Patrícia Cruz Bergami-Santos

Monocyte-derived dendritic cells from breast cancer patients are biased to induce CD4+CD25+Foxp3+ regulatory T cells.

DCs orchestrate immune responses contributing to the pattern of response developed. In cancer, DCs may play a dysfunctional role in the induction of CD4+CD25+Foxp3+ Tregs, contributing to immune evasion. We show here that Mo‐DCs from breast cancer patients show an altered phenotype and induce preferentially Tregs, a phenomenon that occurred regardless of DC maturation stimulus (sCD40L, cytokine cocktail, TNF‐α, and LPS). The Mo‐DCs of patients induced low proliferation of allogeneic CD3+CD25negFoxp3neg cells, which after becoming CD25+, suppressed mitogen‐stimulated T cells. Contrastingly, Mo‐DCs from healthy donors induced a stronger proliferative response, a low frequency of CD4+CD25+Foxp3+ with no suppressive activity. Furthermore, healthy Mo‐DCs induced higher levels of IFN‐γ, whereas the Mo‐DCs of patients induced higher levels of bioactive TGF‐β1 and IL‐10 in cocultures with allogeneic T cells. Interestingly, TGF‐β1 blocking with mAb in cocultures was not enough to completely revert the Mo‐DCs of patientsˈ bias toward Treg induction. Altogether, these findings should be considered in immunotherapeutic approaches for cancer based on Mo‐DCs.

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-α in lung cancer

IntroductionAntigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-α induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-α within the microenvironment of primary lung carcinomas.ResultsHigher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-α-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry.ConclusionThese data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.

Tolerogenic iDO + Dendritic cells are induced by PD-1-expressing Mast cells

Mast cells (MCs) are tissue resident cells, rich in inflammatory mediators, involved in allergic reactions, and with an increasingly recognized role in immunomodulation. Dendritic cells (DCs), on the other hand, are central to the determination of immune response patterns, being highly efficient antigen-presenting cells that respond promptly to changes in their microenvironment. Here, we show that direct cell contact between immature monocyte-derived DCs (iDCs) and MC bends DCs toward tolerance induction. DCs that had direct contact with MC (MC-iDC) decreased HLA-DR but increased PD-L1 expression and stimulated regulatory T lymphocytes, which expresses FoxP3+, secrete TGF-β and IL-10, and suppress the proliferation of mitogen-stimulated naïve T lymphocytes. Furthermore, MC-iDC expressed higher levels of indoleamine-2,3-deoxigenase (IDO), a phenomenon that was blocked by treatment of MC with anti-PD-1 or by the treatment of DCs with anti-PD-L1 or anti-PD-L2, but not by blocking of H1 and H2 histamine receptors on DCs. Contact with MC also increased phosphorylated STAT-3 levels in iDCs. When a STAT-3 inhibitor, JSI-124, was added to the DCs before contact with MC, the MC-iDC recovered their ability to induce allogeneic T cell proliferation and did not increase their IDO expression.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

T cell stimulation by dendritic cell-tumor cell hybrids is enhanced in the presence of free dendritic cells

Dendritic cell-tumor cell hybrids (heterokaryons) are under investigation as a means for tumor immunotherapy since they should express MHC class I molecules from both cell types and, thus, present tumor antigens within an immunogenic context. The aim of this work was, therefore, to evaluate, in vitro, the immunostimulatory potential of these heterokaryons. To generate the hybrids, mature dendritic cells (mDCs) differentiated from peripheral blood of healthy donors were fused with cells from the breast cancer cell line MDA-MB-231 by applying an electric pulse. The hybrids expressed both the tumor antigen Her2 and the mDC marker CD11c (percentage of double-positive cells; Mix: 2.4 ± 0.5; Fusion: 13.2 ± 3.0; p<0.0001; n=8) and the molecules CD40, CD83, CD80 and CD86. They also expressed more MHC class I molecules at their surface (median fluorescence intensity; Mix: 17.8 ± 3.4; Fusion: 33.2 ± 7.2; p<0.05; n=4) which was proved to be due to the co-expression of molecules from both cell types. When co-cultured with autologous T lymphocytes for 5 days, the hybrids induced the same percentage of T cells positive for Foxp3 and T-bet but a higher percentage of GATA-3 positive T cells, when compared to the mix. Intriguingly, the hybrids induced lower T cell proliferation, when compared to the mix. However, when non-fused mDCs were added to the co-culture, T cell activation was significantly more intense than the sum of the activation induced by each cell type individually, as measured by T cell proliferation (percentage of cells with CFSE dilution; mDC: 9.7; Fusion: 6.3; mDC + Fusion: 47.8) and CD25 expression (percentage of positive cells; mDC: 9.9; Fusion: 9.7; mDC + Fusion: 57.4). Furthermore, addition of these free mDC to co-culture also induced more T-bet positive T cells. So, these data indicate that the induction/direction of immune responses by dendritic-tumor cell hybrids may be modified by the addition of non-fused DCs to the preparation, a strategy that may have clinical relevance.

Tumour Cells Incorporate Exosomes Derived from Dendritic Cells Through a Mechanism Involving the Tetraspanin CD9

Exosomes (Exos) are secreted nanovesicles that contain membrane proteins and genetic material, which can be transferred between cells and contribute to their communication in the body. We show that Exos, obtained from mature human dendritic cells (DCs), are incorporated by tumour cells, which after Exos treatment, acquire the expression of HLA-class I, HLA-class II, CD86, CD11c, CD54 and CD18. This incorporation reaches its peak eight hours after treatment, can be observed in different cell tumour lines (SK-BR-3, U87 and K562) and could be a means to transform non-immunogenic into immunogenic tumour cells. Interestingly, tetraspanins, which are expressed by the tumour cells, have their surface level decreased after Exo treatment. Furthermore, the intensity of Exo incorporation by the different tumour cell lines was proportional to their CD9 expression levels and pre-treatment of Exos with anti-CD9 decreased their incorporation (by SK-BR-3 cells). This modification of tumour cells by DC-derived Exos may allow their use in new immunotherapeutic approaches to cancer. Furthermore, by showing the involvement of CD9 in this incorporation, we provide a possible selection criterion for tumours to be addressed by this strategy.

Cationic liposomes produced via ethanol injection method for dendritic cell therapy

Abstract Cationic liposomes can be designed and developed in order to be an efficient gene delivery system for mammalian cells. Dendritic cell (DC) vaccines can be used to treat cancer, as cationic liposomes can deliver tumor antigens to cells while cells remain active. However, most methods used for liposome production are not able to reproduce in large scale the physicochemical and biological properties of liposomes produced in laboratory scale. In this context, ethanol injection method achieved promising results, although requiring post-treatment for size reduction and/or to remove residual ethanol. Thus, the purpose of this study was to generate cationic liposomes suitable for gene therapies via ethanol injection method in only one step (VEI) and compared to those submitted to a size reduction processes by microfluidization (MFV). For this, the method to produce cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoylphosphatidylethanolamine (DOPE) was optimized using a statistical design approach. As a result, the size of VEI decreased from 290 nm to 110 nm and the polydispersity from 0.54 to 0.17. In the case of MFV, size decreased from 128 nm to 107 nm and polydispersity from 0.40 to 0.18. ST and MFV before and after optimization were also characterized in terms of morphology by transmission electron microscopy (TEM) and structure by differential scanning calorimetry (DSC). Finally, to show their potential in gene/immune therapies applications, DCs were stimulated by such liposomes. Cells internalized liposomes, increasing expression of the costimulatory molecule CD86 and inducing T lymphocyte proliferation.