Chemoattractants MDC and TARC are secreted by malignant B-cell precursors following CD40 ligation and support the migration of leukemia-specific T cells.

The use of tumor cells as vaccines in cancer immunotherapy is critically dependent on their capacity to initiate and amplify tumor-specific immunity. Optimal responses may require the modification of the tumor cells not only to increase their immunogenicity but also to improve their ability to recruit effector cells to the tumor sites or sites of tumor antigen exposure. It has been reported that CD40 cross-linking of acute lymphoblastic leukemia (ALL) cells significantly increases their immunogenicity and allows the generation and expansion of autologous antileukemia cytotoxic T lymphocytes. This study demonstrates that the CD40 ligation of these tumor cells also induces the secretion of the CC-chemokines MDC and TARC. Supernatants from malignant cells cultured in the presence of sCD40L promote the migration of activated T cells that express CCR4, the common specific receptor for MDC and TARC. More importantly, the supernatants from CD40-stimulated tumor cells also support the transendothelial migration of autologous CCR4(+) antileukemia T cells. Therefore, the results demonstrate that the delivery to leukemia cells of a single physiologic signal, that is, CD40 cross-linking, simultaneously improves tumor cell immunogenicity and induces potent chemoattraction for T cells. (Blood. 2001;98:533-540)

Pax5 determines the identity of B cells from the beginning to the end of B-lymphopoiesis.

Despite being one of the most intensively studied cell types, the molecular basis of B cell specification is largely unknown. The Pax5 gene encoding the transcription factor BSAP is required for progression of B-lymphopoiesis beyond the pro-B cell stage. Pax5-deficient pro-B cells are, however, not yet committed to the B-lymphoid lineage, but instead have a broad lymphomyeloid developmental potential. Pax5 appears to mediate B-lineage commitment by repressing the transcription of non-B-lymphoid genes and by simultaneously activating the expression of B-lineage-specific genes. Pax5 thus functions both as a transcriptional repressor and activator, depending on its interactions with corepressors of the Groucho protein family or with positive regulators such as the TATA-binding protein. Once committed to the B-lineage, B cells require Pax5 function to maintain their B-lymphoid identity throughout B cell development.

Selection events operating at various stages in B cell development.

B cells have to progress through various checkpoints during their process of development. The three transcription factors E2A, EBF (early B cell factor) and Pax5 play essential roles in B cell commitment checkpoints. The various forms of the BCR and their downstream signaling molecules, which are expressed at different stages of B cell development, act as critical checkpoint guards allowing (positive selection) or preventing (negative selection) developmental progression. The recent advances on the molecular mechanisms operating at these various checkpoints are here summarized and discussed.

B cell development and immunoglobulin gene transcription in the absence of Oct-2 and OBF-1.

Oct-2 and OBF-1 (also called OCA-B or Bob-1) are B cell-specific transcription factors that bind to the conserved octamer site of immunoglobulin promoters, yet their role in immunoglobulin transcription has remained unclear. We generated mice in which the lymphoid compartment was reconstituted with cells that lack both Oct-2 and OBF-1. Even in the absence of these two transcription factors, B cells develop normally to the membrane immunoglobulin M-positive (IgM+) stage and immunoglobulin gene transcription is essentially unaffected. These observations imply that the ubiquitous factor Oct-1 plays a previously unrecognized role in the control of immunoglobulin gene transcription and suggest the existence of another, as yet unidentified, cofactor. In addition, both factors are essential for germinal center formation, although OBF-1 is more important than Oct-2 for IgG production after immunization.

Within the hemopoietic system, LAR phosphatase is a T cell lineage-specific adhesion receptor-like protein whose phosphatase activity appears dispensable for T cell development, repertoire selection and function.

Expression of the receptor-type tyrosine phosphatase LAR was studied in cells of the murine hemopoietic system. The gene is expressed in all cells of the T cell lineage but not in cells of any other hemopoietic lineage and the level of expression in T cells is developmentally regulated. The CD4(-)8(-)44(+) early thymic immigrants and mature (CD4(+)8(-)/CD4(-)8(+)) thymocytes and T cells express low levels, whereas immature (CD4(-)8(-)44(-) and CD4(+)8(+)) thymocytes express high levels of LAR. Among bone marrow cells only uncommitted c-kit(+)B220(+)CD19(-) precursors, but not B cell lineage committed c-kit(+)B220(+)CD19(+) precursors, express low levels of LAR. In contrast to the c-kit(+)B220(+)CD19(+) pre-BI cells from normal mice, counterparts of pre-BI cells from PAX-5-deficient mice express LAR, indicating that PAX-5-mediated commitment to the B cell lineage results in suppression of LAR. During differentiation of PAX-5-deficient pre-BI cell line into non-T cell lineages, expression of LAR is switched off, but it is up-regulated during differentiation into thymocytes. Thus, within the hemopoietic system, LAR appears to be a T cell lineage-specific receptor-type phosphatase. However, surprisingly, truncation of its phosphatase domains has no obvious effect on T cell development, repertoire selection or function.

Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells.

During B-cell development the surrogate light (SL) chain is selectively expressed in progenitor and precursor B cells during the developmental stages of D(H) to J(H) and V(H) to D(H)J(H) rearrangements. Approximately half of all muH chains produced by these rearrangements cannot pair with SL chains and cannot form a pre-B-cell receptor (pre-BCR). A spectrum of affinities between VpreB and individual V(H) domains generates preB cells with pre-BCR of different fitness which, in turn, determines the extent of the pre-B II-cell proliferation and the fidelity of allelic exclusion of the H chain locus. Once pre-BCR is expressed, SL chain expression is turned off. As pre-B II cells proliferate, SL is diluted out, thus limiting pre-BCR formation. As a consequence, pre-B II cells stop proliferating, become small and resting and begin to rearrange the L chain loci. Multiple rearrangements of the kappaL chain alleles are often detected in wild-type small pre-B II cells. Around 20% of the muH chain-expressing small pre-B II cells also express L chains but do not display the Ig on the surface. Hence, it is likely that not all L chains originally generated in resting pre-B II cells can pair with the muH chain previously present in that cell. The best fitting ones are selected preferentially to generate sIg+ B cells. Furthermore, the transition of immature B cells from the bone marrow to spleen and their development to mature cells appear as two separate steps controlled by different genes.

Fidelity and infidelity in commitment to B-lymphocyte lineage development.

During B-lymphocyte development in mouse fetal liver and bone marrow, a pre-B I cell stage is reached in which the cells express B-lineage-specific genes, such as CD19, Ig alpha and Igbeta and VpreB and lambda5, which encode the surrogate light (SL) chain. In these pre-B I cells both alleles of the immunoglobulin heavy (IgH) chain locus are D(H)J(H) rearranged. Transplantation of pre-B I cells from wild-type (e.g. C57Bl/6) mice in histocompatible RAG-deficient hosts leads to long-term reconstitution of some of the mature B-cell compartments and to the establishment of normal IgM levels, a third of the normal serum IgA levels, and IgG levels below the detection limit. Neither T-lineage nor myeloid cells of donor origin can be detected in the transplanted hosts, indicating that the pre-B I cells are committed to B-lineage differentiation. Consequently, the B-cell-reconstituted hosts respond to T-cell-independent antigens but not to T-cell-dependent antigens. Responses to T-cell-dependent antigens can be restored in the pre-B I-cell-transplanted, RAG-deficient hosts by the concomitant transplantation of mature CD4+ T cells. The transplanted wild-type pre-B I cells do not home back to the bone marrow and become undetectable shortly after transplantation. B-lymphocyte development in Pax-5-deficient mice becomes arrested at the transition of pre-B I to pre-B II cells i.e. at the stage when V(H) to D(H)J(H) rearrangements occur and when the pre-B-cell receptor, complete with muH chains and SL chains, is normally formed. T-lineage and myeloid cell development in these mice is normal. Pre-B I cells of Pax-5-deficient mice have a wild-type pre-B I-cell-like phenotype: while they do not express Pax-5-controlled CD19 gene, and express Ig alpha to a lesser extent, they express Igbeta, VpreB and lambda5, and proliferate normally in vitro on stromal cells in the presence of interleukin (IL)-7. Clones of these pre-B I cells carry characteristic D(H)J(H) rearrangements on both IgH chain alleles. However, removal of IL-7 from the tissue cultures, unlike wild-type pre-B I cells, does not induce B-cell differentiation to surface IgM-expressing B cells, but induces macrophage differentiation. This differentiation into macrophages requires either the presence of stromal cells or addition of macrophage colony-stimulating factor (M-CSF). Addition of M-CSF followed by granulocyte-macrophage colony-stimulating factor induces the differentiation to MHC class II-expressing, antigen-presenting dendritic cells. In vitro differentiation to granulocytes and osteoclasts can also be observed in the presence of the appropriate cytokines. Moreover, transplantation of Pax-5-deficient pre-B I clones into RAG-deficient hosts, while not allowing B-cell differentiation, leads to the full reconstitution of the thymus with all stages of CD4-CD8- and CD4+CD8+ thymocytes, to normal positive and negative selection of thymocytes in the thymus, and to the development of normal, reactive mature CD4+ and CD8+ T-cell compartments in the peripheral lymphoid tissues, all carrying the clone-specific D(H)J(H) rearrangements. On the other hand, Ig alpha, Igbeta, VpreB and lambda5 are turned off in the thymocytes, demonstrating that the expression of these genes does not commit cells irreversibly to the B lineage. Further more, Pax-5-deficient pre-B I cells are long-term reconstituting cells. They home back to the bone marrow of the RAG-deficient host, can be reisolated and regrown in tissue culture, and can be retransplanted into a secondary RAG-deficient host. This again develops thymocytes and mature T cells and allows the transplanted clonal pre-B I cells to home to the bone marrow.

Age-dependent changes in B lymphocyte development in man and mouse.

In recent years, detailed analyses of B cell development in both humans and mice have revealed similar subsets of precursors along the same pathway of differentiation. From these studies it also became clear that both species undergo age related changes in this B lymphocyte development program. In this review we summarize these findings and discuss, potential mechanisms underlying these age related changes, and possible causative correlations between these changes and age related B cell abnormalities.

Lineage commitment in lymphopoiesis.

The mechanisms controlling the commitment of hematopoietic progenitor cells to the lymphoid lineages are still mostly unknown. Recent findings indicate that the earliest phase of B cell development may proceed in two steps. At the onset of B-lymphopoiesis, the transcription factors E2A and EBF coordinately activate the B-cell-specific gene expression program. Subsequently, Pax5 appears to repress the promiscuous transcription of lineage-inappropriate genes and thus commits progenitor cells to the B-lymphoid pathway by suppressing alternative cell fates. B-lineage commitment by Pax5 seems to occur in a stochastic manner in the bone marrow, as indicated by the random activation of only one of the two Pax5 alleles in early pro-B cells. In contrast, loss- and gain-of-function analyses have implicated the Notch1 receptor in the specification of the T cell fate, which may thus be controlled by instructive signals in the thymus.

The B cell receptor, but not the pre-B cell receptor, mediates arrest of B cell differentiation.

B cell development in organ cultures of fetal liver from mice at day 14 of gestation resembles in kinetics and cell numbers generated the one observed in vivo. This development in vitro can be blocked by an IL-7 receptor-specific monoclonal antibody. Monoclonal antibodies specific for the pre-B cell receptor, i. e. for VpreB, lambda5, or muH chains, do not perturb B cell development in these organ cultures up to and including the CD25+ small pre-BII cell stage. However, muH chain-specific antibodies inhibit the appearance of the subsequent surface IgM+ immature B cells. In organ cultures of muH chain allotype heterozygous (muHa x muHb)F1 fetal livers a dose-dependent inhibition by allotype-specific monoclonal antibodies of sIgM+ immature B cells expressing the corresponding, but not the other, allotype was observed. By combining cell sorting with limiting dilution analysis of lipopolysaccharide-reactive cells, the probable target cell of this muH chain-specific inhibition was identified as an IgM+, CD23-immature B cell. Hence, engagement of the pre-B cell receptor by specific antibodies does not influence B cell development, while engagement of the B cell receptor on immature B cells does.

Precursor B cell receptor-dependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment.

The capacity of precursor B (pre-B) I cells from fetal liver and bone marrow to proliferate and differentiate into surface immunoglobulin-positive immature B cells in vitro was analyzed. Both fetal liver- and bone marrow-derived progenitors do so in a pre-B cell receptor (pre-BCR)-dependent manner in tissue culture medium alone, without addition of other cells or cytokines. Approximately 20% of the initial pre-B I cells enter more than one division. Analyses at the single-cell level show that approximately 15% divide two to five times. Coculture of pre-B I cells with stromal cells did not enhance proliferation or differentiation, whereas the presence of interleukin 7, especially in combination with stromal cells, resulted mainly in the expansion of pre-B I cells and prevented their further differentiation. Thus, the environment of fetal liver or bone marrow is not required for the pre-BCR to exert its function, which is to select and expand cells that have undergone an inframe V(H)-D(H)J(H) rearrangement that produces a pre-BCR-compatible muH chain. It appears unlikely that a ligand for the pre-BCR drives this pre-B cell proliferation.

Commitment to the B-lymphoid lineage depends on the transcription factor Pax5.

The Pax5 gene encoding the B-cell-specific activator protein (BSAP) is expressed within the haematopoietic system exclusively in the B-lymphoid lineage, where it is required in vivo for progression beyond the pro-B-cell stage. However, Pax5 is not essential for in vitro propagation of pro-B cells in the presence of interleukin-7 and stromal cells. Here we show that pro-B cells lacking Pax5 are also incapable of in vitro B-cell differentiation unless Pax5 expression is restored by retroviral transduction. Pax5-/- pro-B cells are not restricted in their lineage fate, as stimulation with appropriate cytokines induces them to differentiate into functional macrophages, osteoclasts, dendritic cells, granulocytes and natural killer cells. As expected for a clonogenic haematopoietic progenitor with lymphomyeloid developmental potential, the Pax5-/- pro-B cell expresses genes of different lineage-affiliated programmes, and restoration of Pax5 activity represses this lineage-promiscuous transcription. Pax5 therefore plays an essential role in B-lineage commitment by suppressing alternative lineage choices.

Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors.

The mechanisms controlling the commitment of haematopoietic progenitors to the B-lymphoid lineage are poorly understood. The observations that mice deficient in E2A and EBF lack B-lineage cells have implicated these two transcription factors in the commitment process. Moreover, the expression of genes encoding components of the rearrangement machinery (RAG1, RAG2, TdT) or pre-B-cell receptor (lambda5, VpreB, Igalpha, Igbeta) has been considered to indicate B-lineage commitment. All these genes including E2A and EBF are expressed in pro-B cells lacking the transcription factor Pax5. Here we show that cloned Pax5-deficient pro-B cells transferred into RAG2-deficient mice provide long-term reconstitution of the thymus and give rise to mature T cells expressing alpha/beta-T-cell receptors. The bone marrow of these mice contains a population of cells of Pax5-/- origin with the same phenotype as the donor pro-B cells. When transferred into secondary recipients, these pro-B cells again home to the bone marrow and reconstitute the thymus. Hence, B-lineage commitment is determined neither by immunoglobulin DJ rearrangement nor by the expression of E2A, EBF, lambda5, VpreB, Igalpha and Igbeta. Instead, our data implicate Pax5 in the control of B-lineage commitment.

Three chemokines with potential functions in T lymphocyte-independent and -dependent B lymphocyte stimulation.

Three clustered mouse chemokine genes, ABCD-1, -2 and -3, are all expressed highly in dendritic cells and, at various levels, in activated B cells. T cell-independently activated B cells express ABCD-1 and -2, but not -3. T cell-dependently activated B cells express all three. ABCD-1 attracts activated CD8+ cytotoxic T cells and CD4+ helper T cells of type 1 and 2. ABCD-2 preferentially attracts type 2 helper T cells, while ABCD-3 does not attract T cells at all. Both ABCD-1 and ABCD-2 bind to the same receptor (CCR4). In addition, ABCD-1 binds to a second, unknown, receptor on a separate T cell population. The three chemokines might guide T cell-independent as well as -dependent responses with two types of CD4+ T cells.