Cytokine Regulation of Early Lymphohematopoietic Development

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1 Concise Review Cytokine Regulation of Early Lymphohematopoietic Development Fumiya Hirayama, Makio Ogawa The Department of Medicine, Medical University of South Carolina and the Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina, USA Key Words. Lymphohematopoietic progenitors Early lymphopoiesis Cytokine regulation Abstract. A two-step methylcellulose culture provided a method to study the differentiation of murine lymphohematopoietic progenitors. In the presence of two cytokines, one from a group consisting of Steel factor (SF) and flt3/flk2 ligand (FL) and the other from a group consisting of interleukin 6 (IL-6), G- CSF, IL-11 and IL-12, murine lymphohematopoietic progenitors proliferated and generated not only myeloid lineage cells but also committed B cell progenitors. Although somewhat less effectively than SF and FL, IL-4 also synergized with IL-6 or IL-11 in support of B lymphopoiesis. This early process of B lymphopoiesis appears to proceed through three stages: lymphohematopoietic proliferative stage, commitment stage and early B lymphoid proliferative stage. Surprisingly, IL-3 could neither replace nor act synergistically with SF, IL-4 or FL in maintaining the B lymphoid potential of the cells in the primary culture, although IL-3 was very effective in support of multilineage myeloid colony formation. In addition, when added to permissive cytokine combinations, IL-3 inhibited development of the B cell lineage. After screening available lymphohematopoietic cytokines, it was found that IL-1 (both α and β) also has similar inhibitory effects on early B lymphopoiesis. Studies using in vivo transfer of primary colonies suggested that cytokine regulation of commitment to T cell lineage may also be similar to that of B cell lineage. Stem Cells 1996;14: Introduction In adults, the entire process of B cell development takes place in the bone marrow, whereas T cell development starts in the bone marrow from hematopoietic stem cells but later moves to the thymus. Although the sites are different, at a certain point in the development, Correspondence: Dr. Makio Ogawa, VA Medical Center, 109 Bee Street, Charleston, SC , USA. Accepted for publication February 21, AlphaMed Press /96/$5.00/0 both B cell progenitors and T cell progenitors undergo a sequential gene rearrangement cascade of immunoglobulin (Ig) or T cell receptor (TCR) [1,2]. While our understanding of the mechanisms of the gene rearrangement and subsequent repertoire selection processes has been greatly advanced at the cellular and molecular levels [3-11], the mechanisms regulating the earlier stages of B and T cell development from lymphohematopoietic stem cells remain unclear. In order to study the very early stages of B and T cell development, it is necessary to develop culture assays which can support the differentiation of lymphohematopoietic progenitors along lymphocyte lineages. Recently, investigators in three laboratories described such culture assays. Baum et al. [12] established a coculture system in which fetal human primitive hematopoietic progenitors are allowed to differentiate into B lymphoid and myeloid lineages on cloned murine stromal cells. Cumano et al. [13] developed a stromal cell-dependent clonal culture assay in which bipotential progenitors from murine day 12 fetal liver differentiate along B cell and macrophage lineages in the presence of stromal cells and interleukin 7 (IL-7). Subsequently, they found that the stromal cells can be replaced by Steel factor (SF, also called c-kit ligand) and IL-11 [14]. We developed a two-step methylcellulose clonal culture assay in which murine primitive lymphohematopoietic progenitors are allowed to proliferate and differentiate into committed B cell and myeloid lineage cells [15]. More recently we found that the T cell potential is maintained in the primary culture of this two-step assay [16]. Using this culture assay, we were able to elucidate the cytokine interactions regulating the early stages of B and T cell development. STEM CELLS 1996;14:

2 370 Cytokine Regulation of Early Lymphopoiesis Two-Step Clonal Culture Assay for Murine Lymphohematopoietic Progenitors Since semisolid culture assays for murine [17, 18] and human [19] multipotential progenitors had been reported, a number of investigators had observed the presence of lymphocytes or precursors of lymphocytes in murine and human multilineage colonies. However, the clonal origin of these colonies was never firmly established in these studies. Therefore, in order to ensure the clonal origin of the lymphohematopoietic progenitors, we used micromanipulation of highly enriched mouse marrow cells [15]. Density-separated, lineage-negative, Ly-6A/E (Sca-1) positive cells were prepared from bone marrow cells of 5-fluorouracil (5-FU)-treated mice. Cells were individually plated in primary methylcellulose culture containing a combination of a medium conditioned by pokeweed mitogen-stimulated spleen cells (PWM-SCM), SF, IL-7 and erythropoietin (EPO) and incubated for 11 days. As summarized in Table 1, approximately 45% of the enriched progenitors yielded primary colonies. Upon cytological examination, some of the colonies expressed myeloid lineage cells, but the majority of the colonies consisted mostly of blast cells. Primary colonies were individually examined for full myeloid lineage expression and for analysis of B cell potential by replating into secondary myeloid suspension culture and lymphocyte methylcellulose culture containing SF and IL-7, respectively. All primary colonies revealed myeloid differentiation consisting of various combinations of lineages. Sixty percent of the primary colonies expressed erythroid and/or megakaryocyte lineages in addition to granulocytes and/or monocytes (Table 2). When recultured into secondary lymphocyte culture, approximately 40% of primary colonies yielded compact unicentric colonies consisting of small round cells. The majority of those cells were identified to be pre-b cells based on the following findings: 1) the cells were positive for B220 and dimly positive for Thy-1, but they were all negative for other lineage markers; 2) most of the cells expressed immunoglobulin µ chain mrna; 3) the cells did not differentiate into myeloid cells in culture, and 4) the cells reconstituted serum IgM and spleen B cells but not spleen T cells upon injection into severe combined immunodeficient (SCID) mice [15]. These results confirmed that a significant number of the hematopoietic progenitors in the bone marrow of 5-FU-treated mice are lymphohematopoietic progenitors. There were no obvious differences in the myeloid potentials between progenitors possessing B cell capability and those revealing no lymphoid potentials (Table 2). Cytokine Regulation of Lymphohematopoietic Progenitors Although we established the primary culture assay with PWM-SCM, SF, IL-7 and EPO, we subsequently observed that IL-7 and EPO are not necessary ingredients. We then tested whether combinations of early-acting cytokines [20] can replace PWM-SCM in support of the B cell potential of the primary colonies. Single cytokines, except IL-3, did not support primary colony formation. Two-factor combinations consisting of one from a cytokine group of SF [21], IL-4 [22] and flt3/flk2 ligand (FL) [23], and the other from a cytokine group consisting of IL-6 [24], G-CSF [25], IL-11 [26] and IL-12 [27] supported the proliferation of lymphohematopoietic progenitors and their differentiation along myeloid and B cell lineages, with a few exceptions. The results are summarized in Table 1. Lymphohematopoietic potentials of individual progenitors Exp. No. of Single Cells Cultured No. of Single Cells Producing Colonies in 1 Culture No. of Single Cells Producing Pre-B Cell Colonies in 2 Culture Means (Ranges) of the No. of Pre-B Cell Colonies Derived from Single Cells (26-540) (4-420) (12-308) (16-168) Total (4-540)

3 Hirayama, Ogawa 371 Table 2. Myeloid and B lymphoid potentials of individual progenitors B lymphoid potential Myeloid potential * + m 2 nm 9 8 nm 1 nmmast 2 7 nme 1 nmemast 2 1 nmmastm 3 6 nmmaste 5 3 nmem 2 nmemaste 1 nmemastm 1 1 nmmastem 9 13 nmemastem 1 3 Total Of the 87 progenitors that gave rise to primary colonies, 80 yielded a sufficient number of cells for two or more cytological examinations. * Based on a serial cytological examination of cells from primary culture and secondary myeloid culture. Abbreviations of lineage: n = neutrophile; m = macrophage; e = eosinophile; mast = mast cell; E = erythroblast; M = megakaryocyte. Table 3. Among the cytokines in the first group, SF and IL-4 were equally effective in support of primary colony formation and differentiation along myeloid lineages. However, cytokine combinations based on SF supported the generation of precursors for pre-b cell colonies in the primary culture better than those based on IL-4. FL was less effective than SF and IL-4 in support of primary colony formation, but allowed the generation of as many precursors for pre-b cell colonies as SF-containing combinations. Among the second group of cytokines, IL-6, G-CSF and IL-11 were equally effective, but IL-12 was somewhat less effective than the others. In contrast, IL-3 either alone or in combination with SF, IL-6, G-CSF, IL-11 or IL-12, did not support the B cell potential of the primary colonies even though the number and size of the primary colonies were comparable to those present in the SF-based cultures. We then studied the effects of addition of IL-3 to permissive cytokine combinations on B cell potential of the primary colonies. As shown in Table 4, addition of IL-3 abrogated the B cell potential of the primary colonies [23, 28]. The inhibition of the B cell potential by IL-3 was not an artifact of the detection method for B cell progenitors Table 3. B cell potential of primary colonies supported by combinations of cytokines Relative No. of Relative No. of Pre-B Cell Colonies SF IL-4 FL IL-3 IL-6 IL-11 G-CSF IL-12 Primary Colonies Produced Produced per Primary Colony NA + + NA NA Summarized from earlier publications [15, 23, 27]. : No colony formation. NA: Not applicable.

4 372 Cytokine Regulation of Early Lymphopoiesis Table 4. Effects of addition of IL-3 or IL-1α on the size and B cell potential of the primary colonies supported by SF and IL-11 Test Cytokine No. of No. of in Primary Primary Pre-B Cell Culture Colonies Colonies None 17 ± ± 6 IL-3 20 ± 4 0 IL-1α 20 ± 1 0 Fifty enriched marrow cells were cultured in the presence of SF, IL-11, IL-7, EPO and the cytokine to be tested. Primary colonies were scored and replated with SF and IL-7 on day 13. The number of pre-b cell colonies was derived from 1/40 of 20 pooled primary colonies. because colony transplantation into SCID mice also demonstrated the abrogation of the B cell potential of the primary colonies [28]. A doseresponse study revealed that IL-3 at a concentration as low as 0.1 ng/ml causes over 95% inhibition [28]. This inhibitory effect of IL-3 was very surprising to us since stimulatory roles of IL-3 in B cell development have been reported. For example, several investigators have established IL-3-dependent B220 + pre-b cell lines [29, 30] and pro-b cell lines [31-33]. It was recently reported that IL-3 can support the proliferation of B220 + c-kit + pre-b cells in the presence of stromal cells [34]. Therefore, these results indicate that IL-3 may be stimulatory to the B220 + stage of B cell development. Our results suggest that IL-3 is inhibitory to the cells at earlier stages of B lymphopoiesis, that is, commitment to B cell lineage and the subsequent early B lymphoid proliferation stage. There are other lines of evidence indicating negative effects of IL-3 on B cell development. Cockayne et al. [35] reported that transgenic mice expressing IL-3 antisense RNA develop B cell lymphoproliferative disorders at three to nine months of age. B220 + sigm pre-b cells accumulated in the peripheral lymphoid organs of those mice. Nolta et al. [36] observed that when human CD34 + cells were transplanted into immunodeficient mice together with human marrow stromal cells that had been engineered to produce human IL-3, human cells of all lineages except B cells were detected in the recipients. After screening all available lymphohematopoietic cytokines, we found that IL-1 also has similar negative effects on early B cell development (Table 4) [23, 28]. Lymphohematopoietic progenitors have also been identified in the bone marrow of normal mice [37] and fetal liver [38]. Their responses to the early-acting cytokines were similar to those of progenitors isolated from bone marrow of 5-FU-treated mice, although FL appeared to be more effective than SF in support of the B cell potential of fetal liver progenitors [38]. Effects of IL-3 on Early B Lymphopoiesis Establishment of the permissive clonal culture assay for early B cell development from lymphohematopoietic progenitors made it possible to characterize the kinetics and stages of B cell commitment. Bone marrow cells from 5-FU-treated mice were enriched for lymphohematopoietic progenitors and cultured in the presence of SF, IL-11, IL-7 and EPO. We analyzed the kinetics of appearance and disappearance of committed B cell progenitors and uncommitted progenitors by serial reculture of developing cells at one- or two-day intervals [39]. This study revealed that early B cell development from lymphohematopoietic progenitors can be divided into three stages: 1) proliferation of uncommitted B lymphoid/myeloid progenitors; 2) commitment to B cell lineage, and 3) proliferation of early committed B cell progenitors, which results in the production of B220 + pre-b cells. The commitment to B cell lineage by lymphohematopoietic progenitors in 5-FU-treated mice is well-synchronized and takes place on or around day 7 of culture. By day 9 the commitment to B cell lineage appears to be completed. Short-term exposure of the cells to IL-3 at each stage of early B cell development revealed stage-specific effects of IL-3 [39]. IL-3 was not inhibitory to the cells at the first stage. When added in the early phase of the first stage, IL-3 slightly enhanced the proliferation of lymphohematopoietic progenitors. On the contrary, IL-3 exerted strong inhibitory effects on the cells at the second stage and in the very early phase of the third stage. However, inhibitory effects of IL-3 were not observed on continued proliferation of pre-b cells. This apparent

5 Hirayama, Ogawa 373 bidirectional effect of IL-3 appears to be caused by kinetic alteration induced by IL-3. IL-3 hastens and simultaneously suppresses the peak of lymphohematopoietic proliferation [39]. Effects of IL-3 on Early T Lymphopoiesis We have also analyzed the same culture conditions for support of the early stages of T cell development from lymphohematopoietic progenitors [16]. Bone marrow cells from 5-FU-treated mice were enriched for lymphohematopoietic progenitors and plated individually by micromanipulation in methylcellulose culture containing SF and IL-11. The resulting primary colonies, all of which revealed differentiation along myeloid lineages in the secondary myeloid culture, were individually injected into SCID mice. Mice were sacrificed three months later and thymi and spleens were examined for the presence of donor-derived lymphocytes. In approximately 10% of the recipients, B and T cell reconstitution was observed. B cell potential of the same progenitors was also assessed using the two-step methylcellulose culture. The culture studies identified twice as many progenitors as having B cell potential as the in vivo transfer experiments. Therefore, the incidence of pluripotent lymphohematopoietic progenitors having B and T cell potentials may be higher than the 10% that was estimated through the in vivo transfer experiments. At this time, however, we are not certain about the exact nature of the cells in the primary colonies that reconstituted T cells in SCID mice. There are a few possibilities. Because committed B cell progenitors develop in the primary colonies ([15] and unpublished data), it is possible that committed T cell progenitors reconstituted the T cells in SCID mice. It is also possible that common T/B lymphoid progenitors developed in the primary colonies and were responsible for T cell reconstitution. Wu et al. [40] and Matsuzaki et al. [41] reported that the earliest-defined thymocyte population has both T and B cell potentials, suggesting the presence of common T/B lymphoid progenitors. Those intrathymic common T/B lymphoid progenitors might be analogous to the putative common T/B lymphoid progenitors in the primary colonies. Alternatively, totipotent hematopoietic stem cells with self-renewal capability may be responsible for the reconstitution of T lineage cells in SCID mice. Despite the ambiguities of the origin of the transplanted T cells, this clonal culture system appears to provide a new method for the study of early T cell development. Interestingly, IL-3 and IL-1 again independently inhibited T cell potential of the primary colonies when added to the primary culture [16]. Acknowledgments This work has been performed with support from a number of investigators. We would like to express our appreciation to Drs. Steven C. Clark, Debra Donaldson, Stanley F. Wolf and Paul Schendel (Genetics Institute, Cambridge, MA), Drs. Stewart D. Lyman and Linda S. Park (Immunex Corporation, Seattle, WA), Drs. Akihiro Shimosaka and Takamoto Suzuki (Kirin Brewery Co., Tokyo, Japan), Drs. Yoshikatsu Hirai and Yasuo Irie (Otsuka Pharmaceutical, Tokushima, Japan), Dr. Atsushi Miyajima (Tokyo University, Tokyo, Japan), Drs. Tetsuo Sudo and Masanobu Naruto (Toray Industries, Kamakura, Japan), Dr. Connie R. Faltynek (Sterling Winthrop Inc., Collegeville, PA), Dr. Lawrence M. Souza (Amgen, Thousand Oaks, CA), Dr. Por-Hsiung Lai (Protein Institute, Broomall, PA) and Dr. Makoto Kawakita (Kumamoto University, Kumamoto, Japan) for kind gifts of cytokines. The authors are also grateful to Drs. Toshiyuki Hamaoka and Shiro Ono (Osaka University, Osaka, Japan), Dr. Paul W. Kincade (Oklahoma Medical Research Foundation, Oklahoma City, OK), Dr. Robert L. Coffman (DNAX, Palo Alto, CA), Dr. Tatsuo Kina (Kyoto University, Kyoto, Japan) and Dr. Shin-Ichi Nishikawa (Kyoto University, Kyoto, Japan) for providing monoclonal antibodies. This research was supported by National Institutes of Health Grant DK and DK 48714, Office of Research and Development, Medical Research Service, Department of Veterans Affairs. References 1 Tonegawa S. Somatic generation of antibody diversity. Nature 1983;302: Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature 1988;334:

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