Human pre-B cells differentiate into Ig-secreting plasma cells in the presence of interleukin-4 and activated CD4+ T cells or their membranes

Studies on human B-cell development have been hampered by the lack of reproducible culture techniques to induce pre-B cells to differentiate into Ig-secreting plasma cells. Here, we describe that highly purified surface (s) mu-, cytoplasmic (c) mu+, CD10+, CD19+ human pre-B cells derived from fetal bone marrow (BM) differentiate with high frequencies into Ig-secreting plasma cells, when cocultured with activated, cloned CD4+ T cells and with interleukin-4 (IL-4). Production of IgM, total IgG, IgG4, and IgE in pre-B-cell cultures was detected, indicating that the cells also underwent Ig isotype switching. Pre-B-cell differentiation occurred in the absence of BM stromal cells, IL-7, and stem cell factor (SCF). However, IL-7 significantly enhanced the levels of Ig produced, whereas SCF was ineffective. Neutralizing anti-IL-4 monoclonal antibodies (MoAbs) completely inhibited pre-B-cell differentiation showing the specificity of the reaction. Intact CD4+ T- cell clones could be replaced by membrane preparations of these cells, indicating that the costimulatory signals provided by the activated CD4+ T cells are contact-mediated. In contrast, anti-CD40 MoAbs failed to provide the costimulatory signal required for pre-B-cell differentiation, which may be related to the very low expression of CD40 on fetal BM B cells. Activated CD4+ T cells and IL-4 also induced s mu expression and Ig synthesis in cultures initiated with pre-B cells that had been preincubated in medium for 2 days, and from which spontaneously emerging s mu+ B cells were removed by using a fluorescence-activated cell sorter. These results support the notion that the Ig synthesis observed in pre-B-cell cultures was not caused by outgrowth and differentiation of cells that spontaneously matured into s mu+ B cells. In addition, IL-4 and CD4+ T cells strongly enhanced CD40 and HLA-DR expression on the majority of cultured pre-B cells, further indicating that CD4+ T cells and IL-4 activate bona fide pre-B cells. Taken together, these data indicate that activated CD4+ T cells and IL-4 can provide all the necessary signals required for human pre-B cells to differentiate into Ig-secreting plasma cells.     

The effects of interleukin 7 (IL-7) on human bone marrow in vitro.

Interleukin 7 (IL-7) stimulates the proliferation of pre-B cells from long-term murine lymphoid cultures and normal bone marrow. In addition, IL-7 stimulates the proliferation of murine T cells, including fetal and adult thymocytes as well as peripheral T cells. Flow cytometry and cell enumeration analyses were carried out on light-density human bone marrow cells incubated in the presence or absence of IL-7. The data showed no evidence for a proliferative effect of IL-7 on B-lineage cells expressing CD24 or on myeloid cells expressing CD15; however, IL-7 did stimulate the growth of T cells expressing CD3. After 16 days of stimulation the number of CD3+ cells in marrow cultures increased 350% in the presence of IL-7. In contrast, cultures incubated in the absence of IL-7 showed a 50% decrease in the number of T cells, with a preponderance of myeloid lineage cells. Flow cytometry indicated that cells from IL-7-stimulated cultures were mature T cells because they also expressed cell surface antigens for either CD4 or CD8. These studies show that in contrast to the murine system, IL-7 does not appear to stimulate the growth of human pre-B cells from adult human bone marrow. This is consistent with other experiments that suggest that human pro-B cells and not human pre-B cells respond to IL-7. It appears that IL-7 preferentially promotes the growth of T cells from human marrow.     

Stochastic rearrangement of immunoglobulin variable-region genes in chicken B-cell development.

The molecular mechanism by which immunoglobulin (Ig) gene rearrangement occurs is highly conserved between mammalian and avian species. However, in avian species, an equivalent to the mammalian pre-B cell, which has undergone Ig heavy-chain gene rearrangement and expresses mu heavy chains in the absence of Ig light-chain rearrangement, has not been convincingly demonstrated. It is consequently unclear whether an ordered progression of gene rearrangement events leading to functional Ig expression occurs in avian species. To examine the sequence of Ig gene rearrangement events in chicken B-cell development, we transformed day 12 embryo bursal cells with the REV-T(CSV) retrovirus. More than 100 clones were analyzed by Southern blotting and polymerase chain reaction for the presence of Ig gene rearrangements. The majority of these clones contained only germline Ig sequences. Several clones contained complete heavy- and light-chain rearrangements and 13 clones contained only heavy-chain rearrangements analogous to stages of mammalian B-cell development. However, 5 clones contained rearrangements of light-chain genes in the absence of complete heavy-chain rearrangement. Consequently, we conclude that rearrangement of chicken Ig light-chain genes does not require heavy-chain variable-region rearrangement. This observation suggests that chicken Ig gene rearrangement events required for Ig expression occur stochastically rather than sequentially.     

Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells

By using two-color phenotypic analysis with fluorescein isothiocyanate- anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) signifcantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM- 102), or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-beta1 (TGF-beta1) and macrophage inflammatory protein- 1beta (MIP-1beta) could suppress survival of pre-B cells even in the presence of IL-7. Plasma cells alone could not suppress survival of pre- B cells in the presence of IL-7, but coculture of plasma cells with KM- 102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF-beta1, and MIP-1beta mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF- beta1 and MIP-1beta mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated to secrete lesser levels of supporting factor (IL- 7) and higher levels of inhibitory factors (TGF-beta1 and MIP-1beta) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.     

A recombinant single-chain IL-7/HGFbeta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S12, CLPs, and pre-pro-B cells

A novel recombinant interleukin-7/hepatocyte growth factor beta-chain (IL-7/HGFbeta) hybrid cytokine was constructed as a single chain (sc) composed of IL-7 and HGFbeta connected by a flexible linker. Unlike recombinant (r) IL-7, which stimulated pro-B cells and pre-B cells only, scIL-7/HGFbeta stimulated the proliferation of pre-pro-B cells, common lymphoid progenitors (CLPs), and colony-forming unit (CFU)-S12 in cultures of IL-7-/- mouse BM cells. When injected in vivo, 3- to 4-fold more splenic B-lineage cells appeared in recipients of bone marrow (BM) cells from the scIL-7/HGFbeta-stimulated cultures than from rIL-7-stimulated cultures. Moreover, on a per-cell basis, scIL-7/HGFbeta culture-generated cells produced 16- to 20-fold more BM and splenic B-lineage cells than did normal BM cells. Antibody blocking, receptor phosphorylation, and confocal microscopy demonstrated that scIL-7/HGFbeta signals though both the IL-7 and HGF (c-Met) receptors, which form IL-7R/c-Met complexes on the surface of CLPs and pre-pro-B cells. In addition, the IL-7Ralpha chain, gammac chain, and c-Met were coisolated from purified CLPs and pre-pro-B cells on scIL-7/HGFbeta affinity gels, indicating that they are major components of the IL-7/HGFbeta receptor. Hence, the present results demonstrate that the IL-7/HGFbeta hybrid cytokine efficiently and selectively stimulates the most primitive B-lineage precursors in BM by inducing juxtacrine interactions between the IL-7 and c-Met receptors.     

Growth inhibitory and agonistic signals of interleukin-7 (IL-7) can be mediated through the CDw127 IL-7 receptor

The present study was aimed at identifying surface-membrane molecules involved in the regulation of human B-cell ontogeny. For this purpose, murine monoclonal antibodies (MoAbs) were generated against Pre-Alp, a pre-B acute lymphoblastic leukemia (ALL) cell line, and MoAb R34.34 was selected for further characterization. R34.34 recognized a molecule expressed on normal B-cell precursors (BCP) but not on mature B cells. The antibody also reacted with T lymphocytes, a subpopulation of monocytes from peripheral blood, and a subset of CD34+ cells. Immunoprecipitation analysis indicated that R34.34 recognizes an 80-kD molecular weight antigen. Antibody R34.34 was further found to be directed against an epitope interfering with binding of interleukin-7 (IL-7) to Pre-Alp cells. Expression cloning from a Pre-Alp cDNA library showed that R34.34 antigen is CDw127, the 75- to 80-kD IL-7 receptor. Proliferation of the B-lineage ALL cell lines Reh and Mieliki was inhibited by IL-7, and this effect was specifically reverted by MoAb R34.34. In addition, antibody R34.34 specifically inhibited IL-7- dependent proliferation of normal BCP, Pre-Alp cells, and peripheral T cells. These results imply that both inhibitory and proliferative effects of IL-7 can be mediated through the same receptor on various lineages. R34.34 antibody should be important for the analysis of signal transduction through CDw127.     

Interleukin-7 induces the proliferation of normal human B-cell precursors.

In the present study, we investigated the effects of human recombinant interleukin-7 (IL-7) on the proliferation of enriched hematopoietic cells isolated from human adult and fetal bone marrow (BM). In cultures of CD34+ cells, IL-7 was found to induce dose-dependent incorporation of 3H-thymidine (3H-TdR), but had no demonstrable effect on the development of myeloid colony-forming cells. Numbers of B-cell precursors (BCP), initially present within CD34+ populations and which included a CD34+CD20+ subset, were significantly increased when CD34+ BM cells were cultured in the presence of IL-7. This effect was most striking on CD20+ BCP, and resulted at least partly from higher numbers of cycling cells as indicated by Hoechst 33342 fluorescence (Calbiochem, Behring Diagnostics, La Jolla, CA). These results indicate that IL-7 promotes the growth of BCP within the CD34+ compartment. In line with the B-lineage affiliation of CD34+ target cells, committed BCP (CD10+ CD19+ surface IgM-) isolated from BM were also found to proliferate in response to IL-7. Interestingly, this effect of IL-7 was strongly potentiated by the addition of IL-3. Taken together, and in accordance with previous observations on murine cells, our data indicate that IL-7 acts as a growth factor during the ontogeny of human B lymphocytes.     

Insulin-like growth factor-1 potentiates expansion of interleukin-7- dependent pro-B cells

Commitment to B-lymphocyte differentiation is characterized by expression of the B220 form of the common leukocyte antigen (Ly-5) and D-JH rearrangement of the Ig heavy chain gene complex. B-lineage progenitor cells, or pro-B cells, that have initiated Ig gene rearrangement, but do not express detectable Ig heavy or light chain protein, have recently been shown to retain substantial capacity for expansion in vitro in the presence of bone marrow (BM) stromal cells and interleukin-7 (IL-7). Although the potentiating effect of stromal cells on pro-B-cell proliferation can be partially attributed to the ligand for the proto-oncogene receptor c-kit (c-kit ligand [KL] or stem cell factor), several lines of evidence suggest that c-kit-mediated cell signalling is not required for pro-B-cell expansion. Previous studies from this laboratory demonstrated that insulin-like growth factor-1 (IGF-1) potentiated the proliferative effect of IL-7 on nonadherent cells from lymphoid long-term BM cultures in a manner similar to that shown for KL. To further delineate specific cell stages that respond to lymphopoietic cytokines, we derived continuously proliferating pro-B-cell lines from day-14 murine fetal liver in the presence of IL-7 and BM stromal cell clone S10. Initial expansion and continued proliferation of these pro-B-cell lines was absolutely dependent on the presence of both IL-7 and stromal cells. In the absence of KL, IL-7-stimulated proliferation of these cells in short- term cultures and addition of either recombinant IGF-1 or KL significantly potentiated this proliferative response. Although IGF-2 and insulin also potentiated the effect of IL-7, our data suggest that neither IGF-2 nor insulin represent normal regulators of intramyeloid lymphocyte development. IGF-1 and KL activate unique cascades of intracellular signalling events and inclusion of both cytokines in cultures of IL-7-stimulated pro-B cells resulted in additive potentiation of the proliferative response. Taken together, these results suggest that expansion of pro-B cells in vivo is maintained by at least three stromal cell-derived cytokines. IL-7 appears to be unique in delivering the primary proliferative signal for pro-B-cell expansion; however, both KL and IGF-1 potentiate the proliferative effect of IL-7 on these cells. The functional redundancy and additive effects of IGF-1 and KL as amplification signals for developing B- lineage cells underscore the essential nature of clonal expansion and diversification in development of immunocompetent lymphoid cells.     

Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH7-27 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor alpha beta lineage.

Rearranged IGH genes were detected by Southern blotting in 22% of 118 cases of T-cell acute lymphoblastic leukemia (ALL) and involved monoallelic and biallelic rearrangements in 69% (18/26) and 31% (8/26) of these cases, respectively. IGH gene rearrangements were found in 19% (13/69) of CD3(-) T-ALL and in 50% of TCRgammadelta+ T-ALL (12/24), whereas only a single TCRalpha beta+ T-ALL (1/25) displayed a monoallelic IGH gene rearrangement. The association with the T-cell receptor (TCR) phenotype was further supported by the striking relationship between IGH and TCR delta (TCRD) gene rearrangements, ie, 32% of T-ALL (23/72) with monoallelic or biallelic TCRD gene rearrangements had IGH gene rearrangements, whereas only 1 of 26 T-ALL with biallelic TCRD gene deletions contained a monoallelic IGH gene rearrangement. Heteroduplex polymerase chain reaction (PCR) analysis with VH and DH family-specific primers in combination with a JH consensus primer showed a total of 39 clonal products, representing 7 (18%) VH-(DH-)JH joinings and 32 (82%) DH-JH rearrangements. Whereas the usage of VH gene segments was seemingly random, preferential usage of DH6-19 (45%) and DH7-27 (21%) gene segments was observed. Although the JH4 and JH6 gene segments were used most frequently (33% and 21%, respectively), a significant proportion of joinings (28%) used the most upstream JH1 and JH2 gene segments, which are rarely used in precursor-B-ALL and normal B cells (1% to 4%). In conclusion, the high frequency of incomplete DH-JH rearrangements, the frequent usage of the more downstream DH6-19 and DH7-27 gene segments, and the most upstream JH1 and JH2 gene segments suggests a predominance of immature IGH rearrangements in immature (non-TCRalpha beta+) T-ALL as a result of continuing V(D)J recombinase activity. More mature alpha beta-lineage T-ALL with biallelic TCRD gene deletions apparently have switched off their recombination machinery and are less prone to cross-lineage IGH gene rearrangements. The combined results indicate that IGH gene rearrangements in T-ALL are postoncogenic processes, which are absent in T-ALL with deleted TCRD genes and completed TCR alpha (TCRA) gene rearrangements.     

Development of a Model for Evaluating the Interaction Between Human Pre-B Acute Lymphoblastic Leukemic Cells and the Bone Marrow Stromal Cell Microenvironment

Clonal expansion of B-cell precursor acute lymphoblastic leukemia(ALL) is potentially regulated by survival, growth, and death signalstransduced by the bone marrow (BM) microenvironment. Using a human BMstromal cell culture that supports the growth of normal human B-cellprecursors, we established a pre-B ALL cell line designated BLIN-2.BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, adic(9;20) chromosomal abnormality, and a bi-allelic deletion of thep16^INK4a and p19^ARF genes. Themost interesting feature of BLIN-2 is an absolute dependence onadherent human BM stromal cells for sustained survival and growth.BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis,and direct contact with viable BM stromal cells is essential foroptimal growth. BLIN-2 cells also grow on vascular cell adhesionmolecule-1 (VCAM-1)-negative human skin fibroblasts, making itunlikely that a very late antigen-4 (VLA-4)/VCAM-1interaction is required for BLIN-2 growth. Western blot analysis ofBLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16^INK4ap19^ARF but does not require BM stromal cells for growth,does not undergo Rb phosphorylation after BM stromal cell contact. TheBLIN-2 cell line will facilitate identification of ligand/receptorinteractions at the B-cell precursor/BM stromal cell interface and mayprovide new insight into microenvironmental regulation of leukemic cell survival and growth.     

Survival of human leukaemic B-cell precursors is supported by stromal cells and cytokines: association with the expression of bcl-2 protein

We searched for cytokines with the potential to support the survival of human B-cell precursor acute lymphoblastic leukaemia (pre-B ALL) cells. 47 patients with pre-B ALL were classified into four stages: stage I, CD19+CD10-CD20-; stage II, CD19+CD10+CD20-; stage III, CD19+CD10+CD20+cytoplasmic mu-heavy chain (cmu)-; stage IV, CD19+CD10+CD20+cmu. Interleukin (IL)-3 receptor alpha chain (IL-3Ralpha) was expressed in all stages, whereas the expressions of IL-7Ralpha and IL-2Ralpha were pronounced in stages IV and II, respectively. Neither IL-3, IL-7 nor IL-2 supported the survival of pre-B ALL cells. When pre-B ALL cells were layered on stromal, MS-10, cells, viability of the pre-B ALL cells increased. Addition of IL-3 to culture containing MS-10 cells enhanced the survival of pre-B ALL cells in all cases, whereas addition of IL-7 augmented the survival of pre-B ALL cells of some cases of stage III and all cases of stage IV. The survival of pre-B ALL cells was also supported by the conditioned media of MS-10 cells. Stromal-cell-derived factor 1 (SDF-1) supported the survival of pre-B ALL cells. Effects of the conditioned media of MS-10 cells were abrogated by an anti-SDF-1 neutralizing antibody. The extent of survival of pre-B ALL cells supported by stromal cells and IL-3 and IL-7, correlated with the expression level of bcl-2 protein. The effects of stromal cells may be in part related to SDF-1.     

Pro-B cells sense productive immunoglobulin heavy chain rearrangement irrespective of polypeptide production

B-lymphocyte development is dictated by the protein products of functionally rearranged Ig heavy (H) and light (L) chain genes. Ig rearrangement begins in pro-B cells at the IgH locus. If pro-B cells generate a productive allele, they assemble a pre-B cell receptor complex, which signals their differentiation into pre-B cells and their clonal expansion. Pre-B cell receptor signals are also thought to contribute to allelic exclusion by preventing further IgH rearrangements. Here we show in two independent mouse models that the accumulation of a stabilized μH mRNA that does not encode μH chain protein specifically impairs pro-B cell differentiation and reduces the frequency of rearranged IgH genes in a dose-dependent manner. Because noncoding IgH mRNA is usually rapidly degraded by the nonsense-mediated mRNA decay machinery, we propose that the difference in mRNA stability allows pro-B cells to distinguish between productive and nonproductive Ig gene rearrangements and that μH mRNA may thus contribute to efficient H chain allelic exclusion.     

Insulin-like growth factor-I regulates pro-B cell differentiation.

Progression of B-lymphocyte development in the bone marrow of postnatal mammals is marked by progressive rearrangement and expression of immunoglobulin (Ig) heavy- and light-chain genes. Following productive VHDJH gene rearrangement in the Ig heavy-chain gene complex, mu-heavy chain is the first Ig gene product expressed in cells committed to the B-lymphoid differentiation pathway. Interleukin (IL)-7 has been shown to stimulate proliferation of pre-B cells following c mu expression and this proliferative stimulus is potentiated by kit ligand (KL). However, it appears that neither of these cytokines contributes to differentiation of pro-B cells or initiation of expression of Ig gene products. We previously demonstrated that differentiation of pro-B cells and expression of mu-heavy chain is stimulated by either bone marrow stromal cell line S17 or cell-free supernatants from that line. This biological activity was attributed to molecules with an apparent M(r) of less than 10 Kd and approximately 40 to 60 Kd. We now report that this biological activity resides with stromal cell-derived insulin-like growth factor-I (IGF-I). Recombinant IGF-I stimulated the expression of cytoplasmic mu-heavy chain in short-term bone marrow cultures and this stimulus was abrogated in the presence of anti-IGF-I antibody. We also demonstrate that either anti-IGF-I antibody or pretreatment of S17 cells with antisense oligonucleotide for IGF-I abrogated the pro-B cell differentiation activity of S17 stromal cell supernatants. Although IGF-I did not directly stimulate proliferation of B-lineage cells, like KL, it potentiated the proliferative stimulus provided by IL-7. Taken together, these data strongly suggest that IGF-I produced by bone marrow stromal cells in the hematopoietic microenvironment plays a key role in regulating primary B lymphopoiesis.     

Ambiguous phenotypes and genotypes in 16 children with acute leukemia as characterized by multiparameter analysis

Ambiguous phenotypes and genotypes were observed in 16 children with acute leukemia. Surface marker, cytogenetic, molecular genetic, and DNA flow cytometric analyses as well as standard morphologic and cytochemical studies were used to divide the patients into three groups. The first group comprised five children with acute leukemia whose blast cells were morphologically lymphoid, while immunophenotyping disclosed simultaneous expression of early pre-B cell and myeloid features. Molecular genetic studies showed evidence of heavy-chain immunoglobulin (Ig) gene rearrangements in all patients. Cytogenetic data, available in three of these children, revealed t(4;11). In five of the 16 patients, morphologic and surface marker analyses indicated the coexistence of two separate cell populations, one with myeloid and the other with early pre-B cell features. Further evidence of B cell commitment in these patients was provided by demonstration of Ig heavy-chain gene rearrangements in all five patients. Surprisingly, one of the five patients showed oligoclonal Ig heavy-chain as well as monoclonal gene rearrangement for the beta chain of the T cell receptor (beta-TCR). The last group consisted of four cases with otherwise typical acute lymphoblastic leukemia (ALL), early pre-B cell phenotype, and coexpression of myeloid or T cell-associated antigens, and two children with unequivocal acute myeloid leukemia (AML) and coexpression of T cell antigens. Gene rearrangement of Ig heavy-chain could be demonstrated in five of six patients, additional Ig light-chain gene rearrangement in two children with ALL, and bigenotypic features (Ig heavy-chain and beta-TCR gene rearrangement) in one patient. In none of the 16 patients did flow cytometry disclose clonal abnormalities of leukemic cell DNA content. Based on these findings, we suggest that malignant transformation in the first and second group of patients took place at a stage ontogenetically close to the pluripotent stem cell, whereas ambiguous phenotypes in the third group resulted from aberrant gene expression or insufficient reagent specificity.     

Loss of IL-7 receptor alpha-chain (CD127) expression in acute HCV infection associated with viral persistence

Interleukin-7 (IL-7) is required for the establishment and maintenance of memory CD4(+) and CD8(+) T lymphocytes, and cells lacking IL-7Ralpha (CD127) demonstrate impaired IL-2 secretion and have a short life-span. Chronic HCV is characterized by T cells that are functionally impaired and exhibit an immature phenotype. To investigate the potential role of IL-7/IL-7Ralpha in the outcome of HCV infection, we used multiparameter flow cytometry to characterize patients with acute infection (n = 24), long-term chronic infection (12) and normal subjects (13). HCV infection per se resulted in downregulation of CD127 on total CD4(+) and CD8(+) T lymphocytes as compared to normal controls. Total expression was lowest in those patients who subsequently developed persistence and intermediate in those patients with acute-resolving infection. This reduction affected both na?ve and effector/memory T cells. CD127 correlated phenotypically with upregulation of chemokine receptors CCR7 and CXCR4, expression of the anti-apoptotic molecule B cell leukemia/lymphoma 2 (Bcl-2), and enhanced IL-2 production. In six HLA A2-positive patients, we longitudinally tracked tetramer responses to HCV and CMV epitopes; at baseline, reflecting the expression of CD127 on whole T cell populations, viral-specific CTLs in patients who became chronic demonstrated lower CD127. In conclusion, CD127 is a useful marker of functional CD4(+) and CD8(+) T cells and its expression correlates with virologic outcome of acute HCV. These data provide a mechanistic basis for the observation that CTLs generated in early infection rapidly decline as chronicity is established; CD127 expression should be considered in the design of novel immunotherapeutic approaches.     

Changing antigen receptor gene rearrangements in a case of early pre-B cell leukemia: evidence for a tumor progenitor cell with stem cell features and implications for monitoring residual disease.

A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.     

The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal- cell-independent expansion and differentiation of human fetal pro-B cells in vitro

The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow-derived CD34+CD19+ pro-B cells were analyzed in a stromal- cell-independent, serum-deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34-CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte- macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL-7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B- cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro- B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor.