Dendritic cells and immunity against cancer.

T cells can reject established tumours when adoptively transferred into patients, thereby demonstrating the power of the immune system for cancer therapy. However, it has proven difficult to maintain adoptively transferred T cells in the long term. Vaccines have the potential to induce tumour-specific effector and memory T cells. However, clinical efficacy of current vaccines is limited, possibly because tumours skew the immune system by means of myeloid-derived suppressor cells, inflammatory type 2 T cells and regulatory T cells (Tregs), all of which prevent the generation of effector cells. To improve the clinical efficacy of cancer vaccines in patients with metastatic disease, we need to design novel and improved strategies that can boost adaptive immunity to cancer, help overcome Tregs and allow the breakdown of the immunosuppressive tumour microenvironment. This can be achieved by exploiting the fast increasing knowledge about the dendritic cell (DC) system, including the existence of distinct DC subsets that respond differentially to distinct activation signals, (functional plasticity), both contributing to the generation of unique adaptive immune responses. We foresee that these novel cancer vaccines will be used as monotherapy in patients with resected disease and in combination with drugs targeting regulatory/suppressor pathways in patients with metastatic disease.

Naked antigen-presenting molecules on dendritic cells.

Antigen-presenting cells work to present peptides derived from exogenous and endogenous antigens to circulating T cells, sparking off an immune response. Dendritic cells are unique amongst antigen-presenting cells, not least for their newly described ability to circumvent the need to internalize exogenous antigens before presenting them.

Dendritic cells: On the move from bench to bedside.

As dendritic cells increasingly become the adjuvant of choice in new approaches to cancer immunotherapy, a degree of protocol standardization is required to aid future large-scale clinical trials.

Human B cell precursors proliferate and express CD23 after CD40 ligation.

The CD40 surface membrane molecule plays an important role in the activation of mature human B cells, but its role in earlier stages of B lineage development is unknown. Here, we have investigated the effects of triggering the CD40 antigen on B cell precursors (BCP) by crosslinking with anti-CD40 antibody presented by Fc gamma-receptor type II-transfected murine Ltk- cells (CD40 system). CD10+ surface immunoglobulin negative (sIg-) BCP, freshly isolated from fetal bone marrow or precultured on stromal cells, proliferated in the CD40 system. This effect required the presence of IL-3, which acted as a specific cosignal among a panel of cytokines examined. The association of IL-10 and IL-7 potentiated the observed IL-3 and CD40-dependent BCP proliferation, demonstrating that IL-10 can act on early B lineage cells. CD40-dependent activation of fetal BCP did not favor maturation to sIg+ B cells, but resulted in the induction of high levels of surface membrane CD23. The emerging CD23+ BCP lacked sIg and CD10, and represented an important proportion of the cycling cells in the CD40-dependent cultures. Taken together, our data demonstrate that stimulation of the CD40 antigen induces expression of the CD23 gene, and regulates cell proliferation during normal human B cell ontogeny.

Interleukin 10 induces apoptotic cell death of B-chronic lymphocytic leukemia cells.

Recent studies have established that interleukin (IL)-10 induces growth and most notably differentiation of normal human B lymphocytes. We studied here the effects of IL-10 on the proliferation and survival of B-chronic lymphocytic leukemia (B-CLL) cells. IL-10 was found to inhibit 54-96% of the spontaneous tritiated thymidine incorporation observed in 3 of 12 B-CLL samples. Furthermore, IL-10 decreased the viable cell recovery of all five B-CLL samples tested, irrespective of whether cells were spontaneously synthesizing DNA or not. After 1 wk, B-CLL populations cultured with IL-10 were lost while those cultured without IL-10 survived. Flow cytometric analysis, DNA gel electrophoresis, and Giemsa staining all revealed that IL-10 induced B-CLL cells to die from apoptosis. This IL-10-mediated apoptosis was dose dependent and specific as it could be inhibited by a neutralizing anti-IL-10 antibody. B-CLL cells undergoing apoptosis in response to IL-10 showed decreased Bcl-2 protein levels. Addition of IL-2, IL-4, interferon gamma, and anti-CD40 monoclonal antibody prevented the IL-10-mediated apoptosis of B-CLL cells. None of the malignant B cell populations obtained from eight non-Hodgkin's lymphomas and three hairy cell leukemias underwent apoptosis after IL-10 treatment, thus suggesting that the apoptotic effect of IL-10 is specific for B-CLL cells. Thus, IL-10 inhibits the DNA synthesis and most notably the survival of B-CLL cells, findings that call for considering IL-10 in the immunotherapy of chemoresistant B-CLL.

Cytokine-induced proliferation and immunoglobulin production of human B lymphocytes triggered through their CD40 antigen.

Human resting B lymphocytes enter a state of sustained proliferation when incubated with both mouse fibroblastic L cells stably expressing Fc gamma RII/CDw32 and anti-CD40 antibodies. We have explored the effects of 11 recombinant human cytokines (CKs) on induced cell proliferation and immunoglobulin (Ig) production. Interleukin 4 (IL-4) was the only CK able to enhance anti-CD40-induced B cell multiplication as measured by enumeration of viable cells, and interferon gamma (IFN-gamma) further stimulated this induced proliferation. IL-4 enhanced the production of IgM and IgG by B cells and induced them to produce IgE. Combinations of IL-4 and IL-2 resulted in the production of large amounts of IgM and IgA. Interestingly, IFN-gamma did not inhibit the production of IgE by cells stimulated with anti-CD40 and IL-4. None of the tested CK combinations resulted in the production of large quantities of IgG. Therefore, this new culture system represents a unique model to study isotype regulation in highly purified human B lymphocytes, in addition to allowing the generation of long-term factor-dependent human B cell lines.

Memory B cells are biased towards terminal differentiation: a strategy that may prevent repertoire freezing.

Isolation of large numbers of surface IgD+CD38- naive and surface IgD-CD38- memory B cells allowed us to study the intrinsic differences between these two populations. Upon in vitro culture with IL-2 and IL-10, human CD40-activated memory B cells undergo terminal differentiation into plasma cells more readily than do naive B cells, as they give rise to five- to eightfold more plasma cells and three- to fourfold more secreted immunoglobulins. By contrast, naive B cells give rise to a larger number of nondifferentiated B blasts. Saturating concentrations of CD40 ligand, which fully inhibit naive B cell differentiation, only partially affect that of memory B cells. The propensity of memory B cells to undergo terminal plasma cell differentiation may explain the extensive extra follicular plasma cell reaction and the limited germinal center reaction observed in vivo after secondary immunizations, which contrast with primary responses in carrier-primed animals. This unique feature of memory B cells may confer two important capacities to the immune system: (a) the rapid generation of a large number of effector cells to efficiently eliminate the pathogens; and (b) the prevention of the overexpansion and chronic accumulation of one particular memory B cell clone that would freeze the available peripheral repertoire.

Epstein-Barr virus transformation induces B lymphocytes to produce human interleukin 10.

Interleukin 10 (IL-10) is a pleiotropic factor that enhances proliferation of activated human B lymphocytes and induces them to secrete high amounts of immunoglobulins. Here we show that several human B cell lines were able to constitutively secrete human (h)IL-10. Whereas none of the pre-B nor the plasmocytic cell lines tested produced hIL-10, 25 of the 36 tested mature B cell lines (lymphoblastoid and Burkitt lymphoma cell lines) secreted hIL-10. Moreover, 24 of these 25 hIL-10-producing B cell lines contained the Epstein-Barr virus (EBV) genome, suggesting a relationship between hIL-10 production by human B cell lines and EBV expression. Accordingly, whereas polyclonal activation via triggering of surface immunoglobulins or CD40 antigen induced highly purified normal human B lymphocytes to produce only low (0.3-0.4 ng/ml) but significant amounts of hIL-10, EBV infection induced them to secrete high amounts of hIL-10 (4-9 ng/ml). Furthermore, addition of exogenous hIL-10, simultaneously to EBV infection, potentiated cell proliferation, whereas a blocking anti-IL-10 antiserum inhibited it. Thus, hIL-10 produced by infected human B lymphocytes appears to be involved in the mechanisms of EBV-induced B cell proliferation.

Interleukin 4 inhibits the proliferation but not the differentiation of activated human B cells in response to interleukin 2.

The combined effect of IL-4 and IL-2 on proliferation of anti-IgM antibody or Staphylococcus aureus strain Cowan I (SAC)-preactivated B cells was investigated. It was observed that in most cases, rIL-2 used at optimal concentration induced higher levels of tritiated thymidine ([3H]TdR) uptake than rIL-4 used at optimal concentration. When rIL-4 and rIL-2 were added together, it was repeatedly found that B cell proliferation induced by rIL-2 was significantly reduced and was, in most cases, comparable with the proliferation induced by rIL-4 alone. Cell cycle studies demonstrated that rIL-4 significantly reduced the number of cells entering S and G2/M phases of the cell cycle upon rIL-2 stimulation. B cell blasts preincubated for 24 or 48 h with rIL-4 displayed a reduced proliferation in response to rIL-2. In contrast, preculture of resting B cells with rIL-4 did not impair their subsequent proliferation in response to rIL-2 plus insolubilized anti-IgM antibody. This suggests that rIL-4 can only exert its inhibitory effect once B cells have received an activation signal. The differentiative activity of rIL-2 measured on B cell blasts preactivated for 2 d with SAC was not altered by rIL-4, which suggests that rIL-4 did not exert its inhibitory activity on rIL-2-induced B cell proliferation by enhancing rIL-2-mediated differentiation. Delayed addition of a neutralizing anti-IL-4 antiserum demonstrated that a period of contact of at least 24 h between IL-4 and B cell blasts was necessary for the development of the antagonistic effect of IL-4 on IL-2-mediated growth of activated B cells. These data demonstrate that IL-4 antagonizes the B cell growth-promoting effect of IL-2 without affecting the differentiation of preactivated B cells in response to IL-2.

Interferon gamma inhibits interleukin 10 production by monocytes.

Interleukin 10 (IL-10) was first described for its ability to inhibit interferon gamma (IFN-gamma) production. Herein, we studied the balance between IFN-gamma and IL-10 production by human peripheral blood mononuclear cells (PBMC) in response to Staphylococcus aureus Cowan (SAC) or lipopolysaccharide (LPS). Monocyte depletion reduced IL-10 production by 90% and resulted in an increased IFN-gamma production. Addition of anti-IL-10 antibody to PBMC cultures also strongly increased IFN-gamma production. In contrast, among various cytokines, only IFN-gamma strongly reduced IL-10 synthesis by SAC- or LPS-activated PBMC and monocytes. Thus, IFN-gamma has proinflammatory effects through the combination of two mechanisms: (a) induction of early tumor necrosis factor alpha (TNF-alpha) and IL-1 beta synthesis; and (b) inhibition of the delayed production of IL-10, an inhibitor of TNF-alpha and IL-1 beta synthesis. Taken together, the present data indicate that IFN-gamma and IL-10 antagonize each other's production and function.

Interleukin 4 counteracts the interleukin 2-induced proliferation of monoclonal B cells.

B cells from patients suffering from B-type chronic lymphocytic leukemia (B-CLL) are susceptible to the effects of several interleukins. Using the cells from 12 different patients we show that IL-4 does not synergize with anti-mu antibody for the enhancement of DNA synthesis. Moreover IL-4 profoundly (90%) suppresses the response to IL-2 in the 10 patient responders to this interleukin. This suppression occurs whether IL-2 is used alone, in costimulation with anti-mu antibody, or in synergy with IFN-gamma. In no instance did IL-4 induce terminal differentiation. This negative effect of IL-4 can take place in monoclonal B-CLL cells where IL-4 enhances the expression of CD23. IL-4 does not interfere with the upregulation of CD25 by IL-2. Thus, IL-4 may display inhibitory effects on the proliferative response of selected B cell populations. The antagonism between IL-4 and IL-2 has important implications for the potential use of cytokines in the management of B-CLL patients.

The low-affinity receptor for IgE (CD23) on B lymphocytes is spatially associated with HLA-DR antigens.

Two hybridomas that produce the mAbs 135 and 449 B4 were obtained that inhibited the binding of IgE to the Fc epsilon RL/CD23 on the EBV-transformed B cell line RPMI 8866. mAb 135 was obtained from a mouse immunized with RPMI 8866 cells, whereas mAb 449B4 was obtained from a mouse immunized with a partially purified preparation of Fc epsilon RL/CD23 obtained as the eluate of an IgE immunoabsorbent loaded with a soluble extract of RPMI 8866 cells. These two mAbs bound to Fc epsilon RL/CD23- cell lines and precipitated two polypeptides with 36,000 Mr and 28,000 Mr, which were the HLA-DR alpha and beta chains, respectively. Immunoprecipitation with mAb 135 of NP-40 lysates from dithio-bis(succinimidyl propionate) (DSP) crosslinked 125I-labeled RPMI 8866 or normal B cells incubated with rIL-4 showed three polypeptides with 42,000, 36,000, and 28,000 Mr. The 42,000 Mr polypeptide is identical to the Fc epsilon RL/CD23 since it could be precipitated by the anti-Fc epsilon RL/CD23 mAb 25 after resolubilization from the SDS-PAGE gel. Immunoprecipitations of the crosslinked cell extracts carried out with the anti-Fc epsilon RL/CD23 mAb 25 yielded the same three polypeptides. Furthermore, when RPMI 8866 or rIL-4 preincubated normal B cells were solubilized with a digitonin buffer, which prevents the dissociation of noncovalently linked polypeptide complexes, mAb 135 and mAb 25 precipitated complexes composed of three molecules with 42,000, 36,000, and 28,000 Mr. The well-characterized anti-HLA-DR mAb L243 was unable to block the binding of either IgE or mAb 135 to RPMI 8866 cells, although it could immunoprecipitate the complex (HLA-DR-Fc epsilon RL/CD23) from crosslinked cell lysates. Since mAb 135 and L243 were able to both bind the RPMI 8866 cells, it demonstrates that they bind to different epitopes of the HLA-DR complex, the mAb 135 epitope of the HLA-DR molecule being close to the IgE binding site of the Fc epsilon RL/CD23. These data demonstrated that the Fc epsilon RL/CD23 and HLA-DR antigens are spatially associated on the B cell membrane.

Germinal center founder cells display propensity for apoptosis before onset of somatic mutation.

B lymphocytes undergo affinity maturation of their antigen receptors within germinal centers. These anatomical structures develop in secondary lymphoid organs from the clonal expansion of a few antigen-specific founder B cells, whose isolation and characterization are reported here. Human germinal center founder cells express the naive B cell markers surface IgM and IgD as well as the germinal center B cell markers CD10 and CD38. They express low levels of Bcl-2, high levels of Fas, and undergo rapid apoptosis in culture. The smaller nonproliferating sIgM+IgD+CD38+ B cells displayed a lower level of somatic mutation in their immunoglobulin variable region genes compared with the large proliferating ones. Unmutated sIgM+IgD-CD38+ tonsillar B cells may thus represent germinal center founder cells in which the program for apoptotic cell death is triggered before the onset of somatic mutation, allowing the selection of the germline antibody repertoire at an early stage.

Tumor necrosis factor alpha cooperates with interleukin 3 in the recruitment of a primitive subset of human CD34+ progenitors.

We have recently demonstrated that tumor necrosis factor alpha (TNF-alpha) potentiates interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor-induced growth of CD34+ hematopoietic progenitor cells (HPC), and favors the generation of dendritic/Langerhans cells. The stimulatory effect of TNF-alpha was detailed in the present study. Thus, CD34+ HPC entering in cycle (S/G2M) after a 48-h pulse with IL-3 expressed the transferrin receptor (TfR), and fluorescence-activated cell sorter-separated TfR+ HPC, but not TfR-HPC, showed a high proliferative response to IL-3. In contrast, TfR-HPC were found to undergo strong proliferation in response to IL-3 + TNF-alpha. Limiting dilution experiments indicated that TNF-alpha increased both the frequency and the average size of clones generated from TfR-HPC as a result of the development of a higher number of large clones. In contrast, TNF-alpha did not enhance the IL-3-dependent proliferation of TfR+ HPC. Preculturing CD34+ HPC for 48 h with TNF-alpha enhanced the subsequent generation of IL-3-dependent colony-forming units. Precultures with TNF-alpha or cultures with suboptimal doses of TNF-alpha allowed the recruitment of cells with both granulocytic and monocytic differentiation potential. Taken together, our results indicate that TNF-alpha recruits a subpopulation of CD34+ HPC hyposensitive to IL-3, with high proliferative capacity and some features of multipotential progenitors, that are likely to be more primitive than those responding to IL-3 alone.

CD40 ligation on human cord blood CD34+ hematopoietic progenitors induces their proliferation and differentiation into functional dendritic cells.

Human CD34+ multilineage progenitor cells (CD34HPC) from cord blood and bone marrow express CD40, a member of the tumor necrosis factor-receptor family present on various hematopoietic and nonhematopoietic cells. As hyper-IgM patients with mutated CD40 ligand (CD40L) exhibit neutropenia, no B cell memory, and altered T cell functions leading to severe infections, we investigated the potential role of CD40 on CD34HPC development. CD40-activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells. DC generated via the CD40 pathway displayed strong major histocompatibility complex class II DR but lacked detectable CD1a and CD40 expression. These features were shared by a dendritic population identified in situ in tonsillar T cell areas. Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L+ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.

The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand.

A subset of CD4+CD11c-CD3- blood cells was recently shown to develop into dendritic cells when cultured with monocyte conditioned medium. Here, we demonstrate that CD4+ CD11c-CD3- cells, isolated from tonsils, correspond to the so-called plasmacytoid T cells, an obscure cell type that has long been observed by pathologists within secondary lymphoid tissues. They express CD45RA, but not markers specific for known lymphoid- or myeloid-derived cell types. They undergo rapid apoptosis in culture, unless rescued by IL-3. Further addition of CD40-ligand results in their differentiation into dendritic cells that express low levels of myeloid antigens CD13 and CD33.

Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1 and IgG3.

During antigen-induced immune responses, human B cells switch isotype from immunoglobulin M (IgM)-IgD to IgG1-4, IgA1-2, or IgE. In the human, no cytokines have yet been demonstrated to act as switch factors for IgG1, IgG2, and IgG3. In this paper, we report that in response to interleukin 10 (IL-10), anti-CD40 activated tonsillar surface IgD+ (sIgD+) B cells are induced to secrete large amounts of IgM, IgG1, and IgG3 but neither IgG2 nor IgG4. Cord blood purified B cells and lymphocytes from Hyper-IgM patients also produced IgG1 and IgG3 after culture with anti-CD40 and IL-10. In contrast, sIgD- isotype-committed B cells produce IgG1, IgG2, and IgG3 when activated through CD40 in the presence of IL-10. Thus, in addition to its growth-promoting and differentiating activities on human B cells, IL-10 may represent a switch factor for IgG1 and IgG3.

Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells.

Measles causes a profound immune suppression which is responsible for the high morbidity and mortality induced by secondary infections. Dendritic cells (DC) are professional antigen-presenting cells required for initiation of primary immune responses. To determine whether infection of DC by measles virus (MV) may play a role in virus-induced suppression of cell-mediated immunity, we examined the ability of CD1a+ DC derived from cord blood CD34+ progenitors and Langerhans cells isolated from human epidermis to support MV replication. Here we show that both cultured CD1a+ DC and epidermal Langerhans cells can be infected in vitro by both vaccine and wild type strains of MV. DC infection with MV resulted within 24-48 h in cell-cell fusion, cell surface expression of hemagglutinin, and virus budding associated with production of infectious virus. MV infection of DC completely abrogated the ability of the cells to stimulate the proliferation of naive allogeneic CD4+ T cell as early as day 2 of mixed leukocyte reaction (MLR) (i.e., on day 4 of DC infection). Mannose receptor-mediated endocytosis and viability studies indicated that the loss of DC stimulatory function could not be attributed to the death or apoptosis of DC. This total loss of DC stimulatory function required viral replication in the DC since ultraviolet (UV)-inactivated MV or UV-treated supernatant from MV-infected DC did not alter the allostimulatory capacity of DC. As few as 10 MV- infected DC could block the stimulatory function of 10(4) uninfected DC. More importantly, MV-infected DC, in which production of infectious virus was blocked by UV treatment or paraformaldehyde fixation, actively suppressed allogeneic MLR upon transfer to uninfected DC-T-cultures. Thus, the mechanisms which contribute to the loss of the allostimulatory function of DC include both virus release and active suppression mediated by MV-infected DC, independent of virus production. These data suggest that carriage of MV by DC may facilitate virus spreading to secondary lymphoid organs and that MV replication in DC may play a central role in the general immune suppression observed during measles.

Receptor revision of immunoglobulin heavy chain variable region genes in normal human B lymphocytes.

Contrary to the general precepts of the clonal selection theory, several recent studies have provided evidence for the secondary rearrangement of immunoglobulin (Ig) genes in peripheral lymphoid tissues. These analyses typically used transgenic mouse models and have only detected secondary recombination of Ig light chain genes. Although Ig heavy chain variable region (V(H)) genes encode a substantial element of antibody combining site specificity, there is scant evidence for V(H) gene rearrangement in the periphery, leaving the physiological importance of peripheral recombination questionable. The extensive somatic mutations and clonality of the IgD(+)Strictly-IgM(-)CD38(+) human tonsillar B cell subpopulation have now allowed detection of the first clear examples of receptor revision of human V(H) genes. The revised VDJ genes contain "hybrid" V(H) gene segments consisting of portions from two separate germline V(H) genes, a phenomenon previously only detected due to the pressures of a transgenic system.

Analysis of somatic mutation in five B cell subsets of human tonsil.

Using a series of phenotypic markers that include immunoglobulin (Ig)D, IgM, IgG, CD23, CD44, Bcl-2, CD38, CD10, CD77, and Ki67, human tonsillar B cells were separated into five fractions representing different stages of B cell differentiation that included sIgD+ (Bm1 and Bm2), germinal center (Bm3 and Bm4), and memory (Bm5) B cells. To establish whether the initiation of somatic mutation correlated with this phenotypic characterization, we performed polymerase chain reaction and subsequent sequence analysis of the Ig heavy chain variable region genes from each of the B cell subsets. We studied the genes from the smallest VH families (VH4, VH5, and VH6) in order to facilitate the mutational analysis. In agreement with previous reports, we found that the somatic mutation machinery is activated only after B cells reach the germinal center and become centroblasts (Bm3). Whereas 47 independently rearranged IgM transcripts from the Bm1 and Bm2 subsets were nearly germline encoded, 57 Bm3-, and Bm4-, and Bm5-derived IgM transcripts had accumulated an average of 5.7 point mutations within the VH gene segment. gamma transcripts corresponding to the same VH gene families were isolated from subsets Bm3, Bm4, and Bm5, and had accumulated an average of 9.5 somatic mutations. We conclude that the molecular events underlying the process of somatic mutation takes place during the transition from IgD+, CD23+ B cells (Bm2) to the IgD-, CD23-, germinal center centroblast (Bm3). Furthermore, the analysis of Ig variable region transcripts from the different subpopulations confirms that the pathway of B cell differentiation from virgin B cell throughout the germinal center up to the memory compartment can be traced with phenotypic markers. The availability of these subpopulations should permit the identification of the functional molecules relevant to each stage of B cell differentiation.