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.

We show here that mouse interferon-alpha (IFN-alpha)-producing cells (mIPCs) are a unique subset of immature antigen-presenting cells (APCs) that secrete IFN-alpha upon stimulation with viruses. mIPCs have a plasmacytoid morphology, can be stained with an antibody to Ly6G and Ly6C (anti-Ly6G/C) and are Ly6C+B220+CD11cloCD4+; unlike other dendritic cell subsets, however, they do not express CD8alpha or CD11b. Although mIPCs undergo apoptosis in vitro, stimulation with viruses, IFN-alpha or CpG oligonucleotides enhanced their survival and T cell stimulatory activity. In vivo, mIPCs were the main producers of IFN-alpha in cytomegalovirus-infected mice, as depletion of Ly6G+/C+ cells abrogated IFN-alpha production. mIPCs produced interleukin 12 (IL-12) in response to viruses and CpG oligodeoxynucleotides, but not bacterial products. Although different pathogens can selectively engage various APC subsets for IL-12 production, IFN-alpha production is restricted to mIPCs' response to viral infection.

Sllmm~'yDendritic cells, the professional antigen-presenting cells (APC) involved in T cell priming, express CD40, a molecule which triggering plays a key role in B cell growth and differentiation as well as monocyte activation. Herein we demonstrate that dendritic Langerhans cells (D-I.c) generated by culturing cord blood CD34 + progenitor cens with granulocyte/macrophage colony-stimulating and tumor necrosis factor oe (TNF-c~) express functional CD40 at a density higher than that found on B cells. Culturing D-IX on CD40-ligand (CD40L) transfected L cells allowed D-IX survival as 50 • 15% of seeded cells were recovered after 4 d while only 5% survived over control L cells. CD40 activation induced important morphological changes with a reduction of cytoplasmic content and a remarkable increase of dendrite development as well as an altered phenotype. In particular, CD40 triggering induced maintenance of high levds of major histocompatibility complex class II antigens and upregulation of accessory molecules such as CD58, CD80 (B7-1) and CD86 (B7-2). CD40 engagement also seems to turn on D-IX maturation as illustrated by upregulation of CD25, a molecule usually expressed on interdigitating dendritic cells of secondary lymphoid organs. Finally, CD40 activated D-IX secreted a limited set of cytokines (TNF-c~, IL-8, and macrophage inflammatory protein 1,', [MIP-loc]) whereas a similar activation induced dutriated monocytes to secrete IL-lot, IL-1/5, IL-6, IL-8, IL-10, TNF-~x, and MIP-lo~. As D-Ix activated T cells upregulated CD40L, it is likely that CD40 activation olD-ix observed herein with a fibroblast cell line stably expressing CD40L, mimics physiological interactiom between dendritic cells and T cells.T he CD40 antigen (1, for review) was identified by monodonal antibodies reacting with carcinomas and B cells (2) and showing costimulatory effects for B lymphocytes (3). It is a 50-kD glycoprotein which bdongs to the TNF receptor superfamily (4). Cross-linking of CD40, in conjunction with IL-4, was found to induce B cells to undergo long-term growth, as well as isotype switching, whereas addition of IL-10 results in B cell differentiation as well as isotype switch (5-8). The use of a CD40-~ fusion protein allowed the isolation of a cDNA encoding for a CD40 ligand (CD40L) 1, a new member of the TNF superfamily mainly expressed on activated T cells (9). Interaction between CD40 and CD40L has now been shown in vitro to be essential during T cell-de. pendent B cell activation (10,11). In vivo studies in mice have demonstrated that an antibody to CD40L can inhibit primary and secondary antibody production and establish- Functional CD40 molecules were found to be expressed on cells other than mature B cells. In particular, upon CD40 cross-llnklng, human progenitor B lymphocytes express CD23 and proliferate in response to . Thymic epithelial cells secrete GM-CSF in response to CD40 engagement (19). Finally, monocytes express high leveh of CD40 after eq, osure to IFN-% IL-3, and GM-CSF and CD40 cross-llnklng induces cyto...

Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. + , and CD8 + T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem.

SummaryIn the present report, we have investigated the in vitro differentiation of surface(s) slgD + and slgD-human B cells into Ig-secreting calls in response to various stimuli, slgD + B cells homogeneously expressed some of the antigens identifying mantle zone B cells, but lacked expression of germinal center markers, thus confirming that the B cell populations positively selected on the basis of slgD expression were highly enriched for naive B lymphocytes. Conversely, slgD-B cells expressed some of the antigens spedfically associated with germinal center B cells. T cell-independent differentiation of slgD + and sIgD-B cells could be achieved by simultaneous crosslinking of sIgs and CD40 in the presence of a mouse Ltk-cell line stably expressing human CDw32/Fc'yPdI (CDw32 L cells). In this experimental system, sIgD + B cells were exclusively proned for IgM synthesis, whereas sIgD-B ceils produced IgG, IgM, and IgA. Both the human and viral forms of interleukin 10 (ILd0) strongly increased the Ig secretion by sIgD + and slgD-B cells simultaneously activated through slgs and CD40. IgM and IgG constituted the predominant Ig isotype produced by slgD + and slgD-B cells, respectively, in response to IL-10. slgD + B cells could be induced for IgA synthesis upon co-culturing with transforming growth factor (TGF-~) and II.-10, in the presence of an anti-CD40 monodonal antibody presented by the CDw32 L cells. In contrast, TGF-~ suppressed the IL-10-mediated IgG, IgM, and IgA secretions by slgD-B cells, slgD + B cells could not be induced for IgA synthesis by TGF-~ and Ibl0 after crosslinking of their slgs, suggesting that ligation of CD40 was one of the obligatory signals required for commitment of naive B cells to IgA secretion. Limiting dilution experiments indicated that the IgA-potentiating effect of TGF-B was due to its capacity to increase the frequency of IgA-producing cells, most likely as a consequence of class switching. Taken together, our data strongly suggest that TGF-B is involved in the regulation of IgA isotype selection in humans.

Human memory B cells that carry mutated IgV region genes were isolated from tonsils by negative selection of IgD+ naive B cells and CD38+ germinal center B cells and plasma cells. They were mainly found within the intraepithelial areas, but not in the B cell follicles of human tonsils. Memory B cells but not naive B cells have the capacity to present antigen directly to T cells, owing to the constitutive expression of the accessory molecules B7-1/CD80 and B7-2/CD86. Signals through antigen receptors and CD40 antigen result in these two molecules being further up-regulated more rapidly and strongly on memory B cells than on naive B cells. The unique anatomical localization of memory B cells beneath the surface of mucosa, together with their strong APC capacity, may explain the well-known prompt and robust secondary antibody responses.

Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N ¼ 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-a following toll-like receptor activation in vitro compared with patients' blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-a production endowed TApDC with the unique capacity to sustain FoxP3 þ Treg expansion, a capacity that was reverted by the addition of exogenous IFN-a. These findings indicate that IFNa-deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-a production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer. Cancer Res; 72(20); 5188-97. Ó2012 AACR.

In ovarian cancer, the immune system fails to eradicate established tumors partly due to the induction of immune tolerance within tumor microenvironment. In this study, we investigated the contribution of plasmacytoid dendritic cells (pDC) in the establishment of immune tolerance in a cohort of 44 ovarian cancer patients. In the tumor and malignant ascites, CD4 þ CD123 þ BDCA2 þ pDC were the most abundant dendritic cell subset; however, they were profoundly depleted in peripheral blood. The presence of pDC in primary ovarian cancer, but not ascites, was an independent prognostic factor associated with early relapse. Following chemotherapy, we observed a partial restoration of blood pDC levels in patients in complete remission. These findings show preferential recruitment of pDC into tumors where they express a partially mature phenotype that may reflect an in situ activation. Importantly, compared with pDC found in ascites or blood, tumor-associated pDC (TApDC) produced less IFN-a, TNF-a, IL-6, macrophage inflammatory protein-1b, and RANTES in response to toll-like receptor stimulation, and alterations in pDC functions were mainly mediated through tumor-derived TNF-a and TGF-b. Unlike ascites-derived pDC, TApDC induced IL-10 production from allogeneic naive CD4 þ T lymphocytes, suggesting the existence of a paracrine immunosuppressive loop. Taken together, our findings indicate that both local and systemic dysfunction of pDC play a critical role in the progression of ovarian cancer via induction of immune tolerance. Cancer Res; 71(16); 5423-34. Ó2011 AACR.