CD4 T cell activation by B cells in human Leishmania (Viannia)infection
© Rodriguez-Pinto et al.; licensee BioMed Central Ltd. 2014
Received: 8 January 2014
Accepted: 21 February 2014
Published: 25 February 2014
An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated.
CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC.
Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells.
The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to activate CD4 T cells and induce cytokine secretion is similar to that of all cell populations present in PBMCs. This capacity implicates B cells as a plausible target for modulation of the immune response to Leishmania infection as a therapeutic strategy.
KeywordsB cells Cutaneous leishmaniasis Leishmania (Viannia)
Leishmaniasis is a parasitic disease endemic in 88 countries that affects an estimated 12 million people in the rural areas of the tropical and subtropical regions of the world. In South America, Leishmania species of the Viannia subgenus cause cutaneous and muco-cutaneous disease that can become chronic and cause severe disfigurement. In spite of the advances in knowledge of parasite biology and the host immune response, effective and safe treatment remains a challenge and there is yet no approved vaccine [1, 2]. These needs may be addressed by manipulating the host immune response to obtain parasite elimination without tissue damage.
Professional antigen presenting cells (APCs) initiate the adaptive immune response by activating CD4 T cells. Activation of APCs, in the form of MHCII molecule upregulation and costimulatory molecule expression, is essential for induction of immunity, and the cytokines secreted during antigen presentation shape the ensuing response. In the murine model of cutaneous leishmaniasis (CL) caused by L. major, IL-12 secretion by APCs is crucial for generation of IFN-γ-secreting Th1 cells, macrophage activation and parasite elimination . Failure to generate a Th1 response leads to chronic disease. In the human disease caused by L. (Viannia), cells from patients with chronic disease secrete large amounts of IFN-γ and TNF-α but also Th2 and regulatory cytokines such as IL-13 and IL-10, resulting in an unbalanced immune response, excessive inflammation, tissue damage, and parasite survival [4–8]. A similar picture was observed in a murine model of chronic L. (V.) panamensis infection where IL-13 was shown to be crucial for development of pathology . Since CD4 T cell activation by APCs leads to this harmful response, modulation of this event could promote healing or prevent disease.
Three types of APCs are recognized: dendritic cells (DCs), macrophages and B cells. As the natural host of Leishmania, macrophages would be the ideal candidates for initiating the adaptive immune response in CL. However, it is well documented that infection of macrophages with Leishmania does not induce MHCII molecule upregulation, costimulatory molecule expression or IL-12 secretion, but rather inhibits these processes, shutting down antigen presentation by macrophages. These effects have been shown in a range of Leishmania species in both animal models and human cells [9–12]. On the other hand, DC function has been more difficult to determine, as both activation and inhibition of APC function have been found. In the murine L. major model it is well recognized that DCs initiate the immune response and secrete IL-12 in the resistant phenotype . However, studies with other Leishmania species have shown that infection with parasites does not lead to DC activation [14–18]. Notably, L. (V.) braziliensis infection of DCs inhibits cell activation and antigen presentation while uninfected neighboring DCs are able to upregulate MHCII and costimulatory molecules and induce T cell activation . Thus, it seems that induction of immunity by DCs in CL depends on their avoidance of infection. In summary, both macrophage and DC APC function can be inhibited by L. (Viannia). Therefore, the participation of B cells in CD4 T cell activation in CL caused by species of this subgenus may be crucial.
The role of B cells in human CL caused by L. (Viannia) has not been defined. Histological studies in Colombian patients infected with L. (Viannia) have revealed prominent B cell infiltration of skin lesions and leishmanin skin test reaction sites [19, 20], and a study from Brazil showed a significant increase in B cell frequency in lymph node aspirates of patients that presented lymphadenopathies associated with the late stage of lesion development . These findings suggest that B cells may play an important role in the immune response to L. (Viannia). In mice, lesion development caused by L. panamensis was found to be delayed in the absence of B cells, although final lesion size and parasite load were not affected . B cells have been shown in murine models of leishmaniasis to contribute to immunologic regulation through production of cytokines and immunoglobulins and as a result of antigen presentation [22–29]. However, variation occurs that may depend upon the species, specific Leishmania strain and mouse genetics [27, 29].
The contribution of B cells in CD4 T cell activation in human CL or its pathogenesis is yet unexplored. B cells specific for a particular antigen have an exquisite capacity to concentrate and direct the antigen to the appropriate processing compartments. Furthermore, BCR signaling induces optimal APC activation, making B cells competent APCs capable of activating naïve CD4 T cells. Hence, the participation of B cells as APCs has been shown to be essential in many different contexts [30–35]. Furthermore, their capacity to secrete several cytokines capable of inducing Th1, Th2 and regulatory responses has been well documented . For these reasons, and in order to identify immunomodulatory intervention strategies we have examined the capacity of B cells to activate CD4 T cells in CL.
In the present study, CD4 T cell activation by B cells was assessed in cells from patients with CL caused by L. (Viannia) species. We found that B cells upregulated the costimulatory molecule CD86 in PBMC cultures stimulated with L. panamensis and were able to activate CD4 T cells and induce cytokine secretion in the absence of other recognized APCs. Furthermore, we show that exposure to L. panamensis antigen enhances BCR-mediated endocytosis by the Ramos human B cell line. Taken together these results indicate a role for B cells in the modulating the immune response to L. panamensis.
We evaluated the response of cells from CL patients from the southwestern region of Colombia where most cases are caused by L. panamensis or L. braziliensis. We have previously shown that there is a strong recall response to L. panamensis antigens in patients from this region regardless of the infecting species [37–39]. Thus, we stimulated cells with promastigote L. panamensis antigen (pLAg) obtained from L. panamensis strain MHOM/COL/81/L13. First, we compared CD4 T cell and APC activation in CL patients and healthy subjects by culturing PBMCs with pLAg and measuring CD4 T cell, B cell and DC activation markers. We then determined the competence of B cells for CD4 T cell activation by culturing them with CD4 T cells and pLAg and evaluating activation of both cell types. We evaluated the cytokine secretion profile of PBMC and B cell/CD4 T cell cultures to establish whether the cytokine profile induced by B cell-CD4 T cell interaction is similar to that induced when all PBMCs participate. Finally, we examined the effects of exposure to pLAg on a human B cell line in relation with induction of activation markers and BCR-mediated endocytosis.
Human subjects and cells
Peripheral blood samples (60 to 100 mL) were obtained from CL patients from the southwestern region of Colombia (Departments of Valle del Cauca and Nariño). Participants included 8 males and 2 females of 20 to 65 years of age (median = 27), who had typical cutaneous lesions, were parasitologically diagnosed by smear, culture or biopsy, and had not received anti-leishmanial treatment before enrollment. Blood samples from four healthy subjects from non-endemic areas were obtained as controls. All subjects had negative serology for HIV and HTLV-1. All participants provided written informed consent. The study protocol, consent forms and all procedures were approved by the CIDEIM Institutional Review Board for the ethical conduct of research involving human subjects.
PBMCs were isolated by centrifugation over Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). B cells and CD4 T cells were isolated by MACS using the B cell isolation and CD4+ T cell isolation kits II, respectively (Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer’s instructions. Purity assessed by staining with anti-CD20 or anti-CD4 was ≥95% for both populations. Ramos cells were obtained from the American Type Culture Collection (Rockville, MD).
pLAg was prepared by suspending promastigotes of the L. panamensis strain MHOM/COL/81/L13 at a concentration of 1 × 107 parasites/mL, freezing in liquid nitrogen and thawing at 37°C four times. Cells were suspended in RMPI 1640 (Sigma-Aldrich) with 10% FBS (Gibco, Carlsbad, CA), 2 mM L-glutamine, penicillin (100 U/mL) and streptomycin (100 mg/mL) and distributed in 96 well plates in 200 μL per well. For PBMC cultures, 4 × 105 PBMCs and for B cell/CD4 T cell co-cultures, 2 × 105 B cells or 2 × 105 T cells or both cell types were plated per well. Then 8 μL of pLAg were added to the appropriate wells to reach a parasite:cell ratio of 0.2:1. The cells were incubated for 5 days at 37°C with 5% CO2. Cells and supernatants were harvested for evaluation of cell surface markers and cytokine secretion, respectively.
Ramos cells were cultured in RPMI 1640 with 10% heat-inactivated FBS at a concentration of 106 cells/mL with pLAg (107 parasites/mL final concentration), 5 μM CpG ODN 2006 (InvivoGen, San Diego, CA) or no stimulus for 48 hours. Cells were harvested for evaluation of cell surface markers or BCR-mediated endocytosis.
Ramos cells were incubated with goat F(ab’)2 anti-human IgM-biotin (Invitrogen, Camarillo, CA) at 4°C for 20 minutes, washed twice with PBS, resuspended in media and incubated with the Ova Antigen Delivery Reagent (Ova-FITC conjugated to an anti-biotin antibody, Miltenyi Biotec, Bergisch-Gladbach, Germany) at 37°C for different time periods, at which cells were harvested and fluorescence evaluated by flow cytometry.
The following antibodies were used for cell surface marker evaluation: anti-CD4-PE-Cy5, anti-CD69-PE, anti-CD127-PE-Cy7, anti-CD20-PE-Cy7, anti-CD86-PE, anti-CD80-FITC (eBioscience, San Diego, CA), anti-CD25-ECD, anti-HLA-DR-ECD, and anti-CD11c-PC5 (Beckman Coulter, Brea, CA). Primary human cells or Ramos cells were suspended in FACS buffer (1× PBS, 1% BSA, 0.1% NaN3) and incubated for 20 minutes with panels of the indicated antibodies at 4°C. After washing, cells were analyzed in a Navios flow cytometer (Beckman Coulter, Brea, CA) and data was analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR). Gates used for analysis were set using appropriate isotype controls.
Cytokines were measured in culture supernatants using the Human Th1/Th2/Th9/Th17/Th22 13plex FlowCytomix Multiplex (eBioscience) following the manufacturer’s instructions. Data were analyzed using FlowCytomix Pro Software (eBioscience).
The Kolmogorov-Smirnov test was used to determine parametric or non-parametric distribution of the data. Thereafter, for comparisons between healthy subjects and CL patients, parametric data were analyzed using student t-test, and the Mann–Whitney test was applied for non-parametric data. For comparisons between CL patient cell cultures with L. panamensis- and control cultures, parametric and non-parametric data were analyzed using the paired t-test and the Wilcoxon signed-rank test, respectively. Statistical significance was defined as p < 0.05. All data were analyzed using Prism 5 software (GraphPad Software, Inc., La Jolla, CA).
Results and discussion
CD4 T cells and B cells of CL patients are concomitantly activated by L. panamensisin PBMC cultures
Purified B cells are capable of activating CD4 T cells and inducing cells with a regulatory phenotype in the absence of other APCs
These results demonstrate that the capacity of isolated B cells from CL patients to activate CD4 T cells is similar to that of all APCs present in PBMCs. We performed a paired analysis to compare the net increase for each CD4 T cell activation parameter between PBMC and B cell/CD4 T cell cultures and found no significant differences. Likewise, net cytokine secretion did not reveal significant differences except for IL-12p70 and IL-1β (which were secreted in low albeit significant amounts only in PBMC cultures), IL-6 (which was secreted in both types of cultures but in significant larger amounts in PBMC cultures) and IL-2 (which was only secreted in B cell/CD4 T cell cultures). Thus, 10 of the 13 cytokines measured had similar levels in both types of cultures in spite of the fact that PBMCs contain several cytokine-producing cell types besides B cells and CD4 T cells. The differences observed in IL-12p70, IL-1β and IL-6 can be explained by the presence of mononuclear phagocytes in PBMCs that are known sources of these cytokines. Significant IL-2 production only in B cell/CD4 T cell cultures suggests that secretion of this cytokine is efficiently induced by interaction of these cell types.
The known mechanisms for CD4 T cell activation by T cells are antigen presentation and cytokine secretion. Since B cells stimulated with pLAg did not produce any significant amount of 13 cytokines that are prominent in antigen induced recall responses, our data strongly suggest that presentation of L. panamensis antigens by B cells is responsible for CD4 T cell activation. B cells can encounter soluble antigen soon after its entry into the body by several mechanisms and internalize it efficiently by BCR-mediated endocytosis . Therefore, the fact that B cells are not phagocytic cells, and thus not a natural cellular host for Leishmania, does not diminish the relevance of their CD4 T cell activation function in CL. On the contrary, this characteristic may be crucial for their role as APCs in this disease since processing and presentation of particulate antigens on MHCII requires phagosome maturation, a process that can be inhibited by Leishmania , whereas presentation of soluble antigens on MHCII requires their correct targeting by the endocytic receptor, a process that is highly efficient after BCR-mediated endocytosis [30, 48]. Accordingly, a study by Carvalho et al. found that DCs that were infected by L. braziliensis did not upregulate costimulatory and MHCII molecules, while bystander DCs were activated and presented soluble antigen efficiently . An earlier study by Fruth et al. also showed that infection of macrophages with L. major inhibited APC function, but pulsing with Leishmania antigens did not . Consequently, since infection of APCs with live parasites inhibits their function, APCs such as B cells that do not harbor the parasite and are capable of handling soluble antigens may be ideally poised to activate the immune response.
Cultures of purified lymphocytes from CL patients also showed a small but significant increase in the expression of CD25 and CD69 and in the frequency of CD25hiCD127- cells among CD4 T cells when these cells were co-cultured with B cells in the absence of pLAg (Figure 4B-D). We also found a significant induction of CD86 and HLA-DR in B cells under the same conditions (Figure 5A and C). These results suggest that non-cognate interactions between B cells and CD4 T cells can generate activating signals in this setting. The induction of homeostatic lymphocyte proliferation by MHCII/self-peptide presented by APCs is well documented [32, 49, 50]. Hence, the activation observed in B cell/CD4T cell cultures in the absence of antigen may represent this type of interaction. Alternatively, the activation found in the absence of added antigen may be due to B cells from infected individuals already being loaded with Leishmania antigens, since we have found evidence of parasites in blood, during active CL and even after treatment of the disease .
Treatment with L. panamensisupregulates MHCII and costimualtory molecules independently of BCR signaling and enhances BCR-mediated endocytosis in human B cells
To test the effect of L. panamensis antigen on BCR-mediated endocytosis in Ramos cells, we targeted the model antigen ovalbumin (Ova) to the BCR using an anti-IgM antibody. BCR-mediated endocytosis of fluorescent Ova was evaluated in cells previously cultured for 48 hours with pLAg or CpG or without stimulus, as described in Materials and Methods. We found that Ova endocytosis by Ramos cells increased over the first two hours and then reached a plateau. When these cells were treated with CpG or pLAg, Ova endocytosis was more efficient and continued to increase throughout the period of evaluation (Figure 7C and D). A statistically significant difference was detected for both pLAg and CpG when compared to untreated cells both with respect to fluorescence intensity and the percentage of Ova-FITC positive cells at the 1 hour time point (Figure 7E). When OVA antigen was not targeted to the BCR, fluorescence did not increase at any time point, confirming that the assay evaluated BCR-mediated endocytosis (Figure 7D). This experimental strategy allowed us to show that L. panamensis antigen promotes specific antigen uptake by human B cells. The expression of surface IgM did not change significantly after treatment with CpG or pLAg (data not shown), indicating that the enhanced endocytosis was not due to the availability of more internalizing receptors. Similar enhancement of Ova endocytosis was previously observed in DCs incubated with L. braziliensis antigens for four days .
These results demonstrate that L. panamensis antigen can induce activation of B cell antigen presenting function without BCR engagement. TLR9 is the innate immune receptor that has the highest expression on human B cells [52–54] and it can be stimulated by Leishmania DNA [55, 56]. Since the pLAg preparation includes the parasite DNA, it is likely that TLR9 signaling can mediate activation of the antigen presenting function of human B cells, a question that we will address in future studies. In humans, B cells and plasmacytoid DCs are the only cell types responsive to TLR9 [57, 58]. Because the antigen presenting capacity of plasmacytoid DCs is low  and is further downregulated by TLR9 signaling , B cells constitute the primary APC targeted by TLR9 ligands in humans. In light of the numerous examples of successful protection in animals using CpG as adjuvant in vaccination strategies for CL [61–64], defining the role of TLR9 in B cell antigen presentation is critical to develop efficient strategies that can be translated into clinical applications.
We have shown that B cells from the peripheral blood of CL patients are capable of inducing CD4 T cell activation and cytokine secretion, an effect likely mediated by antigen presentation. The CD4 T cell activating capacity of these B cells was similar in both quantity and quality to that observed after incubation with all cells present in PBMCs. These B cells represent an attractive target for immunomodulatory strategies that aim to redirect the host’s immune response to a healing phenotype in chronic Leishmania (Viannia) infections.
Dr. Rodriguez-Pinto’s current address: Facultad de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas, Quito, Ecuador.
Professional antigen presenting cells
Peripheral blood mononuclear cells
Promastigote L. panamensis antigen.
We thank Liliana Valderrama and Alexandra Cossio for coordination of the study, Olga Fernández, Laura González and Karen Goldsmith-Pestana for technical assistance, Wilson Cortez, Eduardo Ortiz, Lyda Cuervo, Ricardo Márquez, María del Mar Castro, Javier Martínez and Jimena Jojoa for patient evaluation and recruitment, and Beatriz Herrera for assistance with bibliography. This work was supported by Fogarty International Center (NIH) (grant number D43 TW006589), the United States National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (grant number 1R01AI093775) and the 2011 Gorgas Memorial Institute Research Award to D.R-P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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