falcon 3040, days. The X-ray machine (Stabilipan, Siemens, Germany) Oxnard, CA, USA) in RPMI 1640 medium supplemented was operated at 250 kV, 15 mA, 0.5 mm Cu filtration, dose with 10% fetal calf serum (Flow Laboratories, Irvine, UK), rate 1.4 Gy/min in air, focus distance 50 cm. 2mml-glutamine (GIBCO), 10 ␮g/ml gentamycin (Shering, Kenilworth, NY, USA) and 2 × 10−5 m 2-mercap- Bone marrow transplantation toethanol. This supplemented medium is hereafter referred to as complete medium. Bone marrow cells were harvested from C57BL/6 Thy1.2 mice by gently flushing their femurs with RPMI 1640 medium (GIBCO, Grand Island, NY, USA). Cells were Thymocyte mitotic response to Concanavalin A (ConA) centrifuged, resuspended in phosphate-buffered saline The number of cells per thymus was determined and then (PBS) and counted in a hemocytometer. From 105 to 107 single thymocyte suspensions were cultured in triplicate at ° cells in 0.2 ml were intravenously injected into C57BL/6 37 C in a 5% CO2 humidified incubator, for 48 h. Each Thy1.1 recipient mice, immediately after irradiation. Alter- culture contained 106 cells in 0.2 ml complete medium natively, T cells were removed from the bone marrow cell alone or containing 2 ␮g ConA. Cultures received 18.5 kBq suspension by negative selection using the Macs system. of tritiated thymidine (specific activity 1.739 GBq/mmol) Briefly, bone marrow cells were washed with PBS and (Amersham International, Amersham, UK) in 20 ␮l, 4 h incubated for 20 min on ice with goat anti-mouse Thy1.2 before harvesting with an automated cell harvester MoAb-coated MicroBeads (Miltenyi Biotech, Bergisch- (Micromate 196; Packard, Meriden, CT, USA). Radio- Gladbach, Germany). One hundred ␮l of MicroBeads were activity was measured in a Matrix 96 direct betacounter used for magnetic staining of 107 cells. After incubation (Packard) and expressed as c.p.m. per culture. with MicroBeads, cells were washed with PBS, resuspended in 500 ␮l and applied to the top of a prefilled and washed A2 column (Miltenyi), provided with a G22 needle Cytofluorimetric analysis of thymocytes for flow regulation. Columns were washed with 2 ml of The reagents used to detect Thy1.1 and Thy1.2 antigens PBS. The negative fraction was collected and analyzed by were anti-Thy1.1 fluorescein isothiocyanate (FITC)-conju- cytofluorimetry for the presence of T cells. gated and anti-Thy1.2 phycoerythrin (PE)-conjugated MoAbs (Becton Dickinson Immunocytometry Systems, San 6 ␮ Cytokine treatment Jose, CA, USA). Thymocytes (10 cells/100 l) were incubated with 4 ␮l of a titrated mixture of the two MoAbs for Irradiated mice were injected subcutaneously, once a day 30 min on ice and finally washed with PBS. Samples were for 5 consecutive days starting immediately after analyzed using a FACStar Plus (Becton Dickinson Immu- irradiation, with 0.1 ml PBS, supplemented with 0.1% bov- nocytometry Systems) flow cytometer with standard optical ine serum albumin, alone or containing 8 ␮g of murine configuration. The argon laser was tuned at 488 nm, 200 recombinant IL-3 (320 ␮g/kg/day), and/or 5 ␮g of human mW power output. Fluorescence was collected in log mode. recombinant IL-11 (200 ␮g/kg/day), and/or 5 ␮g of rat After color compensation adjustment, data were acquired recombinant SCF, and/or 5 ␮g of human recombinant wild- in list mode with the FACStar Plus Research Software type IL-6 (200 ␮g/kg/day), and/or 5 ␮g of K-7/D-6. IL- (Becton Dickinson Immunocytometry Systems). Low for- 3, provided by Genzyme (Cambridge, MA, USA), had a ward scatter signals were gated out and at least 10000 biological activity of 7 × 106 U/mg. IL-11, provided by events from viable cells were accumulated. Thy1.1/Thy1.2 Genetics Institute (Cambridge, MA, USA), had a biological subset analysis was carried out by quadrant analysis on the activity of 2.3 × 107 U/mg. SCF, provided by Amgen resulting cytograms. (Thousand Oaks, CA, USA), was at the concentration of 1 mg/ml. Wild-type IL-6 or the IL-6 variant K-7/D-6, pro- Splenocyte mitotic responses to ConA and duced by IRBM (Pomezia, Rome, Italy), was injected lipopolysaccharide (LPS) together with 10 ␮g/day of human soluble IL-6R (sIL- 6R␣). K-7/D-6 is an IL-6 super-agonist displaying 70-fold The number of cells per spleen was determined and then higher affinity than wild-type IL-6 for sIL-6R␣.18,19 All single splenocyte suspensions were cultured in triplicate as

Bone Marrow Transplantation Hematopoietic reconstitution after lethal irradiation and BMT D Frasca et al 429 described above for thymocytes. Brieﬂy, each triplicate cul- dose. Thereafter, these values increase with the time from ture well contained 4 × 105 splenocytes in 0.2 ml complete irradiation and with the number of injected bone marrow medium alone or with 1 ␮g ConA or 0.2 ␮g LPS. Spleno- cells. At 28 days after irradiation and injection of 107 bone cyte cultures were harvested and processed as described for marrow cells, mitotic responses in the thymus and in the thymocyte cultures. spleen are still impaired, whereas the recovery of cell counts is complete. 7 Hematological analyses Thereafter, the dose of 10 bone marrow cells was chosen to evaluate the effects of hematopoietic cytokines Erythrocyte and leukocyte counts were performed on blood at 7 days after irradiation. individually collected by decapitation.

Effect of different cytokines on the reconstitution of Statistical analysis thymus, spleen and blood cells after irradiation and bone Two-tailed Student’s t-test was carried out to evaluate the marrow transplantation statistical significance of the difference between means. C57BL/6 Thy1.1 mice were 9.5 Gy irradiated and injected Significance levels: P Ͻ 0.05 (*); P Ͻ 0.01 (**); not significant (NS). with IL-3 and IL-11, or IL-3 and SCF, or IL-3 and IL-6, or K-7/D-6 alone, over a period of 5 consecutive days, starting immediately after irradiation. Alternatively, mice received Results whole bone marrow cells or T cell-depleted bone marrow cells from C57BL/6 Thy1.2 mice, alone or with the hemato- Kinetics of the reconstitution of thymus and spleen cells poietic cytokines. Mice were killed 7 days later. Results in after lethal irradiation and bone marrow transplantation Table 2 show that irradiation 7 days prior to sacrifice mark- edly reduces the cell number in the thymus. Treatment of C57BL/6 Thy1.1 mice were whole-body exposed to 9.5 Gy irradiated mice with cytokines is unable to improve the thy- and injected with different doses of bone marrow cells from mus cellularity. Injection of whole bone marrow cells or T C57BL/6 Thy1.2 mice, immediately after irradiation. Mice cell-depleted bone marrow cells into irradiated mice is (six per group) were killed 7, 14, 21 or 28 days later. At slightly effective but yields insufficient thymocyte numbers 28 days, the survival was 60% after transplantation of 105 to perform all tests. However, the combination of whole bone marrow cells; 70% after transplantation of 106 bone bone marrow cells or T cell-depleted bone marrow cells marrow cells and 100% after transplantation of 107 bone with hematopoietic cytokines greatly increases the thymus marrow cells. Results in Table 1 show thymus and spleen cell number. In particular, IL-11 or IL-6 in combination cell repopulation and mitotic responses. At day 7 after with IL-3 was able to significantly accelerate the recovery irradiation the cell count and mitotic responses in both thy- of thymus cells, whereas SCF was only slightly effective. mus and spleen are negligible regardless of the injected cell Conversely, K-7/D-6 was found to be a very efficient mol-

Table 1 Recovery of the immune system after bone marrow transplantation

Radiation Days after Bone marrow Cells/thymus Mitotic response Cells/spleen Mitotic response of splenocytes dose (cGy) irradiation cells (×106) of thymocytes (×106) injected to ConA to ConA to LPS ± c.p.m. s.e. c.p.m. ± s.e. c.p.m. ± s.e.

0 7 none 67 Ϯ 2 8233 Ϯ 180 85 Ϯ 8 8226 Ϯ 192 4081 Ϯ 170 950 7 105 2 Ϯ 1 not done 1 Ϯ 0 not done not done 950 7 106 4 Ϯ 1 not done 3 Ϯ 1 not done not done 950 7 107 7 Ϯ 3 not done 18 Ϯ 2 102 Ϯ 15 96 Ϯ 12

0 14 none 78 Ϯ 6 7962 Ϯ 238 90 Ϯ 3 7938 Ϯ 180 3719 Ϯ 71 950 14 105 12 Ϯ 5 160 Ϯ 35 12 Ϯ 4 197 Ϯ 82 90 Ϯ 21 950 14 106 20 Ϯ 4 469 Ϯ 97 15 Ϯ 2 104 Ϯ 22 133 Ϯ 26 950 14 107 32 Ϯ 3 864 Ϯ 125 24 Ϯ 3 175 Ϯ 11 180 Ϯ 92 0 21 none 73 Ϯ 8 8638 Ϯ 325 87 Ϯ 12 8533 Ϯ 205 3664 Ϯ 427 950 21 105 16 Ϯ 3 805 Ϯ 271 21 Ϯ 3 1023 Ϯ 182 381 Ϯ 162 950 21 106 25 Ϯ 8 1017 Ϯ 182 31 Ϯ 4 1488 Ϯ 372 417 Ϯ 138 950 21 107 39 Ϯ 5 2566 Ϯ 427 58 Ϯ 9 2031 Ϯ 198 975 Ϯ 102 0 28 none 80 Ϯ 7 10137 Ϯ 502 93 Ϯ 10 10684 Ϯ 366 5027 Ϯ 116 950 28 105 32 Ϯ 6 1124 Ϯ 99 42 Ϯ 8 1614 Ϯ 175 1062 Ϯ 75 950 28 106 42 Ϯ 4 2628 Ϯ 301 70 Ϯ 9 2017 Ϯ 81 2117 Ϯ 84 950 28 107 77 Ϯ 4 NS 6128 Ϯ 180 88 Ϯ 5 NS 7233 Ϯ 90 4032 Ϯ 184

Two-tailed Student’s t-test was carried out to evaluate the difference between means of irradiated vs unirradiated. All differences were signiﬁcant at P Ͻ 0.01 except those indicated by NS. Mean ± s.e. from six mice per group.

Bone Marrow Transplantation Hematopoietic reconstitution after lethal irradiation and BMT D Frasca et al 430 Table 2 Thymocytes from lethally irradiated Thy1.1+ mice 7 days after transplantation of whole Thy1.2+ bone marrow cells (BMC) or T cell- depleted BMC

Radiation Cells injected Cytokines Cells/thymus Thy1.1+ cells Thy1.2+ cells Mitotic response dose (cGy) injected (×106) (%) (%) of thymocytes to ConA c.p.m. ± s.e.

0 none none 69.9 Ϯ 6.4 93.0 Ϯ 0.9 0.0 Ϯ 0.0 8453 Ϯ 315 950 none PBS 0.9 Ϯ 0.1 — — not detectable 950 none IL-3 + IL-11 1.2 Ϯ 0.1 NS — — not detectable 950 BMC PBS 2.7 Ϯ 0.4** — — not detectable 950 BMC IL-3 + IL-11 18.5 Ϯ 2.4** 3.3 Ϯ 2.7 86.2 Ϯ 3.6 963 Ϯ 154 950 T-depleted BMC none 3.1 Ϯ 1.2** — — not detectable 950 T-depleted BMC IL-3 + IL-11 16.0 Ϯ 3.3** 1.9 Ϯ 0.6 92.3 Ϯ 4.1 937 Ϯ 144 0 none none 62.6 Ϯ 4.2 95.4 Ϯ 2.6 0.0 Ϯ 0.0 6998 Ϯ 126 950 none PBS 0.9 Ϯ 0.6 — — not detectable 950 none IL-3 + SCF 2.2 Ϯ 1.6 NS — — not detectable 950 BMC PBS 4.5 Ϯ 2.3** — — not detectable 950 BMC IL-3 + SCF 11.3 Ϯ 6.2 NS 3.1 Ϯ 1.1 92.2 Ϯ 2.8 928 Ϯ 112 950 T-depleted BMC PBS 3.9 Ϯ 1.9** — — not detectable 950 T-depleted BMC IL-3 + SCF 7.7 Ϯ 4.3 NS 3.5 Ϯ 1.7 93.4 Ϯ 2.2 762 Ϯ 298 0 none none 72.3 Ϯ 6.5 94.3 Ϯ 0.6 0.0 Ϯ 0.0 7107 Ϯ 399 950 none PBS 1.2 Ϯ 0.3 — — not detectable 950 none IL-3 + IL-6 2.1 Ϯ 0.6 NS — — not detectable 950 BMC PBS 3.5 Ϯ 1.7 NS — — not detectable 950 BMC IL-3 + IL-6 9.6 Ϯ 1.3** 2.5 Ϯ 1.1 97.6 Ϯ 2.5 1133 Ϯ 201 950 T-depleted BMC PBS 2.1 Ϯ 0.2 NS — — not detectable 950 T-depleted BMC IL-3 + IL-6 5.8 Ϯ 0.6* 2.8 Ϯ 1.7 95.8 Ϯ 0.4 1247 Ϯ 324 0 none none 76.0 Ϯ 4.2 96.2 Ϯ 2.3 0.0 Ϯ 0.0 7523 Ϯ 322 950 none PBS 1.5 Ϯ 0.9 — — not detectable 950 none K-7/D-6 1.8 Ϯ 1.0 NS — — not detectable 950 BMC PBS 3.7 Ϯ 0.6* — — not detectable 950 BMC K-7/D-6 13.1 Ϯ 2.9** 3.1 Ϯ 0.4 93.4 Ϯ 2.3 849 Ϯ 122 950 T-depleted BMC PBS 3.1 Ϯ 1.1 NS — — not detectable 950 T-depleted BMC K-7/D-6 9.7 Ϯ 0.8** 2.9 Ϯ 1.1 92.7 Ϯ 1.8 912 Ϯ 203

Two-tailed Student’s t-test was carried out to evaluate the statistical signifiance of the difference between the following means: cytokine vs PBS; BMC, PBS vs PBS; BMC, cytokine vs BMC, PBS; T-depleted BMC, cytokine vs T-depleted BMC, PBS. Significant at P Ͻ 0.05(*) or at P Ͻ 0.01(**). Not significant (NS). Mean ± s.e. from six mice per group.

ecule that was able, when used alone, to induce a significant can be reduced to 0.2% by treating bone marrow cells with recovery of thymus cells. In these experiments, almost all anti-Thy1.2 MoAb. The removal of T cells from the bone thymocytes are of donor type, but have not yet acquired marrow cell suspension before injection abrogates the cyto- full ability to respond to ConA. kine effect on the mitotic response of splenocytes to ConA Table 3 shows the results obtained with spleen cells from but not to LPS, suggesting that the effect observed with these same mice. Lethal irradiation significantly reduces the whole bone marrow cells results from amplification of the spleen cell number and ability of splenocytes to respond to bone marrow-contaminating T cells rather than from fast ConA and LPS. Injection of cytokines alone is ineffective stem cell migration to and maturation in the thymus and/or on the recovery of these parameters. Bone marrow trans- accelerated T cell migration to the spleen. Notably, in these plantation alone improves to some extent the cell number experiments the removal of contaminating T cells had no but not the mitotic responses. If, however, whole bone mar- influence on the cytokine effect on the thymus (Table 2). row cells are injected together with hematopoietic cyto- Blood cells from mice lethally irradiated and reconsti- kines, spleen cell number and mitotic responses to ConA tuted with bone marrow cells, alone or together with hema- and LPS are greatly enhanced, and sometimes fully reco- topoietic cytokines, were examined and results are reported vered. In particular, cytokine treatment with IL-3, together in Table 4. The recovery of leukocyte counts is strongly with IL-11 or with IL-6, induces recovery of cell numbers accelerated by injection of IL-3 in combination with IL-11 and functions in the spleen of these mice at day 7 to a or IL-6, whereas the combination with SCF is ineffective. similar extent to that observed (Table 1) at days 21–28 after K-7/D-6 alone was also able to induce significant recovery irradiation and bone marrow transplantation. K-7/D-6 was of leukocytes of mice lethally irradiated and reconstituted very efficient as it induced significant recovery of spleen with whole bone marrow cells or T cell-depleted bone mar- cell number and mitotic responses. SCF was found to have row cells. As to erythrocytes, the increase in cell number only negligible effects. induced by bone marrow cells and cytokines may result T cell contamination of bone marrow cells is 5% and from precursor regeneration as well as from other factors,

Bone Marrow Transplantation Hematopoietic reconstitution after lethal irradiation and BMT D Frasca et al 431 Table 3 Splenocytes from lethally irradiated Th1.1+ mice 7 days after transplantation of whole Thy1.2+ bone marrow cells (BMC) or T cell- depleted BMC

Radiation Cells injected Cytokines Cells/spleen Mitotic response Mitotic response dose (cGy) injected (×106) of splenocytes of splenocytes to ConA to LPS (c.p.m. ± s.e.) c.p.m. ± s.e.

0 none none 96.3 Ϯ 3.1 7226 Ϯ 160 4432 Ϯ 652 950 none PBS 6.7 Ϯ 0.3 not detectable not detectable 950 none IL-3 + IL-11 4.6 Ϯ 0.3 NS 35 Ϯ 21 60 Ϯ 26 950 BMC PBS 24.6 Ϯ 3.1** 46 Ϯ 10 56 Ϯ 13 950 BMC IL-3 + IL-11 25.4 Ϯ 6.2 NS 4482 Ϯ 1363 4228 Ϯ 711 950 T-depleted BMC none 19.6 Ϯ 2.2** 51 Ϯ 13 39 Ϯ 12 950 T-depleted BMC IL-3 + IL-11 17.2 Ϯ 4.8 NS 97 Ϯ 17 3882 Ϯ 163 0 none none 85.6 Ϯ 9.2 8144 Ϯ 715 4562 Ϯ 213 950 none PBS 7.2 Ϯ 1.6 not detectable not detectable 950 none IL-3 + SCF 6.8 Ϯ 2.3 NS 48 Ϯ 17 57 Ϯ 23 950 BMC PBS 17.9 Ϯ 7.2** 59 Ϯ 24 68 Ϯ 21 950 BMC IL-3 + SCF 18.2 Ϯ 8.6 NS 62 Ϯ 17 49 Ϯ 16 950 T-depleted BMC PBS 15.2 Ϯ 8.4** 72 Ϯ 31 58 Ϯ 24 950 T-depleted BMC IL-3 + SCF 16.0 Ϯ 9.7 NS 97 Ϯ 32 89 Ϯ 31 0 none none 93.4 Ϯ 8.6 7933 Ϯ 324 4881 Ϯ 439 950 none PBS 8.4 Ϯ 3.6 not detectable not detectable 950 none IL-3 + IL-6 7.9 Ϯ 1.6 NS 103 Ϯ 12 115 Ϯ 21 950 BMC PBS 21.7 Ϯ 7.7** 221 Ϯ 23 108 Ϯ 14 950 BMC IL-3 + IL-6 26.7 Ϯ 5.4 NS 3841 Ϯ 623 3378 Ϯ 197 950 T-depleted BMC PBS 19.3 Ϯ 8.6** 301 Ϯ 56 265 Ϯ 75 950 T-depleted BMC IL-3 + IL-6 21.4 Ϯ 7.3 NS 275 Ϯ 58 3762 Ϯ 274 0 none none 85.2 Ϯ 6.3 8035 Ϯ 657 4902 Ϯ 321 950 none PBS 7.9 Ϯ 2.4 not detectable not detectable 950 none K-7/D-6 6.9 Ϯ 2.3 NS 165 Ϯ 97 102 Ϯ 56 950 BMC PBS 18.8 Ϯ 6.2** 223 Ϯ 59 125 Ϯ 45 950 BMC K-7/D-6 19.5 Ϯ 9.4 NS 7934 Ϯ 233 5027 Ϯ 465 950 T-depleted BMC PBS 21.3 Ϯ 4.8** 248 Ϯ 34 181 Ϯ 49 950 T-depleted BMC K-7/D-6 23.4 Ϯ 7.2 NS 301 Ϯ 27 4986 Ϯ 563

Two-tailed Student’s t-test was carried out to evaluate the statistical significance of the difference between the following means: cytokine vs PBS; BMC, PBS vs PBS; BMC, cytokine vs BMC, PBS; T-depleted BMC, cytokine vs T-depleted BMC, PBS. Significant at P Ͻ 0.05(*) or at P Ͻ 0.01(**). Not significant (NS). Mean ± s.e. from six mice per group. such as hemoconcentration, affecting their compartment cytokines, when used alone, are able to support the survival distribution. of HPC without inducing their proliferation22 suggests that they may induce the expression of receptors for other growth factors which are required for cell division. Simi- Discussion larly, the development of erythroid, megakaryocyte, mono- cyte, platelet or neutrophil colonies from HPC requires the In the present paper we report the effects of different hema- combination of different cytokines. topoietic cytokines in mice given a lethal dose of X-rays HPC usually reside in the bone marrow for 2 or 3 weeks and bone marrow transplantation. The combination of IL-3 before migrating to the thymus, where they remain quiesc- with IL-11 or IL-6 was able to elicit significant therapeutic ent for another 10–14 days before cycling.23 Thus, treat- effects, whereas the combination of IL-3 with SCF was ment of irradiated mice with hematopoietic cytokines sig- almost completely ineffective. Conversely, K-7/D-6 alone nificantly shortens the time required for the repopulation of was able to promote rapid recovery of thymus and spleen the thymus by the injected bone marrow cells. Indeed, pre- cells after irradiation, being as potent as the combination vious findings24 suggest that early acting hematopoietic of IL-3 and IL-11 and more potent than IL-3 in combi- growth factors are able to reduce the time required by HPC nation with IL-6. These effects of hematopoietic cytokines to go through the G1 phase of the cell cycle. The repopu- suggest that the bone marrow cell population used to recon- lation of the spleen after lethal irradiation and bone marrow stitute the irradiated mice contains cells which can be transplantation follows different kinetics as compared to the induced by hematopoietic cytokines to proliferate, colonize thymus. Thus, both pluripotent stem cells and HPC present the lymphoid tissues and give rise to differentiated cells. It in the bone marrow cell suspension injected into irradiated has been previously shown that optimal proliferation and mice reach the spleen through the circulation and there are differentiation of HPC require the co-stimulation by two induced to differentiate if hematopoietic growth factors are or more growth factors. The finding that some early-acting present. Moreover, our results indicate that the hematopo-

Bone Marrow Transplantation Hematopoietic reconstitution after lethal irradiation and BMT D Frasca et al 432 Table 4 Blood cells from lethally irradiated Thy1.1+ mice 7 days after transplantation of whole Thy1.2+ bone marrow cells (BMC) or T cell-depleted BMC

Radiation dose (cGy) Cells injected Cytokines injected Leukocytes (/mm3) Erythrocytes (×106/mm3)

0 none none 5200 Ϯ 496 15 Ϯ 1 950 none PBS 532 Ϯ 48 2 Ϯ 1 950 BMC PBS 551 Ϯ 32 NS 4 Ϯ 2NS 950 BMC IL-3 + IL-11 925 Ϯ 55** 10 Ϯ 3* 950 T-depleted BMC PBS 625 Ϯ 50 NS 4 Ϯ 3NS 950 T-depleted BMC IL-3 + IL-11 1148 Ϯ 95** 12 Ϯ 3* 0 none none 4962 Ϯ 501 14 Ϯ 2 950 none PBS 484 Ϯ 75 3 Ϯ 2 950 BMC PBS 421 Ϯ 52 NS 4 Ϯ 2NS 950 BMC IL-3 + SCF 456 Ϯ 62 NS 5 Ϯ 3NS 950 T-depleted BMC PBS 501 Ϯ 66 NS 4 Ϯ 3NS 950 T-depleted BMC IL-3 + SCF 553 Ϯ 71 NS 5 Ϯ 1NS 0 none none 5330 Ϯ 471 13 Ϯ 1 950 none PBS 396 Ϯ 84 4 Ϯ 1 950 BMC PBS 422 Ϯ 75 NS 3 Ϯ 2NS 950 BMC IL-3 + IL-6 855 Ϯ 92* 9 Ϯ 1* 950 T-depleted BMC PBS 429 Ϯ 65 NS 3 Ϯ 1NS 950 T-depleted BMC IL-3 + IL-6 922 Ϯ 84** 11 Ϯ 2* 0 none none 4872 Ϯ 498 14 Ϯ 2 950 none PBS 466 Ϯ 72 3 Ϯ 1 950 BMC PBS 502 Ϯ 68 NS 4 Ϯ 1NS 950 BMC K-7/D-6 1012 Ϯ 99** 12 Ϯ 3** 950 T-depleted BMC PBS 484 Ϯ 68 NS 4 Ϯ 2NS 950 T-depleted BMC K-7/D-6 1168 Ϯ 76** 13 Ϯ 2**

ietic cytokines used in this work also amplify the pool of Acknowledgements mature T cells contaminating the injected bone marrow and homing to the spleen of the irradiated recipient. We thank Dr S Neben (Genetics Institute) for kindly providing The possibility of increasing HPC proliferation by cyto- IL-11, and Dr G Ciliberto (IRBMP Angeletti) for the gifts of hu kine treatment is of clinical relevance, as the success of a r IL-6, K-7/D-6 and human soluble IL-6R. This work was sup- transplant depends mainly on the availability of sufﬁcient ported in part by CNR Progetto Finalizzato Biotecnologie. numbers of HPC. Following chemotherapy, the adminis- tration of hematopoietic growth factors leads to a marked rise in the number of HPC in the peripheral blood, meas- References ured either as colony-forming units or as CD34+ cells. The protocol designed to enrich human CD34+ cells by immun- 1 Metcalf D. The Molecular Control of Blood Cells. Harvard omagnetic separation has raised great interest.25 Treatment University Press: Cambridge, MA, 1988. 2 Nicola NA, Vadas M. Hemopoietic cell growth factors and of volunteers with different combinations of colony-stimul- their receptors. Annu Rev Biochem 1989; 58: 45–77. ating factors for several days before the leukapheresis pro- 3 Metcalf D. Hemopoietic regulators: redundancy or subtlety? cedure made it possible to purify sufﬁcient numbers of Blood 1993; 82: 3515–3523. CD34+ cells from the peripheral blood with concomitant 4 Ogawa M. Differentiation and proliferation of hemopoietic depletion of T cells, thus reducing the risk of severe stem cells. Blood 1993; 81: 2844–2853. GVHD.9 Based on our results, we suggest the use of K- 5 Jacobsen SEW. IL-12, a direct stimulator and indirect inhibitor 7/D-6 as a very powerful molecule to expand HPC through of haematopoiesis. In: Immunoregulation by IL-12, 62nd stimulation of gp130 signaling, as shown in vitro.19 Tox- Forum in Immunology, 1996, p 506. 6 Sato N, Sawada K, Koizumi K et al. In vitro expansion of icity tests are required before clinical applications of K- human peripheral blood CD34+ cells. Blood 1993; 82: 7/D-6 in vivo, both in irradiated and cell grafted recipients 3600–3609. as well as in cell donors. Moreover, human applications 7 Knobel KM, McNally MA, Berson AE et al. Long-term recon- require re-evaluation of the experimental protocol in terms stitution of mice after ex vivo expansion of bone marrow cells. of cell and cytokine doses. Differential activity of cultured bone marrow and enriched stem cell populations. Exp Hematol 1994; 22: 1227–1235.

Bone Marrow Transplantation Hematopoietic reconstitution after lethal irradiation and BMT D Frasca et al 433 8 Lane TA, Law P, Maruyama M et al. Harvesting and enrich- 18 Cabibbo A, Sporeno E, Toniatti C et al. Monovalent phage ment of hematopoietic progenitor cells mobilized into periph- display of human interleukin (hIL)-6: selection of superbinder eral blood of normal donors by GM-CSF or G-CSF: potential variants from a complex molecular repertoire in the hIL-6 D- role in allogeneic marrow transplantation. Blood 1995; 85: helix. Gene 1995; 167: 41–47. 275–282. 19 Toniatti C, Cabibbo A, Sporeno E et al. Engineering human 9 Williams SF, Lee WJ, Bender JG et al. Selection and expan- interleukin-6 to obtain variants with strongly enhanced bio- sion of peripheral blood CD34+ cells in autologous stem cell activity. EMBO J 1996; 15: 2726–2737. transplantation for breast cancer. Blood 1996; 87: 1687–1691. 20 Frasca D, Leter G, Doria G. Murine recombinant interleukin 10 Ihle JN, Weinstein Y. Immunological regulation of 3 induces recovery of T and B cells in irradiated mice. Blood hematopoietic/lymphoid stem cell differentiation by interleu- 1994; 83: 1563–1568. kin-3. Adv Immunol 1986; 39: 1–50. 21 Frasca D, Pioli C, Guidi F et al. IL-11 synergizes with IL-3 11 Wagemaker G, Burger H, van Gils CJM et al. Interleukin-3. on the recovery of the immune system in irradiated mice. Int Biotherapy 1990; 2: 337–340. Immunol 1996; 8: 1651–1657. 12 Paul SR, Bennett F, Calvetti JA et al. Molecular cloning of a 22 Miura N, Okada S, Zsebo KM et al. Rat stem cell factor and cDNA encoding IL-11, a stromal cell-derived lymphopoietic IL-6 preferentially support the proliferation of c-kit positive and hematopoietic cytokine. Proc Natl Acad Sci USA 1990; murine hemopoietic cells rather than their differentiation. Exp 87: 7512–7516. Hematol 1993; 21: 143–149. 13 Yin T, Miyazawa K, Yang YC. Characterization of IL-11 23 Penit C, Ezine S. Cell proliferation and thymocyte subset receptor and protein tyrosine phosphorylation induced by IL- reconstitution in sublethally irradiated mice. Compared kin- 11 in mouse 3T3-L1 cells. J Biol Chem 1992; 267: 8347– etics of endogenous and intrathymically transferred progeni- 8351. tors. Proc Natl Acad Sci USA 1989; 86: 5547–5551. 14 Neben S, Turner K. The biology of IL-11. Stem Cells 1993; 24 Tanaka R, Katayama N, Ohishi K et al. Accelerated cell-cyc- 11: 156–162. ling of hematopoietic progenitor cells by growth factors. 15 Zsebo KM, Wypych J, McNiece IK et al. Identiﬁcation, puri- Blood 1995; 86: 73–79. ﬁcation and biological characterization of hematopoietic stem 25 Lane TA, Low P, Maruyama M et al. Harvesting and enrich- cell factor from buffalo rat liver-conditioned medium. Cell ment of hematopoietic progenitor cells mobilized into the per- 1990; 63: 195–201. ipheral blood of normal donors by granulocyte–macrophage 16 Kishimoto T, Taga T, Akira S. Cytokine signal transduction. colony-stimulating factor (GM-CSF) or G-CSF: potential role Cell 1994; 76: 253–262. in allogeneic marrow transplantation. Blood 1995; 85: 275– 17 Hibi M, Murakami M, Sato M et al. Molecular cloning and 282. expression of an IL-6 signal transducer gp130. Cell 1990; 63: 1149–1157.

Bone Marrow Transplantation