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Peritoneal B-cell development depends on strain, radiation, and time
Experimental Hematology 29 (2001) 221–227
Peritoneal B-cell development depends on strain, radiation, and time Marcia Stickler and Fiona Harding Department of Immunology, Genencor International Inc., Palo Alto, Calif., USA (Received 1 March 2000; revised 30 September 2000; accepted 6 October 2000)
Objective. B-1a, B-1b, and B-2 cells represent the three B-cell subsets in mice. Previous studies have demonstrated that peritoneal B-1a cell development is absent, or nearly so, from adult bone marrow transfers into irradiated adult hosts. The majority of these studies have been performed under a limited set of conditions with irradiated host mice. Here we examined that under a variety of conditions, peritoneal B-1a cells can develop in significant numbers from adult bone marrow transfers into severe combined immunodeficient (SCID) and recombination activation gene 2⫺ (RAG-2⫺) mice. Materials and Methods. Adult bone marrow was transferred into various strains of irradiated and nonirradiated adult immunodeficient RAG-2⫺ and SCID mice. Peritoneal B-cell engraftment was examined by fluorescein-activated cell sorting analysis and unpaired t-tests were used to determine significant differences of B-cell engraftment among the various conditions of cell transfer. Results. The level of B-1a cell engraftment was variously affected by the type of host immunodeficiency, the combination of donor and host strains, and the time allowed for engraftment. Irradiation of SCID, but not RAG-2⫺, host mice inhibited B-1a–cell engraftment. Additionally, decreasing the number of bone marrow progenitor cells transferred was not found to preferentially affect B-1a cell development in irradiated RAG-2⫺ hosts. Conclusion. In the context of these strains, we conclude that adult murine bone marrow contains progenitors that have the capacity to reconstitute peritoneal B-1a cell populations to donor levels. © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: B-1a cells—Engraftment—Adult bone marrow—SCID—RAG-2⫺
Introduction Three distinct B-cell subsets, B-1a, B-1b, and B-2 cells, exist in mice. These populations are distinguished by their phenotype, anatomical location, regulation, development, and function. There is disagreement over the origin of these subsets. One popular theory holds that these cells are derived from distinct lineages primarily because they have been shown to arise at different times during development [1–6]. B-1a and B-1b cells develop primarily from fetally derived precursors that produce a long-lived self-renewing population of mature cells that prevent further development from precursors in the adult animal by feedback inhibition [7–13]. B-2 cells, on the other hand, are relatively shortlived and repopulate themselves from progenitor cells throughout the life of the animal. Additionally, early-maturing B-1a cells have been shown to possess fewer N-region Offprint requests to: Marcia Stickler, M.S., Genencor International, 925 Page Mill Road, Palo Alto, CA 94304 USA; E-mail: [email protected]
insertions due to a lack of terminal deoxytransferase (TdT) expression early in development [14–18]. Evidence supporting the separate lineage theory was derived primarily from adoptive transfer studies. Transfers of fetal liver cells [7,19–21] and fetal spleen cells [7] have been demonstrated to replenish all B-cell subsets in the peritoneal cavity of recipient mice. However, transfers of fetal omentum cells into adult nonirradiated severe combined immunodeficient (SCID) mice have produced B-1a and B-1b cells but no B-2 cells [22]. Transfers of adult bone marrow into irradiated mice have failed to reconstitute B-1a cells in a number of studies [7,13,19–21] but have readily produced B-1b [13,21] and B-2 cells [7,13,19–20]. Other studies, however, have found B-1a cell engraftment from adult bone marrow transfers [12,23]. An alternative explanation of murine B-cell development is that the B-1a phenotype is the product of a differentiation pathway available to any B cell [24,25]. Subpopulations of B cells develop from a common pool of precursors that are selected for by antigen cross-linking of surface IgM. Induc-
0301-472X/01 $–see front matter. Copyright © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(00)0 0 6 4 4 - 5
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tion of the B-1a cell phenotype by treatment with anti-IgM has been demonstrated in vitro [23,26]. While evidence is compelling for both theories, neither one has been definitively proven. CD5 induction by IgM cross-linking has only been demonstrated in vitro, and evidence for separate lineages is conflicting. Studies that have not found B-1a cell reconstitution from adult bone marrow transfers were performed on a limited number of strains of irradiated hosts in which relatively low numbers of cells were transferred or the time allowed for reconstitution was limited. Irradiation of host mice causes a series of events that may affect the ability of B-1a cells to develop, including release of inflammatory cytokines and gut damage [27,28]. Additionally, genetically controlled differences in B-1a levels exist in different strains of mice [3] and this may impact the ability of these cells to engraft after transfer. Here we address some of the variables that may affect peritoneal B-cell engraftment. These include the number of cells transferred, the time allowed for engraftment, radiation conditioning, immunodeficiency induced by SCID vs RAG-2⫺ mutations, and MHC (major histocompatibility complex) haplotype of the host mice. We demonstrate that B-1a cells do in fact engraft in significant numbers from adult bone marrow transfers. However, the level of B-1a cell engraftment is variously affected by the type of host immunodeficiency, the combination of donor and host strains, and the time allowed for engraftment. Surprisingly, irradiation of SCID [29], but not RAG-2⫺ [30], host mice inhibits B-1a cell engraftment.
Materials and methods Mice Adult BALB/c, C57Bl/6, and CB.17 SCID and BALB/c RAG-2⫺ mice were purchased from Taconic (Germantown, NY, USA). C57Bl/6 SCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). 129/RAG-2⫺ mice were bred in house. All mice were kept under pathogen-free conditions. C57Bl/6 (H2Db) mice were used for bone marrow transfers into 129/RAG-2⫺ (H-2Db) mice and C57Bl/6 SCID (H-2Db) mice. BALB/c (H-2Dd) mice were used for transfers into CB.17 SCID (H-2Dd) mice, Balb/ c RAG-2⫺ (H-2Dd) mice, and C57Bl/6 SCID (H-2Db) mice. Twoto 4-month-old donor and recipient mice were used for bone marrow transfers. Bone marrow transfers and cell preparation Bone marrow was obtained from BALB/c and C57Bl/6 donor mice. Femurs and tibias were flushed with Dulbecco’s phosphatebuffered saline (D-PBS) and a single cell suspension was created. T cells were depleted by complement lysis using anti-CD90.2 (Thy 1.2, Pharmingen, San Diego, CA, USA) and rabbit and guinea pig complement (Cedarlane, Ontario, Canada). Briefly, anti-CD90.2 was added to extracted bone marrow at a concentration of 1 g/107 cells. Cells were incubated on ice for 30 minutes, washed with D-PBS, and added to 2.5 to 5 mL of MAR18.5 supernatant (mouse anti-rat IgG) plus a 1 in 20 dilution of rabbit and guinea pig complement. Cells were incubated for one hour at 37⬚C. T-cell–depleted
bone marrow cells were isolated using Histopaque (density ⫽ 1.119; Sigma, St. Louis, MO, USA). Mice receiving radiation were irradiated one day prior to transfers. RAG-2⫺ mice were lethally irradiated with 800 rads and SCID mice were lethally irradiated with 400 rads delivered by a 125Cesium source. Indicated numbers of cells were injected into the tail vein of RAG-2⫺ and SCID recipients. Peritoneal cavity (PerC) cells were harvested at the time of sacrifice. PerC cells were obtained by flushing the peritoneal cavity with 3 mL cold D-PBS with 2% fetal bovine serum. This method allowed for a recovery of approximately 90% of the injected volume. Any red cells present were lysed with ammonium chloride (Sigma). Cells were washed and counted using trypan blue exclusion. Antibodies and staining The frequency of T cells and B-cell subsets in the peritoneal cavity of donor and recipient mice was determined by flow cytometric analysis. PerC cells were stained with each of the following antibodies: anti-CD16/32, to block Fc receptors (clone 2.4G2; Pharmingen, San Diego, CA, USA), anti-IgM fluorescein isothiocyanate (FITC; clone R6-60.2; Pharmingen), anti-CD5 phycoerythrin (PE; clone 53-7.3; Pharmingen), anti-B220 perdinin chloraphyll protein (clone RA3-6B2; Pharmingen), and anti-MAC-1 biotin (clone M1/ 70.15; Caltag, Burlingame, CA, USA), followed by staining with streptavidin allophycocyanin (Caltag). PerC cells were stained with anti–Class II MHC antibodies I-Ad PE (clone AMS-32.1; Pharmingen) and I-Ab FITC (clone AF6-120.1; Pharmingen) to determine the origin of B-1a cells in allogeneic transfers. Analysis of staining data Four-color analysis was performed on the FACSCalibur (BD Immunocytometry Systems, San Jose, CA, USA). Analyses were assessed with CellQuest software (Becton Dickinson, San Jose, CA, USA). B-1a cells were identified by positive expression of IgM, B220, CD5, and MAC-1. B-1b cells were identified by positive expression of B220, IgM, and MAC-1 and lack of CD5 expression. B-2 cells were identified by a positive expression of IgM and B220 and a lack of expression of MAC-1 and CD5. Regions for all lymphocyte subpopulations were drawn after gating on lymphocytes in forward and side scatter. B-cell subsets percentages were reported as a percent of the total peritoneal B-cell population. Total cell numbers for B-cell subsets were determined by multiplying the percent of total leukocytes for each population with the cell counts that were determined by trypan blue exclusion.
Results and discussion Host peritoneal B cells are of donor origin Because SCID mice have a small number of B cells [31], we transferred allogeneic bone marrow (BALB/c) into C57Bl/6 SCID mice to trace the donor- and host-derived B cells. PerC B cells were analyzed for MHC class II markers specific for BALB/c donor (I-Ad) and host C57Bl/6 SCID (I-Ab) B cells 8 weeks after transfer of T-cell–depleted adult BALB/c bone marrow. The majority of PerC B cells (93.8%) in the SCID hosts were of donor origin (I-Ad) (Fig. 1).
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Figure 1. MHC II staining of a BALB/c mouse (left panel), a C57Bl/6 SCID mouse (middle panel), and a BALB/c bone marrow–engrafted C57Bl/6 SCID mouse (right panel). Events shown are gated on lymphocytes on forward and side scatter. BALB/c mice express I-Ad and C57Bl/6 SCID host mice express I-Ab. In the BALB/c bone marrow–engrafted SCID mouse, 93.8% of MHC-positive cells are of donor origin.
Strain differences in B-cell engraftment exist Differences in proportions of B-cell subsets in different strains of mice have been reported [3,4]. Studies that find poor peritoneal B-1a cell engraftment after adoptive transfers have typically been limited to one strain or two congenic strains. To examine the possibility that B-cell engraftment is strain dependent, we performed syngeneic transfers
of BALB/c (H-2Dd) adult bone marrow into BALB/c RAG-2⫺ and CB.17 SCID mice and C57Bl/6 (H-2Db) bone marrow cells into C57Bl/6 SCID and 129/RAG-2⫺ mice. As both RAG-2⫺ and SCID mice are deficient for B and T cells, transfers were made into nonirradiated hosts. A distinct B-1a population developed in all groups of mice (Fig. 2). Interestingly, the type of immunodeficiency, SCID or RAG 2⫺,
Figure 2. Peritoneal B-cell engraftment in nonirradiated SCID and RAG-2⫺ mice. Representative plots are shown. 107 T-cell–depleted adult C57Bl/6 bone marrow cells were transferred into 129/RAG-2 mice and C57Bl/6 SCID mice (top panels) and BALB/c bone marrow was transferred into BALB/c RAG-2 mice and CB.17 SCID mice (bottom panels). Four hosts were engrafted in each group. Mice were analyzed 8 weeks after transfer. These data represent results from a single bone marrow transfer experiment. The boxed region defines B-1a cells as CD5⫹ and IgM⫹, and the percentages of total B cells are reported as an average of the four mice in each group.
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alone did not predict the proportion of B-1a cells that developed. C57Bl/6 SCID mice (H-2Db) developed a significantly higher proportion of B-1a cells than 129/RAG-2⫺ mice (H-2Db) (Fig. 3A, unpaired t-test, p ⬍ 0.01), while proportions of B-1a cells in BALB/c RAG-2⫺ mice (H-2Dd) were not significantly different from CB.17 SCID (H-2Dd) (Fig. 3C). There was, however, a more clear-cut difference with regard to immunodeficiency and the absolute number of PerC B-1a cells that engrafted. Both strains of RAG-2⫺ mice had mean B-1a cell engraftment levels significantly below control levels at 8 weeks after the transfers (129/ RAG-2, p ⬍ 0.05; BALB/c RAG-2⫺, p ⬍ 0.0001). However, there was no significant difference in B-cell engraftment levels with the two strains of SCID mice (Fig. 3B and D). Radiation of host mice has an impact on B-cell engraftment The majority of published adoptive transfer experiments have been performed using irradiated hosts; however, the effects of radiation on B-cell engraftment have not yet been reported. To determine if irradiation of host mice impacts peritoneal B-cell development from adult bone marrow transfers, we transferred 107 BALB/c T-cell–depleted bone marrow cells into irradiated and nonirradiated CB.17 SCID mice and C57Bl/6 bone marrow cells into irradiated and nonirradiated 129/RAG-2 mice. To observe engraftment over time, mice were analyzed at 4, 8, and 12 weeks after transfers.
Differences between irradiated and nonirradiated mice were apparent. With respect to the proportion of the PerC B-cell subsets that engrafted, irradiation had a negative impact on B-1a cell proportions, and a positive impact on B-2 proportions. In both RAG-2⫺ and SCID recipients the proportion of peritoneal B-1a cells that engrafted in the irradiated mice was significantly less than the nonirradiated mice (p ⬍ 0.01, unpaired t-test). The differences were greater at earlier time points. At 4 weeks the mean proportion of B-1a cells was 12% in irradiated and 28% in nonirradiated hosts for the RAG-2⫺ mice and 15% in irradiated and 32% in the nonirradiated hosts for the SCID mice. At 12 weeks the differences were no longer significant for RAG-2⫺ mice, and for the SCID mice the difference was still significant at 13% in irradiated and 21% in nonirradiated hosts (p ⬍ 0.05). Comparisons of the total number of engrafted B-cell populations revealed differences between irradiated and nonirradiated hosts as well as differences between RAG-2⫺ and SCID mice (Fig. 4). Nonirradiated 129/RAG-2⫺ mice engrafted PerC B cells poorly during the entire 12-week period relative to C57Bl/6 donors. At all time points B-1a levels were significantly lower than control levels (p ⬍ 0.05, unpaired t-test). B-1b and B-2 levels were not significantly different from control levels. The irradiated 129/RAG-2⫺ mice, however, engrafted control levels of B-1a cells by 8 weeks and B-1b and B-2 by 4 weeks. Because nonirradiated
Figure 3. Syngeneic B-cell engraftment in nonirradiated RAG-2⫺ and SCID mice. 107 T-cell–depleted adult C57Bl/6 (H-2Db) bone marrow cells were transferred into 129/RAG-2⫺ mice and C57Bl/6 SCID mice and BALB/c (H-2Dd) bone marrow was transferred into BALB/c RAG-2⫺ mice and CB.17 SCID mice. Four hosts were engrafted in each group. PerC cells were analyzed 8 weeks after transfers. The data represent results from a single bone marrow transfer experiment. Mean percentages of total B cells (A and C) and mean absolute numbers (B and D) are shown for the donor mouse strains (C57Bl/6 and BALB/ c) and the two sets of bone marrow recipients. Error bars represent one standard deviation.
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Figure 4. Total number of peritoneal B-1a, B-1b, and B-2 cells in irradiated and nonirradiated RAG-2⫺ and SCID recipients of adult bone marrow. 107 C57Bl/6 (H-2Db) T-cell–depleted adult bone marrow cells were transferred into irradiated (800 rads) and nonirradiated 129/RAG-2 mice (top panels). Adult BALB/c (H-2Dd) bone marrow was transferred into irradiated (400 rads) and nonirradiated CB.17 SCID mice (bottom panels). Cells were analyzed 4, 8, and 12 weeks after transfer. Results from recipients of C57Bl/6 bone marrow represent data from one bone marrow transfer and the results from recipients of BALB/c bone marrow are a compilation of data from two bone marrow transfers. Statistics reported represent significant differences between irradiated and nonirradiated mice using unpaired t-tests: * p ⬍ 0.05, † p ⬍ 0.01, ‡ p ⬍ 0.001.
RAG mice engraft B cells so poorly it is difficult to assess the impact of irradiation on the differential engraftment of the three subpopulations. B-1a cell engraftment in SCID mice, however, was adversely affected by irradiation. Irradiated CB.17 SCID mice engrafted only half the level of B-1a cells of BALB/c donor mice at 12 weeks (p ⬍ 0.001), whereas the B-1a cells in nonirradiated mice reached control levels at 8 weeks. Number of bone marrow cells transferred does not affect the proportion of B-cell subsets that engraft It has previously been demonstrated in SCID mice that transferring limiting numbers of hematopoietic stem cells results in a preferential loss of B-1a cells [20]. If progenitors for B-1a cells are rare in adult bone marrow we would expect that transferring decreasing numbers of bone marrow cells would have a preferentially negative impact on peritoneal B-1a cell engraftment in irradiated RAG mice as well. We tested this hypothesis by transferring decreasing amounts of C57Bl/6 T-cell–depleted bone marrow cells into irradiated 129-RAG-2⫺ mice. Analysis of B-cell subsets in the peritoneal cavity 8 weeks after transfer revealed that there is no relationship between the number of cells transferred and the proportion of B-1a, B-1b, and B-2 cells that engraft. There was no difference in the proportions of B-1a cells in the PerC cavity of hosts receiving 3 ⫻ 107 cells and hosts receiving 105 cells (data not shown). Additionally,
limiting the numbers of bone marrow cells transferred did not preferentially affect the absolute number of B-1a cells that engraft (Fig. 5). When 105 T-cell–depleted bone marrow cells were transferred the engraftment level was onethird of the donor control for B-1a cells, three-quarters for B-1b cells, and only one-fifth for B-2 cells. When 5 ⫻ 106
Figure 5. Absolute numbers of engrafted peritoneal B-1a, B-1b, and B-2 cells. Irradiated 129/RAG-2⫺ mice (800 rads) received 105 (open bars, n ⫽ 5), 5 ⫻ 106 (horizontal striped bars, n ⫽ 5), or 3 ⫻ 107 (vertical striped bars, n ⫽ 3) T-cell–depleted adult C57Bl/6 bone marrow cells. The solid bars represent donor mice. Peritoneal cells were analyzed 8 weeks after transfers.
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cells were transferred both B-1a and B-2 levels reached those of the donor mice and B-1b cells were almost three times that of donor mice. These data contrast somewhat with the previous experiment, where at 8 weeks the mean level of B-1b cells was twice the level in donor mice and the B-2 cells were almost three times the number in donor mice; however, the standard deviations at this time point were very high for the irradiated mice (Fig. 4). These data imply that progenitors for B-1a cells are as numerous in adult bone marrow as are progenitors for B-1b and B-2 cells, or that pluripotent progenitors capable of giving rise to all three subpopulations exist. The separate lineage hypothesis relies largely on evidence obtained from adoptive transfer studies. Studies supporting this hypothesis have demonstrated that progenitor cells in different tissues at different times in ontogeny give rise to different subsets of B cells; specifically, transfers of fetal omentum allow B-1a and B-1b engraftment [22], while adult bone marrow supports B-1b and B-2 engraftment [7,13,19–21]. However, these data have been obtained from a limited number of mouse models under a limited set of conditions. This pattern of engraftment has been shown in particular for irradiated nonimmunodeficient BALB/c congenic mice (H-2Dd) [7,13,21] and irradiated C.B.17 SCID mice (also H-2Dd) [19,20]. Little has been done with adult bone marrow transfers in RAG-2⫺ mice, C57Bl/6 congenic mice (H-2Db), or nonirradiated immunodeficient mice of various strains. One study has shown convincing results for splenic B-1a–cell reconstitution from adult bone marrow in newborn nonirradiated SCID mice [23], but no previous studies have convincingly demonstrated PerC B-1a–cell engraftment from adult bone marrow in adult hosts. Although the data presented here do not directly refute the lineage hypothesis, they do contradict evidence that has been used to support it. The data shown in this report demonstrate that B-1a cells can develop under a variety of conditions from adoptive transfers of adult bone marrow into immunodeficient mice. In fact, transfers into nonirradiated H-2Db SCID mice allow peritoneal B-1a cell engraftment that does not differ significantly from both the proportion and the total number of B-1a cells present in donor mice (Fig. 3A and B). We do not believe that B-1a cell populations found in these mice are developing out of a rare progenitor population or rare circulating B-1a cells found in the bone marrow because limiting the number of bone marrow cells transferred did not result in a preferential decline of the B-1a cell population. No one variable reliably predicted B-1a cell development in these transfer studies, and there was considerable variation from mouse to mouse and between experiments. What tends to decrease the proportion of PerC B-1a cells are irradiation of host mice and/or H-2Dd haplotype, particularly in combination with the SCID defect. These are variables that have been present in a good majority of past studies that have shown reduced or absent B-1a development from adult bone marrow. Because we did not
see a failure of peritoneal B-1a cell engraftment from adult bone marrow, as was demonstrated in previous studies with irradiated immunocompetent H-2Dd mice [7,13,21], a comparison of B-cell engraftment in irradiated congenic immunodeficient and immunocompetent mice is of particular interest for future study. In summary, because there is considerable variability in levels of B-1a cell development depending on the strains used and the conditions of the cell transfer such as irradiation and time allowed for engraftment, caution should be taken when interpreting B-cell engraftment results of any single mouse model. Acknowledgments This work was supported by NIST Cooperative Agreement 70NANB7H3039. The authors would like to thank Manley Huang for his help with the preparation of this manuscript.
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Peritoneal B-cell development depends on strain, radiation, and time Marcia Stickler and Fiona Harding Department of Immunology, Genencor International Inc., Palo Alto, Calif., USA (Received 1 March 2000; revised 30 September 2000; accepted 6 October 2000)
Objective. B-1a, B-1b, and B-2 cells represent the three B-cell subsets in mice. Previous studies have demonstrated that peritoneal B-1a cell development is absent, or nearly so, from adult bone marrow transfers into irradiated adult hosts. The majority of these studies have been performed under a limited set of conditions with irradiated host mice. Here we examined that under a variety of conditions, peritoneal B-1a cells can develop in significant numbers from adult bone marrow transfers into severe combined immunodeficient (SCID) and recombination activation gene 2⫺ (RAG-2⫺) mice. Materials and Methods. Adult bone marrow was transferred into various strains of irradiated and nonirradiated adult immunodeficient RAG-2⫺ and SCID mice. Peritoneal B-cell engraftment was examined by fluorescein-activated cell sorting analysis and unpaired t-tests were used to determine significant differences of B-cell engraftment among the various conditions of cell transfer. Results. The level of B-1a cell engraftment was variously affected by the type of host immunodeficiency, the combination of donor and host strains, and the time allowed for engraftment. Irradiation of SCID, but not RAG-2⫺, host mice inhibited B-1a–cell engraftment. Additionally, decreasing the number of bone marrow progenitor cells transferred was not found to preferentially affect B-1a cell development in irradiated RAG-2⫺ hosts. Conclusion. In the context of these strains, we conclude that adult murine bone marrow contains progenitors that have the capacity to reconstitute peritoneal B-1a cell populations to donor levels. © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: B-1a cells—Engraftment—Adult bone marrow—SCID—RAG-2⫺
Introduction Three distinct B-cell subsets, B-1a, B-1b, and B-2 cells, exist in mice. These populations are distinguished by their phenotype, anatomical location, regulation, development, and function. There is disagreement over the origin of these subsets. One popular theory holds that these cells are derived from distinct lineages primarily because they have been shown to arise at different times during development [1–6]. B-1a and B-1b cells develop primarily from fetally derived precursors that produce a long-lived self-renewing population of mature cells that prevent further development from precursors in the adult animal by feedback inhibition [7–13]. B-2 cells, on the other hand, are relatively shortlived and repopulate themselves from progenitor cells throughout the life of the animal. Additionally, early-maturing B-1a cells have been shown to possess fewer N-region Offprint requests to: Marcia Stickler, M.S., Genencor International, 925 Page Mill Road, Palo Alto, CA 94304 USA; E-mail: [email protected]
insertions due to a lack of terminal deoxytransferase (TdT) expression early in development [14–18]. Evidence supporting the separate lineage theory was derived primarily from adoptive transfer studies. Transfers of fetal liver cells [7,19–21] and fetal spleen cells [7] have been demonstrated to replenish all B-cell subsets in the peritoneal cavity of recipient mice. However, transfers of fetal omentum cells into adult nonirradiated severe combined immunodeficient (SCID) mice have produced B-1a and B-1b cells but no B-2 cells [22]. Transfers of adult bone marrow into irradiated mice have failed to reconstitute B-1a cells in a number of studies [7,13,19–21] but have readily produced B-1b [13,21] and B-2 cells [7,13,19–20]. Other studies, however, have found B-1a cell engraftment from adult bone marrow transfers [12,23]. An alternative explanation of murine B-cell development is that the B-1a phenotype is the product of a differentiation pathway available to any B cell [24,25]. Subpopulations of B cells develop from a common pool of precursors that are selected for by antigen cross-linking of surface IgM. Induc-
0301-472X/01 $–see front matter. Copyright © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(00)0 0 6 4 4 - 5
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tion of the B-1a cell phenotype by treatment with anti-IgM has been demonstrated in vitro [23,26]. While evidence is compelling for both theories, neither one has been definitively proven. CD5 induction by IgM cross-linking has only been demonstrated in vitro, and evidence for separate lineages is conflicting. Studies that have not found B-1a cell reconstitution from adult bone marrow transfers were performed on a limited number of strains of irradiated hosts in which relatively low numbers of cells were transferred or the time allowed for reconstitution was limited. Irradiation of host mice causes a series of events that may affect the ability of B-1a cells to develop, including release of inflammatory cytokines and gut damage [27,28]. Additionally, genetically controlled differences in B-1a levels exist in different strains of mice [3] and this may impact the ability of these cells to engraft after transfer. Here we address some of the variables that may affect peritoneal B-cell engraftment. These include the number of cells transferred, the time allowed for engraftment, radiation conditioning, immunodeficiency induced by SCID vs RAG-2⫺ mutations, and MHC (major histocompatibility complex) haplotype of the host mice. We demonstrate that B-1a cells do in fact engraft in significant numbers from adult bone marrow transfers. However, the level of B-1a cell engraftment is variously affected by the type of host immunodeficiency, the combination of donor and host strains, and the time allowed for engraftment. Surprisingly, irradiation of SCID [29], but not RAG-2⫺ [30], host mice inhibits B-1a cell engraftment.
Materials and methods Mice Adult BALB/c, C57Bl/6, and CB.17 SCID and BALB/c RAG-2⫺ mice were purchased from Taconic (Germantown, NY, USA). C57Bl/6 SCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). 129/RAG-2⫺ mice were bred in house. All mice were kept under pathogen-free conditions. C57Bl/6 (H2Db) mice were used for bone marrow transfers into 129/RAG-2⫺ (H-2Db) mice and C57Bl/6 SCID (H-2Db) mice. BALB/c (H-2Dd) mice were used for transfers into CB.17 SCID (H-2Dd) mice, Balb/ c RAG-2⫺ (H-2Dd) mice, and C57Bl/6 SCID (H-2Db) mice. Twoto 4-month-old donor and recipient mice were used for bone marrow transfers. Bone marrow transfers and cell preparation Bone marrow was obtained from BALB/c and C57Bl/6 donor mice. Femurs and tibias were flushed with Dulbecco’s phosphatebuffered saline (D-PBS) and a single cell suspension was created. T cells were depleted by complement lysis using anti-CD90.2 (Thy 1.2, Pharmingen, San Diego, CA, USA) and rabbit and guinea pig complement (Cedarlane, Ontario, Canada). Briefly, anti-CD90.2 was added to extracted bone marrow at a concentration of 1 g/107 cells. Cells were incubated on ice for 30 minutes, washed with D-PBS, and added to 2.5 to 5 mL of MAR18.5 supernatant (mouse anti-rat IgG) plus a 1 in 20 dilution of rabbit and guinea pig complement. Cells were incubated for one hour at 37⬚C. T-cell–depleted
bone marrow cells were isolated using Histopaque (density ⫽ 1.119; Sigma, St. Louis, MO, USA). Mice receiving radiation were irradiated one day prior to transfers. RAG-2⫺ mice were lethally irradiated with 800 rads and SCID mice were lethally irradiated with 400 rads delivered by a 125Cesium source. Indicated numbers of cells were injected into the tail vein of RAG-2⫺ and SCID recipients. Peritoneal cavity (PerC) cells were harvested at the time of sacrifice. PerC cells were obtained by flushing the peritoneal cavity with 3 mL cold D-PBS with 2% fetal bovine serum. This method allowed for a recovery of approximately 90% of the injected volume. Any red cells present were lysed with ammonium chloride (Sigma). Cells were washed and counted using trypan blue exclusion. Antibodies and staining The frequency of T cells and B-cell subsets in the peritoneal cavity of donor and recipient mice was determined by flow cytometric analysis. PerC cells were stained with each of the following antibodies: anti-CD16/32, to block Fc receptors (clone 2.4G2; Pharmingen, San Diego, CA, USA), anti-IgM fluorescein isothiocyanate (FITC; clone R6-60.2; Pharmingen), anti-CD5 phycoerythrin (PE; clone 53-7.3; Pharmingen), anti-B220 perdinin chloraphyll protein (clone RA3-6B2; Pharmingen), and anti-MAC-1 biotin (clone M1/ 70.15; Caltag, Burlingame, CA, USA), followed by staining with streptavidin allophycocyanin (Caltag). PerC cells were stained with anti–Class II MHC antibodies I-Ad PE (clone AMS-32.1; Pharmingen) and I-Ab FITC (clone AF6-120.1; Pharmingen) to determine the origin of B-1a cells in allogeneic transfers. Analysis of staining data Four-color analysis was performed on the FACSCalibur (BD Immunocytometry Systems, San Jose, CA, USA). Analyses were assessed with CellQuest software (Becton Dickinson, San Jose, CA, USA). B-1a cells were identified by positive expression of IgM, B220, CD5, and MAC-1. B-1b cells were identified by positive expression of B220, IgM, and MAC-1 and lack of CD5 expression. B-2 cells were identified by a positive expression of IgM and B220 and a lack of expression of MAC-1 and CD5. Regions for all lymphocyte subpopulations were drawn after gating on lymphocytes in forward and side scatter. B-cell subsets percentages were reported as a percent of the total peritoneal B-cell population. Total cell numbers for B-cell subsets were determined by multiplying the percent of total leukocytes for each population with the cell counts that were determined by trypan blue exclusion.
Results and discussion Host peritoneal B cells are of donor origin Because SCID mice have a small number of B cells [31], we transferred allogeneic bone marrow (BALB/c) into C57Bl/6 SCID mice to trace the donor- and host-derived B cells. PerC B cells were analyzed for MHC class II markers specific for BALB/c donor (I-Ad) and host C57Bl/6 SCID (I-Ab) B cells 8 weeks after transfer of T-cell–depleted adult BALB/c bone marrow. The majority of PerC B cells (93.8%) in the SCID hosts were of donor origin (I-Ad) (Fig. 1).
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Figure 1. MHC II staining of a BALB/c mouse (left panel), a C57Bl/6 SCID mouse (middle panel), and a BALB/c bone marrow–engrafted C57Bl/6 SCID mouse (right panel). Events shown are gated on lymphocytes on forward and side scatter. BALB/c mice express I-Ad and C57Bl/6 SCID host mice express I-Ab. In the BALB/c bone marrow–engrafted SCID mouse, 93.8% of MHC-positive cells are of donor origin.
Strain differences in B-cell engraftment exist Differences in proportions of B-cell subsets in different strains of mice have been reported [3,4]. Studies that find poor peritoneal B-1a cell engraftment after adoptive transfers have typically been limited to one strain or two congenic strains. To examine the possibility that B-cell engraftment is strain dependent, we performed syngeneic transfers
of BALB/c (H-2Dd) adult bone marrow into BALB/c RAG-2⫺ and CB.17 SCID mice and C57Bl/6 (H-2Db) bone marrow cells into C57Bl/6 SCID and 129/RAG-2⫺ mice. As both RAG-2⫺ and SCID mice are deficient for B and T cells, transfers were made into nonirradiated hosts. A distinct B-1a population developed in all groups of mice (Fig. 2). Interestingly, the type of immunodeficiency, SCID or RAG 2⫺,
Figure 2. Peritoneal B-cell engraftment in nonirradiated SCID and RAG-2⫺ mice. Representative plots are shown. 107 T-cell–depleted adult C57Bl/6 bone marrow cells were transferred into 129/RAG-2 mice and C57Bl/6 SCID mice (top panels) and BALB/c bone marrow was transferred into BALB/c RAG-2 mice and CB.17 SCID mice (bottom panels). Four hosts were engrafted in each group. Mice were analyzed 8 weeks after transfer. These data represent results from a single bone marrow transfer experiment. The boxed region defines B-1a cells as CD5⫹ and IgM⫹, and the percentages of total B cells are reported as an average of the four mice in each group.
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alone did not predict the proportion of B-1a cells that developed. C57Bl/6 SCID mice (H-2Db) developed a significantly higher proportion of B-1a cells than 129/RAG-2⫺ mice (H-2Db) (Fig. 3A, unpaired t-test, p ⬍ 0.01), while proportions of B-1a cells in BALB/c RAG-2⫺ mice (H-2Dd) were not significantly different from CB.17 SCID (H-2Dd) (Fig. 3C). There was, however, a more clear-cut difference with regard to immunodeficiency and the absolute number of PerC B-1a cells that engrafted. Both strains of RAG-2⫺ mice had mean B-1a cell engraftment levels significantly below control levels at 8 weeks after the transfers (129/ RAG-2, p ⬍ 0.05; BALB/c RAG-2⫺, p ⬍ 0.0001). However, there was no significant difference in B-cell engraftment levels with the two strains of SCID mice (Fig. 3B and D). Radiation of host mice has an impact on B-cell engraftment The majority of published adoptive transfer experiments have been performed using irradiated hosts; however, the effects of radiation on B-cell engraftment have not yet been reported. To determine if irradiation of host mice impacts peritoneal B-cell development from adult bone marrow transfers, we transferred 107 BALB/c T-cell–depleted bone marrow cells into irradiated and nonirradiated CB.17 SCID mice and C57Bl/6 bone marrow cells into irradiated and nonirradiated 129/RAG-2 mice. To observe engraftment over time, mice were analyzed at 4, 8, and 12 weeks after transfers.
Differences between irradiated and nonirradiated mice were apparent. With respect to the proportion of the PerC B-cell subsets that engrafted, irradiation had a negative impact on B-1a cell proportions, and a positive impact on B-2 proportions. In both RAG-2⫺ and SCID recipients the proportion of peritoneal B-1a cells that engrafted in the irradiated mice was significantly less than the nonirradiated mice (p ⬍ 0.01, unpaired t-test). The differences were greater at earlier time points. At 4 weeks the mean proportion of B-1a cells was 12% in irradiated and 28% in nonirradiated hosts for the RAG-2⫺ mice and 15% in irradiated and 32% in the nonirradiated hosts for the SCID mice. At 12 weeks the differences were no longer significant for RAG-2⫺ mice, and for the SCID mice the difference was still significant at 13% in irradiated and 21% in nonirradiated hosts (p ⬍ 0.05). Comparisons of the total number of engrafted B-cell populations revealed differences between irradiated and nonirradiated hosts as well as differences between RAG-2⫺ and SCID mice (Fig. 4). Nonirradiated 129/RAG-2⫺ mice engrafted PerC B cells poorly during the entire 12-week period relative to C57Bl/6 donors. At all time points B-1a levels were significantly lower than control levels (p ⬍ 0.05, unpaired t-test). B-1b and B-2 levels were not significantly different from control levels. The irradiated 129/RAG-2⫺ mice, however, engrafted control levels of B-1a cells by 8 weeks and B-1b and B-2 by 4 weeks. Because nonirradiated
Figure 3. Syngeneic B-cell engraftment in nonirradiated RAG-2⫺ and SCID mice. 107 T-cell–depleted adult C57Bl/6 (H-2Db) bone marrow cells were transferred into 129/RAG-2⫺ mice and C57Bl/6 SCID mice and BALB/c (H-2Dd) bone marrow was transferred into BALB/c RAG-2⫺ mice and CB.17 SCID mice. Four hosts were engrafted in each group. PerC cells were analyzed 8 weeks after transfers. The data represent results from a single bone marrow transfer experiment. Mean percentages of total B cells (A and C) and mean absolute numbers (B and D) are shown for the donor mouse strains (C57Bl/6 and BALB/ c) and the two sets of bone marrow recipients. Error bars represent one standard deviation.
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Figure 4. Total number of peritoneal B-1a, B-1b, and B-2 cells in irradiated and nonirradiated RAG-2⫺ and SCID recipients of adult bone marrow. 107 C57Bl/6 (H-2Db) T-cell–depleted adult bone marrow cells were transferred into irradiated (800 rads) and nonirradiated 129/RAG-2 mice (top panels). Adult BALB/c (H-2Dd) bone marrow was transferred into irradiated (400 rads) and nonirradiated CB.17 SCID mice (bottom panels). Cells were analyzed 4, 8, and 12 weeks after transfer. Results from recipients of C57Bl/6 bone marrow represent data from one bone marrow transfer and the results from recipients of BALB/c bone marrow are a compilation of data from two bone marrow transfers. Statistics reported represent significant differences between irradiated and nonirradiated mice using unpaired t-tests: * p ⬍ 0.05, † p ⬍ 0.01, ‡ p ⬍ 0.001.
RAG mice engraft B cells so poorly it is difficult to assess the impact of irradiation on the differential engraftment of the three subpopulations. B-1a cell engraftment in SCID mice, however, was adversely affected by irradiation. Irradiated CB.17 SCID mice engrafted only half the level of B-1a cells of BALB/c donor mice at 12 weeks (p ⬍ 0.001), whereas the B-1a cells in nonirradiated mice reached control levels at 8 weeks. Number of bone marrow cells transferred does not affect the proportion of B-cell subsets that engraft It has previously been demonstrated in SCID mice that transferring limiting numbers of hematopoietic stem cells results in a preferential loss of B-1a cells [20]. If progenitors for B-1a cells are rare in adult bone marrow we would expect that transferring decreasing numbers of bone marrow cells would have a preferentially negative impact on peritoneal B-1a cell engraftment in irradiated RAG mice as well. We tested this hypothesis by transferring decreasing amounts of C57Bl/6 T-cell–depleted bone marrow cells into irradiated 129-RAG-2⫺ mice. Analysis of B-cell subsets in the peritoneal cavity 8 weeks after transfer revealed that there is no relationship between the number of cells transferred and the proportion of B-1a, B-1b, and B-2 cells that engraft. There was no difference in the proportions of B-1a cells in the PerC cavity of hosts receiving 3 ⫻ 107 cells and hosts receiving 105 cells (data not shown). Additionally,
limiting the numbers of bone marrow cells transferred did not preferentially affect the absolute number of B-1a cells that engraft (Fig. 5). When 105 T-cell–depleted bone marrow cells were transferred the engraftment level was onethird of the donor control for B-1a cells, three-quarters for B-1b cells, and only one-fifth for B-2 cells. When 5 ⫻ 106
Figure 5. Absolute numbers of engrafted peritoneal B-1a, B-1b, and B-2 cells. Irradiated 129/RAG-2⫺ mice (800 rads) received 105 (open bars, n ⫽ 5), 5 ⫻ 106 (horizontal striped bars, n ⫽ 5), or 3 ⫻ 107 (vertical striped bars, n ⫽ 3) T-cell–depleted adult C57Bl/6 bone marrow cells. The solid bars represent donor mice. Peritoneal cells were analyzed 8 weeks after transfers.
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cells were transferred both B-1a and B-2 levels reached those of the donor mice and B-1b cells were almost three times that of donor mice. These data contrast somewhat with the previous experiment, where at 8 weeks the mean level of B-1b cells was twice the level in donor mice and the B-2 cells were almost three times the number in donor mice; however, the standard deviations at this time point were very high for the irradiated mice (Fig. 4). These data imply that progenitors for B-1a cells are as numerous in adult bone marrow as are progenitors for B-1b and B-2 cells, or that pluripotent progenitors capable of giving rise to all three subpopulations exist. The separate lineage hypothesis relies largely on evidence obtained from adoptive transfer studies. Studies supporting this hypothesis have demonstrated that progenitor cells in different tissues at different times in ontogeny give rise to different subsets of B cells; specifically, transfers of fetal omentum allow B-1a and B-1b engraftment [22], while adult bone marrow supports B-1b and B-2 engraftment [7,13,19–21]. However, these data have been obtained from a limited number of mouse models under a limited set of conditions. This pattern of engraftment has been shown in particular for irradiated nonimmunodeficient BALB/c congenic mice (H-2Dd) [7,13,21] and irradiated C.B.17 SCID mice (also H-2Dd) [19,20]. Little has been done with adult bone marrow transfers in RAG-2⫺ mice, C57Bl/6 congenic mice (H-2Db), or nonirradiated immunodeficient mice of various strains. One study has shown convincing results for splenic B-1a–cell reconstitution from adult bone marrow in newborn nonirradiated SCID mice [23], but no previous studies have convincingly demonstrated PerC B-1a–cell engraftment from adult bone marrow in adult hosts. Although the data presented here do not directly refute the lineage hypothesis, they do contradict evidence that has been used to support it. The data shown in this report demonstrate that B-1a cells can develop under a variety of conditions from adoptive transfers of adult bone marrow into immunodeficient mice. In fact, transfers into nonirradiated H-2Db SCID mice allow peritoneal B-1a cell engraftment that does not differ significantly from both the proportion and the total number of B-1a cells present in donor mice (Fig. 3A and B). We do not believe that B-1a cell populations found in these mice are developing out of a rare progenitor population or rare circulating B-1a cells found in the bone marrow because limiting the number of bone marrow cells transferred did not result in a preferential decline of the B-1a cell population. No one variable reliably predicted B-1a cell development in these transfer studies, and there was considerable variation from mouse to mouse and between experiments. What tends to decrease the proportion of PerC B-1a cells are irradiation of host mice and/or H-2Dd haplotype, particularly in combination with the SCID defect. These are variables that have been present in a good majority of past studies that have shown reduced or absent B-1a development from adult bone marrow. Because we did not
see a failure of peritoneal B-1a cell engraftment from adult bone marrow, as was demonstrated in previous studies with irradiated immunocompetent H-2Dd mice [7,13,21], a comparison of B-cell engraftment in irradiated congenic immunodeficient and immunocompetent mice is of particular interest for future study. In summary, because there is considerable variability in levels of B-1a cell development depending on the strains used and the conditions of the cell transfer such as irradiation and time allowed for engraftment, caution should be taken when interpreting B-cell engraftment results of any single mouse model. Acknowledgments This work was supported by NIST Cooperative Agreement 70NANB7H3039. The authors would like to thank Manley Huang for his help with the preparation of this manuscript.
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