Interactions between dendritic cells and CD4+ T cells during Plasmodium infection

Background During infection, dendritic cells (DCs) encounter pathogenic microorganisms that can modulate their function and shape the T cell responses generated. During the process of T cell activation, DCs establish strong, long-lasting interactions with naïve T cells. Methods Using a mouse malaria model, the interactions of DCs and naïve CD4+ T cells have been analysed. Results DCs, either incubated in vitro with infected erythrocytes or isolated from infected mice, are able to present exogenous antigens by MHC-II, but are not able to establish prolonged effective interactions with naïve CD4+ T cells and do not induce T cell activation. It was also found that effective T cell activation of naïve CD4+ T cells is impaired during late Plasmodium yoelii infection. Conclusion These data may provide a mechanism for the lack of effective adaptive immune responses induced by the Plasmodium parasite.


Background
Dendritic cells (DCs) are antigen-presenting cells (APC) that play a central role in both innate and adaptive immune responses. To initiate T cell-dependent immune responses to microbial infections, DCs phagocytose antigens in peripheral tissues and migrate to the draining lymph nodes, where they interact with antigen-specific T cells. Maturation of DCs, involving up-regulation of the major histocompatibility complex (MHC) and peptide complexes and the costimulatory molecules at the surface, is required to efficiently prime naïve T cells [1]. Upon maturation, DCs reorganize their actin cytoskeleton, projecting long and motile membrane extensions, called dendrites. The initial encounters between antigen-presenting DCs and specific naïve T cells are characterized by the directional projection of abundant membrane extensions from the DC toward the naïve T cell, followed by entrapping of the T cell within a complex net of membrane extensions [2].
The activation of T cells by DCs during Plasmodium infection has been previously studied. Although different effects have been described depending on the parasite strain used, time after infection or subpopulation of DC analysed, a number of reports found defective activation of T cells [3]. These findings may be related with the low parasite-specific T cell responses induced by human malaria infections [4,5]. and CD11c + DCs from the spleens of groups of three blood stage-infected mice were incubated or not with 10 μg/ml of LPS for 20 h before incubation with OVA peptide 323-339 (ISQAVHAAHAEINEAGR) (Biopeptide Company, San Diego, CA) at 37°C for 1 h. DC-T cell contact was analysed by immunoflurescence, time-lapse video microscopy and FACS as previously described [8]. For immunofluorescence, DCs were washed twice with PBS and immobilized on poly-L-lysine-coated coverslips for 5 min at room temperature (10 5 cells/coverslip). PBS was then removed and replaced with complete medium and the coverslips were incubated for 1 h at 37°C. For DC-T cell conjugate formation, 10 5 naïve DO11.10 T cells isolated from transgenic mice were loaded with CFSE (0.5 μM) and added to DCs and incubated at 37°C. Incubation was stopped after 30 min and coverslips were washed five times with PBS and fixed with 1% paraformaldehyde for 10 min. F-actin was stained using rhodamine-conjugated phalloidin (Molecular Probes) (1:500 in blockingpermeabilization solution). F-actin is used to differentiate DCs from T cells, as the later have much lower levels of Factin [8]. DC-T cell contact was reported as the number of 'engulfed' T cell per 100 DCs in each each coverslip. For time-lapse video microscopy, chambers mounted on a coverglass (Nalge Nunc International) coated with 10 5 DCs were placed on the microscope at 37°C. One minute after addition of 10 5 naïve DO11.10 T cells images were collected every 10 seconds for 20 min. Images were acquired using a 60× oil immersion objective and a Hamamatsu digital camera (Universal Imaging). For FACS, pre-stained naïve DO11.10 T cells with 0.5 μM CFSE and DCs with 1 μM of CellTracker™ Orange (Molecular Probes) were mixed (1:1), spun for 3 min at 500 rpm (4°C) and incubated at 37°C for 30 min. Contact was stopped by transferring the tubes to ice. Analysis by FACS was done immediately after and the results are expressed as percentage of green-red events to total T cells.

Stimulation of naïve DO11.10 CD4 + T cells
Immature and mature bone marrow derived DCs previously incubated with uninfected or P. yoelii-erythrocytes and CD11c + DCs from the spleen of groups of three blood stage-infected mice at different time points were incubated or not with 10 μg/ml of LPS for 20 h before incubation with 10 μg/ml of OVA peptide 323-339 as above. After washing, 2 × 10 5 DCs were incubated with 2 × 10 5 naïve CD4 + T cells in 96 wells plate. T cell activation was measured as up-regulation of CD69 after 12 h of culture.

In vivo activation of naïve CD4 + T cells during malaria blood stage
In order to test the in vivo activation of naïve CD4 + T cells during malaria blood stage, adoptive transfer of DO11.10 cells was performed. 1.6 × 10 6 naïve DO11.10 CD4 + T cells from transgenic mice were labeled with CFSE (10 μM for 45 min) and transferred (i.v.) into groups of three uninfected or infected Balb/c mice 5 or 10 days after infection (sex and age of donor and recipient mice were matched). Mice were immunized with 2 mg/mouse of OVA emulsified in complete Freund's adjuvant (Sigma) by i.p. injection 24 h after transfer. Proliferation and expression of CD11a, CD62L and intracellular IL-2 was determined in transferred CFSE + CD4 + T-cells 3 days after immunization by FACS. Control mice received the same volume of adjuvant alone.

Ethical approval
Experiments performed with mice were approved by the NYU Institutional Animal Care and Use Committee (IACUC).

Results and Discussion
DCs present exogenous antigens in the context of MHC class II molecules for the activation of CD4 + T cells. DCs are also able to cross-present exogenous antigens in the context of MHC-I molecules to activate CD8 + T cells. It was determined whether MHC-II and MHC-I antigen presentation was affected during infection with P. yoelii. For this purpose, specific antibodies were used, that recognize defined peptide epitopes bound to particular MHC molecules. These antibodies do not recognize the antigen or the MHC molecules alone and can be used to determine the presence of MHC-epitope bound complexes in the surface of antigen presenting cells. To determine MHC-I and MHC-II antigen presentation anti-OVA epitope 257-264/H-2 b (25-D1. 16) and anti-LACK epitope 156-173/I-A d (2C44) antibodies were used, respectively. Bone-marrow derived DCs were incubated either alone, with uninfected or P. yoelii-infected erythrocytes for 24 h before addition of purified OVA or LACK proteins for 5 h, followed by stimulation with LPS to induce antigen presentation. It was observed that incubation with P. yoelii-infected erythrocytes inhibits presentation of exogenous antigens on MHC-I ( Figures 1A and  1B), but not on MHC-II molecules ( Figures 1C and 1D).
The activation of T cell hybridomas that recognize specific OVA peptides in the context of MHC-I and II molecules was used as an alternative method to determine antigen presentation. The T cell hybridomas B3Z [9] and DO.11.10 [10] recognize specific OVA peptides on MHC class I (H-2 b ) and II (I-A d ), respectively. T hybridoma cells are less dependent than primary naive T cells on costimulatory molecules and they can be activated by fixed antigen presenting cells. Recognition of peptide-MHC complexes by T hybridoma cells results in increased IL-2 secretion. DCs were incubated with uninfected or P. yoeliiinfected erythrocytes for 24 h before addition of purified OVA proteins for 5 h. DCs were then fixed to prevent secretion of cytokines and incubated with the different hybridomas for 24 h. Activation of hybridomas was determined by detection of IL-2 in the incubation medium. It was found that DCs pre-incubated with P. yoelii-infected erythrocytes activate the T cell hybridoma recognizing OVA-MHC-II, but the activation of the hybridoma recognizing OVA-MHC-I was significantly reduced. The activation of the hybridomas was dose dependent (Figures 2A  and 2B).
Maturation of DCs increases their capacity for antigen presentation, since it increases the expression of MHC molecules on the cell surface [1]. As expected, it was found that the addition of LPS to induce maturation of DCs 5 h after incubation with OVA, resulted in increased activation of both hybridomas recognizing OVA-MHC-I and II ( Figures 2C and 2D). The effect of pre-incubating DCs with P. yoelii-infected or uninfected erythrocytes in antigen presentation of OVA by MHC-I and MHC-II after inducing maturation with LPS was analysed. It was again observed that P. yoelii-infected erythrocytes inhibit the activation of the hybridoma recognizing OVA-MHC-I, but not OVA-MHC-II ( Figures 2E and 2F).
The capacity of CD11c + DCs isolated from P. yoeliiinfected mice to present antigens by MHC-II was analysed. It was found that that MHC-II antigen presentation is maintained during the course of infection, but MHC-I cross-presentation of exogenous antigens is inhibited throughout the course of the disease ( Figures 2G and 2H). These results confirm previous observations [11] that MHC-I antigen presentation is inhibited during Plasmodium infections and indicate that DCs are able to process and present exogenous antigens by MHC-II during P. yoelii infections.
The initial encounters between antigen-presenting DCs and specific naïve T cells include directional projection of abundant membrane extensions from the DC toward the naïve T cell and prolonged interactions between both [2]. To analyse whether Plasmodium interferes with this process, the interactions between bone marrow derived DCs pre-incubated with infected erythrocytes and naïve CD4 + T cells were first studied using time-lapse video microscopy. DCs were pre-incubated with uninfected or P. yoeliiinfected erythrocytes and incubated with naïve anti-OVA Plasmodium yoelii-infected erythrocytes inhibit MHC-I but not MHC-II antigen presentation CD4 + T cells isolated from transgenic mice (DO11.10) recognizing a specific OVA epitope. DCs were loaded with the same peptide epitope (OVA 323-339) before addition of T cells. DCs presenting antigen normally interact with antigen-specific naïve T cells for long periods of time (more than 5 min) with abundant membrane extensions projected in the direction of the T cell [8]. Pre-incubation of DCs with uninfected erythrocytes did not affect DC-T cell interactions, as prolonged interactions with membrane extensions were frequently found in the co-cultures ( Figure 3A and Additional file 1). In contrast, pre-incubation with P. yoelii-infected erythrocytes inhibits the capacity of DCs to maintain prolonged interactions with membrane extensions with naïve T cells ( Figure 3B and Additional file 2). Only short interactions without projection of membrane extensions were observed.
To perform a quantitave analysis of this phenomenon, the formation of stable conjugates between antigen-presenting DCs and specific T cells was observed. Each cell type population was labeled with a different fluorescent dye to allow the determination of dual positives for both labels that correspond to stable conjugates [8]. After a 30 min co-incubation of equal numbers of DCs presenting the ovalbumin epitope and the specific naïve anti-OVA CD4+ T cells formed stable conjugates where DCs 'engulf' T cells ( Figure 4A, upper panels), which are different from loose contacts between these cells ( Figure 4A, lower panel). Quantification of the number of dual positives by fluorescence microscopy revealed that DC-T cell conjugates are formed when control DCs loaded with the specific peptide epitope are incubated with specific T cells and the number of conjugates is increased when DCs had been activated by addition of LPS. Pre-incubation of DCs with P. yoelii-infected erythrocytes significantly inhibited the formation of stable DC-T cell conjugates ( Figure 4B). Analysis of double positives for each fluorescent label by FACs provided similar results ( Figure 4C). Addition of LPS as a maturation signal increased the number of DC-T cell conjugates in the control DCs pre-incubated with uninfected erythrocytes, but did not improve DC-T cell interactions impaired by P. yoelii-infected erythrocytes ( Figures  4B and 4C).

Time-lapse microscopic analysis of the interaction between DCs and T cells after pre-incubation with P. yoelii-infected erythro-cytes Figure 3 Time-lapse microscopic analysis of the interaction between DCs and T cells after pre-incubation with
When a similar analysis was performed using CD11c + DCs isolated from P. yoelii-infected animals at different times after infection, we also found a significant decrease in the numbers of DC-T cell stable conjugates compared to uninfected mice (day 0). Even if lower, detectable levels of stable conjugates formed by DCs from infected mice and specific T cells were found by microscopy and FACs (Figures 4D and 4E), suggesting that DC-T cell interactions are impaired but not completely inhibited by P. yoelii infection. These levels were minimally increased by addition of LPS to DCs (Figures 4D and 4E).
To determine the level of T cell activation in the co-cultures of DC and T cells, DCs were activated by addition of LPS and the surface expression of the early activation marker CD69 in T cells were analysed. In the co-cultures of DCs incubated with P. yoelii-infected erythrocytes or isolated from infected mice, the level of T cells expressing CD69 was greatly decreased compared to co-cultures with control DCs (Figures 4F and 4G).
To study the activation of CD4 + T-cells during P. yoelii infection in vivo, naïve CD4 + T cells were transferred from DO11.10 transgenic mice that are specific for the OVA epitope 323-339 into P. yoelii-infected (day 10 p.i.) or uninfected mice. Mice were immunized with OVA 24 h after transfer and CD4 + T-cell activation was measured 72 h after OVA injection. Naïve CD4 + T-cells were fluorescently labeled before transfer to allow identification. The number of transferred CD4 + T-cells in the spleen after 72 h was similar in uninfected and infected animals ( Figure  5A), but after immunization with antigen, infected mice primed transferred naïve CD4 + T-cells with lower efficiency than uninfected mice. T-cell activation was lower in infected mice as determined by proliferation (decrease in CFSE labeling, Figure 5B), up-regulation of CD11a ( Figure  5C), down-regulation of CD62L ( Figure 5D) and increased intracellular IL-2 ( Figure 5E). The effect of transferring naïve CD4 + T cells from DO11.10 transgenic mice into P. yoelii-infected mice earlier in infection (day 5), was also analysed. Under these conditions, only proliferation (decrease in CFSE labeling) and expression of CD11a were significantly decreased in infected animals. No significant changes were found in IL-2 secretion and expression of CD62L.
The capacity of DCs to provide adequate antigen presentation to T cells in the context of P. yoelii infection was analysed in detail. The results suggest that during P. yoelii infections DCs maintain the ability to process and present exogenous antigens by forming the MHC-II-epitope complex in the surface of DC, however, as shown before for P. berghei [11], impaired cross-presentation of exogenous antigens by MHC-I was found.
Efficient antigen presentation is not sufficient for the activation of naïve CD4 + and CD8 + T cells, which also requires co-stimulatory signals from DCs and specific cytokines [12]. The interactions of naïve T cells with mature priming-inducing DCs are more stable than the contacts with resting tolerance-inducing DCs. It has been proposed that stable DC-T cell interactions participate in the induction of antigen-specific T cell activation through the delivery of an activatory signal by the DCs. In contrast, unstable contacts with resting DCs might induce shortterm activation and proliferation signals in T cells [2], which may explain the lack of T cell activation that we observed during late malaria infection. In the absence of mature DCs, serial unstable contacts between T cells and resting DCs would result in T cell clonal deletion [2], a phenomenon that has also been observed during malaria infections, where there is specific deletion of T cells recognizing Plasmodium antigens [13].
Maturation of DCs increases the duration of DC-T cell interactions and allows the formation of a complex net of membrane extensions in which DCs entrap T cells [8]. The capacity of DCs to establish these effective interactions with naïve CD4 + T cells was found to be inhibited during infection. As actin-mediated reorganization of DC morphology is required to form the strong, long-lasting interactions with T cells [8], it is possible that the parasite may interfere with the cytoskeleton of the DCs. In fact, numerous genes in the DC cytoskeleton are modulated during P. yoelii infection [14].
Previous studies have shown that DCs can process and present antigens associated with MHC class II during blood stage infections, suggesting that efficient activation of CD4 + T cells could take place during murine infection [15], however, defective activation and specific depletion of CD4 + T cells recognizing Plasmodium antigens is observed during murine malaria infections [13,16]. Suppression of OVA-specific CD4 + T cell proliferation has also Activation of naïve CD4 + T-cells is impaired during late Plasmodium blood stage infection been observed in P. chaubaudi-infected mice, but in this model inhibition was evident since early infection [17]. Furthermore, these authors also found that DC and CD4 + T cell interactions are inhibited by P. chaubaudi infection in vitro and in vivo, while antigen presentation by MHC-II is not affected [18].
Recently, two subpopulations of CD11c + DCs with differential abilities to activate antigen-specific T cells have been identified in P. chaubaudi infected mice [19], suggesting that there may be a balance of opposing forces on the host response. Since the interactions between DCs and T cells are decreased, but are still detectable, it is possible that we have observed the sum of different effects contributed by different subpopulations of DCs. It is currently believed that tolerance of DCs, induced by exposure to TLR ligands is induced during malaria infection [20]. In this context, it is likely that tolerized DCs that are found increasingly during late infection would have impaired interactions with T cells leading to decreased activation.

Conclusion
In this work, it was observed that DCs isolated from infected mice at different times after infection are not able to establish strong interactions and prime naïve CD4 + T cells. Protection generated by Plasmodium infections against blood-stage infection is mediated by helper and effector functions of CD4 + T cells [21]. However, this T cell response does not induce complete protection or longterm immunity, suggesting that T cell activation or maintenance is impaired [22]. These results indicate that activation of naïve CD4 + T cells by DCs is impaired during late malaria blood-stage infection in mice.