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We recently found that all trans retinoic acid (ATRA) accelerated B lymphocyte formation, and have now asked if retinoids account for the rapid lymphopoiesis that is characteristic of fetal progenitors. Surprisingly, addition of ATRA to fetal liver cultures actually reduced B lymphopoiesis. A pan-retinoid receptor antagonist selectively suppressed lymphocyte formation from fetal and adult progenitors, suggesting some normal contribution of retinoids to this process. Consistent with this role, B lymphopoiesis was compromised in marrow of mice with prolonged vitamin A deficiency (VAD). Recently identified B1 progenitors from adult marrow were similar to conventional B2 progenitors in being stimulated by ATRA. The inhibitory response observed with fetal cells was seen when adult progenitors were exposed to high doses in culture or when adult mice were treated with ATRA for two weeks. In addition to explosive lymphocyte generation, fetal progenitors tend to be less IL-7 dependent than their adult counterparts, but ATRA did not make cultures IL-7 independent. We conclude that all known categories of B lineage progenitors are responsive to retinoids and probably regulated by these compounds under physiological conditions. Retinoids may account in part for rapid differentiation in fetal life, but not all unique features of fetal progenitors.
Retinoids have been extensively studied in connection with hematopoiesis and immune system development. As one recent area of attention, retinoids control the migration and differentiation of regulatory T cell subsets as well as isotype switching in mucosal B cells . Additional effects of these vitamin A metabolites on stem cells, progenitors and the microenvironment in bone marrow are complex and not well understood. For example, retinoids and their receptors can promote terminal differentiation of normal and malignant granulocytes in culture, but vitamin A deficient (VAD), retinoid antagonist treated or RARγ−/− mice have pronounced granulocytosis [2–4]. Furthermore, a series of papers documented direct inhibition by retinoids of stem and progenitor cell proliferation as well as differentiation [5–8]. There is also evidence for regulation of marrow stromal cell functions by retinoids [4,9]. No doubt the influence of retinoids on blood cell formation is dependent on the nature and environmental context of the target cells.
Further investigation could reveal ways to exploit retinoids for maintaining stem cell integrity, augmenting recovery of immune functions following myeloablation and regulating vaccine responses. In addition, clinical use of these compounds is widespread and undesirable consequences might be avoided. The present study involves a single phenomenon, the ability of retinoids to accelerate B lymphopoiesis. That is, exposure of lymphoid progenitors to substances such as ATRA shortens the time required for them to generate B lineage lymphocytes .
The initial production of lymphocytes during fetal life differs substantially from the process that sustains immune system replenishment in adult bone marrow . For example, fetal lymphocyte production is uniquely independent of the cytokine IL-7 and is biased towards generation of relatively rare B1 type cells  [13, 14]. Moreover, fetal progenitors differentiate much more rapidly in culture . The goal of this study was to learn if retinoids in fetal hematopoietic tissues could be responsible for any of these characteristics. As precedent, the fates of human fetal hematopoietic cells are directed to granulocyte differentiation by retinoids, and primitive hematopoiesis in VAD quail embryos is defective [16, 17]. In the mouse, erythropoiesis is retinoid dependent, but only at a discrete stage of fetal life .
We found that the differentiation of fetal lymphoid progenitors in culture was actually inhibited by ATRA, and in this respect resembled adult cells exposed to high doses or for long intervals. Other results suggest that retinoids regulate fetal lymphopoiesis, but do not determine all unique features of that process.
Production of B lymphocytes from fetal progenitors is more rapid than from their adult counterparts  and we asked if they are similarly responsive to retinoids. That is, the adult process is accelerated by ATRA and it seemed possible that retinoids account for the behavior before birth. A fetal liver fragment culture model was selected in order to approximate conditions in utero. While a variety of retinoids accelerate generation of CD19+ cells from multiple stages of adult bone marrow , 10−7M ATRA actually reduced B lymphopoiesis in the fetal cultures (Fig. 1A).
Using previously described sorting procedures , we isolated Lin− Sca-1+ c-KitHi RAG1/GFP− (LSK), Lin− Sca-1+ c-KitHi RAG1/GFP+ early lymphoid progenitors (ELP) and Lin− Sca-1+ c-KitLo RAG1/GFP+ pro-lymphocytes (Pro-L) from RAG-1/GFP mice, and Lin− c-Kit+ B220+ pro-B cells from C57BL/6 E14.5 fetal liver. These were then placed in co-cultures with OP9 stromal cells. Addition of 10−7M ATRA inhibited B lymphopoiesis in all of these cultures (Fig. 1B). The same was true in time course experiments where 10−8M ATRA was added to fetal LSKs (Fig. 1C).
The inhibitory effect of ATRA could have been caused by influences on stromal cells, lymphoid progenitors or both. Consequently, we performed titration experiments with fetal liver LSKs and using stromal cell free, serum free culture conditions optimized for B lineage differentiation. ATRA inhibited fetal B lymphopoiesis in a dose dependent manner (Fig. 1D).
Ebf1 and Pax5 are essential for B lymphopoiesis, and we previously found that levels of these transcription factors in adult progenitors increased in response to retinoids .Consistent with the rapid differentiation of fetal progenitors, basal levels of these transcription factors were higher than in their adult counterparts (Fig. 2). While exposure to ATRA increased Pax5 transcripts, Ebf1 levels diminished. This analysis was conducted at an 48 hr interval to better appreciate proximal changes in transcription factor expression. No CD19+ cells were present in any cultures at that time.
Thus, fetal progenitors rapidly produce B lineage lymphocytes and we were surprised to find that addition of ATRA suppresses this process. Our experiments suggest the progenitors are directly sensitive to this retinoid and the mechanism could involve down-regulation of Ebf1.
Retinoids are thought to play multiple roles during embryonic/fetal life [1, 5]. Given the complexity and redundancy of retinoid receptors, these phenomena can be hard to appreciate with gene targeting experiments. Consequently, we used the pan RAR antagonist LE540 in our culture systems. As expected, exposure of adult LSKs to LE540 blocked the enhancing effect of ATRA (Fig. 3A, left panel). Furthermore, it inhibited the high constitutive level of B lymphopoiesis in cultures initiated with fetal LSKs (Fig. 3A, middle and right panels). By contrast, myeloid cell production in the same cultures was minimally influenced by ATRA addition and not responsive to LE540 (Fig. 3B). The inhibitory effect of LE540 was partially rescued when ATRA was also present in the fetal liver cultures (Figure 3A, right panel). However, the correct ratio of compounds may not have been optimal, because the rescue was not reflected in absolute numbers of recovered lymphocytes (Figure 3A, middle panel). We conclude that lymphoid progenitors are uniquely sensitive to retinoids that may naturally be present in fetal and adult tissues as well as the serum used for our cultures.
Protocols have been developed for depletion of vitamin A in adult mice, and one study concluded that it caused granulocytosis with slight suppression of B cell numbers in bone marrow . Similar observations were made by treatment with a retinoid antagonist compound, while a significant reduction in pre-B cells was reported in retinoid acid receptor gamma (RARγ−/) mice [3,4]. These experimental designs were not ideal for determining if steady state lymphocyte formation is vitamin A dependent. Therefore, normal pregnant mice were given vitamin A deficient food 10 days after observation of vaginal plugs. The offspring were then weaned at three weeks of age and maintained on the same diet. As previously noted , body weights were slightly sub-normal at 8 weeks of age relative to control diet fed controls, and there was an average 14% difference by 12 weeks of age when all of our measurements were made. Numbers of total bone marrow (Fig. 4A, bone marrow, first panel), as well as the stem and progenitor rich (Lin−Scal1+c-Kithi ; LSK) fraction (Table 1) were within the normal range and additional analysis of this subset was done using RAG1/GFP knock-in mice . This revealed that RAG1+ early lymphoid progenitors (ELP) were significantly elevated (Fig. 4A, bone marrow, second panel). Small, but consistent increases were found in numbers of Lin−Scal1+c-KitLo IL-7Rα+ common lymphoid progenitors (CLP; Fig. 4A, bone marrow, third panel). In contrast to this accumulation of primitive lymphopoietic cells, absolute numbers of CD19+ B220+ lymphocytes were reduced approximately 5 fold (Fig. 4A, bone marrow, fourth panel). Furthermore, small pre-B cells defined as CD19+ CD43− sIgM− were reduced (Table 1). We found no perturbations in mature B cells or T lymphoid lineage cells in either the spleen or thymus.
As noted in a previous report , we found evidence for abnormally elevated cells expressing CD11b/Mac-1 in marrow and spleens of vitamin A deficient mice (Fig. 4A). This abnormality in myelopoiesis was not matched by changes in Lin−Scal1+c-Kitlow common myeloid progenitors (CMP) (Table 1). Natural killer cells marked by expression of NK1.1 were significantly elevated in the spleen (Fig. 4A, spleen, forth panel). The same was true for CD19− B220+ CD11c+ CD11b− Ly6c− NK1.1+ interferon producing killer dendritic cells (IKDC; Table 1) that are developmentally related to NK cells [22,23].
CD19− B220− CD11c+ CD11b+ dendritic cells normally comprise a small fraction of hematopoietic tissues, although their absolute numbers enhanced in bone marrow, they were minimally influenced by vitamin A deficiency in spleen (Table 1). CD19− B220+ CD11c+ CD11b− Ly6c+ NK1.1− plasmacytoid dendritic cells (pDC) are similar in some respects to cells in the B lineage, but pDC cell numbers were unaffected by vitamin A deficiency (Table 1).
LSK and CLP were then recovered from VAD and control mice to test their competence for B lymphopoiesis. LSKs were placed in OP9 stromal cell co-cultures while CLP were held in stromal cell free, serum free B cell cultures (Fig. 4B). These are the same culture conditions we previously used to learn that B lymphopoiesis is accelerated by ATRA . Yields of CD19+ lymphocytes from hematopoietic progenitors recovered from Vitamin A deficient mice were three fold less than those from control diet animals.
We conclude that late stages of B lymphopoiesis and potential for lymphoid progenitors to produce B lineage cells were depressed in bone marrow of vitamin A deficient mice. Numbers of mature lymphocytes in the spleen were within the normal range.
The above findings would be consistent with an influence of retinoids on the initial development of the immune system, and fetal progenitors respond as though they have already been exposed. Among other fetal/adult differences, the first wave of B lymphopoiesis is heavily biased to produce specialized B1 cells . A B1 restricted Lin− B220− CD19+ AA4.1/CD93+ progenitor has been recently identified in fetal liver and bone marrow , and we wondered if it would be responsive to retinoids.
B1 biased progenitors (B1P) require special culture conditions, and we used ones slightly modified from those described by Montecino-Rodriguez and Dorshkind (Materials & methods). Adult marrow B1P cultures produced B220+Mac1LoIgM+ B1 cells, and yields were dramatically increased when ATRA was present (Fig. 5A).
As noted above (Fig. 4), percentage of B cells were reduced in spleens of VAD mice. Further examination of the peritoneal cavity revealed that both IgM+ CD19+ B220+ CD11b/Mac-1Lo CD5+ B1a and the otherwise similar CD5− B1b cells were reduced in VAD mice (Fig. 5B).
We conclude that progenitors of B1 lymphocyte subsets in bone marrow are like those for B2 cells. That is, their differentiation is enhanced by ATRA and mature lymphocyte numbers are reduced in vitamin A deficient mice.
These findings represented a paradox. While adult B lymphopoiesis is accelerated by ATRA, the same compound suppressed the already rapid fetal process. In addition, the retinoid antagonist results shown above (Fig. 3) indicate there was at least a history of endogenous exposure in utero. A possible explanation is suggested by the fact that high retinoid concentrations inhibit the differentiation of adult progenitors in culture . While B cell numbers are elevated in animals treated for one week with time-release ATRA containing pellets, we now show that more prolonged treatment with the same protocol is also inhibitory (Fig. 6A). Bones from long-term treated mice were noticeably fragile, and numbers of total nucleated cells per femur decreased from approximately 25 million to 19 million. Absolute numbers of CLP and B220+ CD19+ lymphoid cells were also significantly reduced. In contrast, numbers of CMP were elevated, accounting for the fact that CD11b+ myeloid cells were within the normal range. Spleens of two week ATRA treated mice contained fewer total cells (Fig. 6B); with the exception of B cells, declines in all cell types were statistically significant.
These findings suggest that high doses or prolonged therapeutic use of retinoids could be detrimental to lymphocyte production in bone marrow. However, we also found that progenitors harvested from two week treated animals were still potent when placed in culture (Fig. 6C). That is, LSK and Lin− Scal1+c-KitLo pro-lymphocyte (Pro-L) survived well and generated CD19+ cells with high yields. Thus, abnormalities associated with prolonged therapy might be readily reversible. The observations indicate that fetal lymphocyte formation may be driven by optimal concentrations of retinoids, and that additional stimulation can be inhibitory.
Fetal lymphopoiesis is known to be relatively independent of IL-7 [12, 13], and we wondered if this might be due to the enhancing influence of endogenous retinoids. Therefore, we initiated stromal cell co-cultures where SCF and Flt3L were the only recombinant cytokines added to adult progenitors. LSKs generated substantial numbers of B220+ CD19+ lymphocytes under these conditions, and again ATRA had a dramatic accelerating effect (Fig. 7A). Limiting dilution experiments revealed that a much higher fraction of LSK generated lymphocytes in ATRA containing cultures (Fig. 7B). That is, precursor frequencies increased from 1/186 to 1/38 when the compound was added. Although no IL-7 was added to these cultures, it seemed possible that small amounts were provided by the OP9 stromal cells. This possibility was investigated by addition of the IL-7 neutralizing M25 monoclonal antibody or initiating cultures with IL-7Rα−/− LSKs (Fig. 7C). We conclude that while ATRA accelerates adult B lymphopoiesis under sub-optimal conditions, it does not replace the requirement for IL-7.
These experiments extended our previous findings suggesting that retinoids are rate limiting for B lymphopoiesis in adult bone marrow. Although addition of ATRA to bone marrow cultures and treatment of adult mice accelerates the process, the same compound actually was inhibitory with fetal lymphoid progenitors. Furthermore, experiments with a retinoid antagonist suggest these compounds are active in fetal life. A possible explanation for this paradox comes from the fact that high retinoid concentrations are suppressive with adult cells and that rapid lymphocyte production cannot be sustained for long periods in adult mice. Other findings relate to unique fetal characteristics that seem not to be retinoid regulated, as well as a reexamination of vitamin A deficiency.
Lymphopoiesis is robust during fetal life, and differs from the adult process in a number of ways [11, 14, 19]. Hematopoietic cells in early embryos are dependent on retinoids [17, 18], and we wondered if these compounds affect initial emergence of the immune system. Surprisingly, ATRA did not promote, and in fact suppressed lymphocyte formation when added to cultures of fetal lymphoid progenitors. It seemed possible that this was because the cells had already been in a retinoid rich environment. Addition of a pan-retinoic acid receptor antagonist to fetal cultures selectively blocked lymphocyte formation. The substance was not toxic to myeloid progenitors, and the results suggest there is a minimal requirement for retinoids in utero.
It has been difficult to assess the importance of retinoids to normal, steady-state blood cell formation, so we reexamined VAD mice. The protocols we used have been well described and result in extensive depletion of the vitamin by 8 weeks of age [2, 20]. Therefore, our determinations of lymphopoiesis were made in 12 week old animals. While primitive lymphoid progenitors were more abundant in VAD mice, their potential for generating lymphocytes in culture was substantially reduced. The five fold reduction in pre-B cell numbers in bone marrow might have resulted from a reduced rate of differentiation beyond the ELP and CLP stages. Mature B cell numbers were unaffected by vitamin A deficiency. This is presumably because retinoid levels are high in young animals, and most mature lymphocytes are long-lived  . Considerable time might be required to completely deplete vitamin A and see changes in peripheral B cell numbers. Granulocyte numbers increase dramatically in VAD and RARγ−/− mice [2, 4], and Fig. 4A), and it is conceivable that phenomenon is linked to decreased B lymphopoiesis. For example, lymphoid progenitor might undergo some type of lineage redirection. However, numbers of myeloid colony forming cells, as well as myeloid progenitors enumerated by flow cytometry were unaffected by VAD. Furthermore, apoptosis was previously found to be reduced in myeloid, but not in lymphoid cells .
Retinoids accelerate the differentiation of highly purified progenitors cultured under defined conditions, while the same substances are inhibitory at high concentrations. Others have found situations where expansion of hematopoietic cells was inhibited by retinoids [6, 7]. We now report that two week treatment of animals is also detrimental to the process, but increased bone fragility and reduced total cellularity might indicate that the environment is harmed by extended exposure to high concentrations of retinoid. Indeed, early and late categories of lymphoid progenitors were functional when harvested from these mice and placed in culture. Interestingly, microenvironmental components rather than hematopoietic cells were defective in the absence of RARγ−/− mediated signaling . These observations indicate that retinoid concentrations must be kept within a critical range, and suggest that suppression of lymphopoiesis in extensively treated patients might be fully reversible. Retinoids might someday be used to promote recovery of humoral immunity following chemotherapy and/or bone marrow transplantation.
IL-7 is essential for B lymphocyte formation in adult bone marrow, but not in embryos . ATRA accelerated B lymphopoiesis in cultures with sub-optimal IL-7 and increased the frequency of functional progenitors. However, dependence on this cytokine was not obviated.
A unique B1 category of lymphocytes is thought to arise first in fetal life and progenitors that are highly biased to produce them have been recently identified . We isolated these B1 progenitors from adult bone marrow and found that their differentiation was enhanced by exposure to ATRA in culture. Furthermore, both of the two known subsets of B1 cells (B1a and B1b) were reduced in the peritoneal cavities of VAD mice. Thus, we believe that retinoids are essential to fetal lymphopoiesis and could account in part for the rapid differentiation of fetal cells in culture. In addition, these substances influence the whole spectrum of B lineage cells in adults. However, exposure to retinoids in utero is unlikely to account for all fetal/adult differences.
C57BL6/J mice, IL-7Rα−/− (Jackson Laboratory, Bar Harbor, ME) or RAG1/GFP knock-in mice  were used for all experiments. Pregnant animals in the second week of gestation were fed with vitamin A deficiency (VAD) diet (TD.88407, Harlan Teklad, Madison, WI) or control diet (TD.88406, Harlan Teklad) as has been described [20,26]. Pups were weaned to the same diet as their dams at age 3 weeks until they were used at 8 – 12 weeks of age.
Time-release pellets of all trans retinoic acid (10 mg/pellet) were purchased from Innovative Research of America (Sarasota, FL) and implanted subcutaneously with a 10-gauge precision trochar. After 2 weeks, mice were killed and bone marrow, splenocytes, thymocytes and B1 cells in the peritoneal cavity were isolated and analyzed by flow cytometry.
Regarding mAbs for murine antigens, anti-CD3, anti-CD8, anti-CD19 (1D3), anti- CD45RA (14.8), anti-Mac-1/CD11b, anti-FcRγ, anti-TER119 mAbs were purified from the cultured supernatants of hybridoma cells grown in our laboratory. Purified anti-Ly-6G (Gr-1) mAbs, Biotin-conjugated anti-CD3 (145-2C11), anti-CD8 (53–6.7), anti-CD19 (1D3), anti-CD45R/B220 (RA3/6B2), anti-Mac-1 (M1/70), anti-Gr1 (RB6/8C5), anti-NK1.1 (PK136), anti-Ter119 (ly-76), and anti-CD43 (S7) mAbs, allophycocyanin-conjugated anti-c-kit (2B8), anti-Mac1 (M1/70), and anti-NK1.1 (PK136), APC-Cy7 conjugated anti-CD19 (1D3), anti-CD4 (GK1.5) and anti-CD25 (PC61), PE-Cy5-confugated-anti CD45RA/B220, PerCp-CY5.5-conjugated anti-CD45.2 (104), and anti-IgM (R6-60.2), PE-conjugated anti-IL-7Rα (SB199), anti-CD11c (HL3), and anti-CD3 (145-2c11) mAbs were all purchased from BD PharMingen (San Diego, CA). PE-Cy5-conjugated anti-Sca1 (D7), anti-Gr-1 (RB6-8C5), anti-CD44 (IM7), allophycocyanin-conjugated anti-CD93 (AA4.1), PE-conjugated anti-IgM (eB121-15F9), anti-CD5 (53-7.3), anti-F4/80 (BM8), and anti-CD11b (M1/70) were obtained from eBioscience (San Diego, CA). PE-Texas red tandem-conjugated streptavidin, allophycocyanin-conjugated streptavidin, and PE-Cy5,5-conjugated streptavidin were purchased from Caltag Laboratories (Burlingame, CA).
Cells recovered from animals or cultures were treated with anti-FCRγ (Fc gamma RIII/II, 2.4G2) antibody to minimize nonspecific binding. Cells were stained with antibodies, and flow cytometry analyses were performed on a LSR II using the BD FACSDiva Software (BD Biosciences, San Jose, CA).
Mouse bone marrow cells collected from 6–12 week old mice or fetal liver cells obtained at embryonic days 14.5 were suspended in PBS buffer supplemented with 3% FCS. Cells were incubated with mAbs to lineage markers (Gr-1 and Mac-1 for myeloid cells, CD19 and CD45RA for B lineage cells, TER-119 for erythroid cells, CD3 and CD8 for T lineage cells, and NK1.1 for NK cells), followed by incubation with goat anti-rat IgG-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD19 and B220 were excluded from the Lin depletion cocktail when pro-B cells were isolated. Lineage cells attached to beads were removed with a magnetic separator. The lineage-depleted bone marrow cells were then incubated with a mixture of labeled Abs to the lineage markers, PE-Cy5-anti-Sca-1 and APC-anti-c-Kit. Cells were sorted on a MoFlo Cell Sorter (Dako, Fort Collins, CO) or FACSAria (BD Biosciences, San Jose, CA). The LSK fraction from RAG1/GFP knock-in mouse bone marrow was defined as Lin−GFP− Scal-1+c-kitHi ,while ELP were defined as Lin− GFP+Sca1+c-kitHi, ProL were defined as Lin−Sca1+c-kitLo, and pro-B cells were defined as Lin− c-Kit+ B220+. Purities of each sorted population were confirmed by post-sorting analyses.
Fetal liver organ culture was conducted as previously described . Briefly, small pieces of fetal liver were put on nuclepore Track Etch membranes (Whatman, Florham Park, NJ) supported with Procare sponges (Medipost Limited, Weymouth, United Kingdom) and cultured in 12-well flat-bottom culture plates containing DMEM-based medium supplemented with 5 × 10−5 M 2-mercaptoethanol and 1 × MEM nonessential amino acids (GIBCO/Invitrogen Corporation, Carlsbad, CA) plus 0.03% Primatone RL (Mediatech/Cellgro, Herndon, VA) with 2% FCS. Fragments were incubated in a humidified CO2 incubator at 37°C with or without ATRA Cells were recovered 6 days later and then evaluated by flow cytometry.
All trans retinoic acid (ATRA) was purchased from Sigma Chemical Co. (St. Louis, MO). LE-540 was from Wako Chemicals USA, Inc. (Richmond, VA). Double sorted progenitor cells were co-cultured with OP9 stromal cells in 96 well plates. The α-MEM medium (GIBCO/Invitrogen Corporation) contained 10% FCS, rm SCF (20 ng/ml), rm FL (10 ng/ml), rm IL-7 (1 ng/ml), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. To investigate the ATRA effect to B lymphopoiesis under sub-optimal conditions, the IL-7 cytokine was deliberately not added in OP9 stromal cell co-culture. At the end of culture, hematopoietic cells were counted excluding stromal cells and then subjected to flow cytometry. We also used anti-CD45.2 mAb to exclude potential contamination of stromal cells. To neutralize endogenous IL-7, purified monoclonal M25 antibody (1 ng/ml) was added to sub-optimal cultures .
Double sorted LSK cells were co-cultured with OP9 stromal cells in 96 well plates for up to 14 days. The α-MEM medium contained 10% FCS, rm SCF (20 ng/ml), rm FL (10 ng/ml), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. The IL-7 cytokine was deliberately not added in this type of culture. At the end of culture, hematopoietic cells were counted excluding stromal cells and then subjected to flow cytometry. We also used anti-CD45.2 mAb to exclude any potential contamination of stromal cells. To neutralize endogenous IL-7, purified monoclonal M25 antibody (1 ng/ml) was added.
Double or triple sorted progenitor cells were put in X-Vivo medium containing 1% detoxified bovine serum albumin, 20 ng/ml SCF, 100 ng/ml Flt3-L, 1ng/ml IL-7, 5 × 10−5 M 2-mercaptoethanol (Sigma), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin as described . Cells were analyzed by flow cytometry 7 to 11 days later.
B1 cell co-cultures were prepared as described  with slight modification. In brief, double sorted B-1P were co-cultured in direct contact with OP9 stromal cells in flat bottom 96 well plates. The α-MEM medium contained 10% FCS, rm SCF (20 ng/ml), rm Flt3-L (10 ng/ml), rh TSLP (10 ng/ml), rm IL-3 (20 ng/ml), rm IL-6 (20 ng/ml), 5 × 10−5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cultures were fed twice weekly and were analyzed for B−1 cell development 2 weeks later.
To analyze the Ebf1 and Pax5 expression pattern affected by ATRA, double sorted fetal liver and adult LSK cells were treated with vehicle (0.1% ethanol) or ATRA in stromal cell free culture for 48 hours. The treated cells were washed, and total RNA was isolated using RNeasy Mini Kit (Qiagen). Total RNA was treated with DNase I (Invitrogen) to remove contaminating genomic DNA, and cDNA was made using random primers (Invitrogen) and moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). The 20 µl real-time PCR amplification mixture contained template cDNA, 1 µl of pre-designed Taqman gene expression assays (Applied Biosystems, Foster City, CA) and 10 µl 2× Taqman PCR Master Mix (ABI). Reactions were 50°C 2 min, 95°C 10 min followed by 40 cycles of 95°C 15 Sec and 60°C 1min. Relative gene expression was calculated as 2−ΔΔCT values with eukaryotic 18sRNA used as an endogenous control. Real-time PCR was performed on an ABI PRISM 7500 (Applied Biosystems, Foster City, CA). Three repeats were run for each sample, and each experiment was performed three times.
The Prism V5.01 software (GraphPad, San Diego, CA) was used for statistical analyses. Student’s t-test was used to compare treated groups with control groups in all case. *, p < 0.05, and **, p < 0.01.
The authors are grateful to Jacob Bass and Diana Hamilton for cell sorting, Tara Khamphanthala for technical assistance, Beverly Hurt for graphics assistance, and Shelli Wasson for editorial assistance. Dr. Fred Finkelman provided important reagents and valuable advice. This work was supported by grants AI20069 and AI58162 from the National Institutes of Health. P.W.K. holds the William H. and Rita Bell Endowed Chair in Biomedical Research.
The authors have no financial or commercial conflict of interest.