implications for T lymphocyte-directed immunotherapy
Montfoort, A.G. van
Citation
Montfoort, A. G. van. (2010, November 25). Antigen targeting to Fc receptors on dendritic cells : implications for T lymphocyte-directed immunotherapy.
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Circulating Specific Antibodies Enhance Systemic T cell Priming by Delivery of Complexed Antigen to Dendritic Cells in vivo
Nadine van Montfoort*, Sara M. Mangsbo*, Marcel G.M. Camps, Ingrid E. C. Verhaart, Aris Waisman, Jan Wouter Drijfhout,
Cornelis J.M. Melief, J.Sjef Verbeek, and Ferry Ossendorp
*contributed equally
Submitted for publication
Chap ter 4
Summary
Increasing evidence suggests that antibodies can have stimulatory effects on T cell immunity, however, the contribution of circulating antigen-specific antibodies on T cell priming has not been conclusively established. To investigate if circulating antibodies aid T cell priming in a host naïve for the T cell antigen, mice with hapten-specific antibodies were infused with haptenated antigen and T cell proliferation was measured. Primary T cell responses were significantly enhanced in mice with circulating antibodies and the response was associated with improved antigen capture by APCs. Importantly, CD11c+ APCs were responsible for the enhanced T cell immunity, albeit CD11c- APCs had captured a significant amount of the injected antigen.
Moreover, the enhanced T cell proliferation was transferable to antigen-naive mice by serum or antigen-specific antibody transfer. Thus, in vivo formation of antigen-antibody immune complexes improves T cell induction demonstrating that humoral immunity can aid the initiation of cellular immunity.
Introduction
The adaptive immune system has two distinct key players, B and T lymphocytes, that play an important and individual role when it comes to combating both infectious diseases and malignant transformations. However, the mutual action of the two distinct cell types may be of importance for the immunological outcome. The Janus face of B lymphocytes in T cell immunity has been debated over the past years. Enhanced anti-tumor T cell responses in B cell deficient mice suggest that B lymphocytes can hamper T cell immunity (1), however, the effect does not appear to be mediated by cognate B cell:T cell interactions or by tumor-specific antibodies (2). Contrasting these data, a recent study by DiLillo et al (3) demonstrates that T cell proliferation as well as Th1 responses were hampered after B cell depletion, indicating a crucial role for B lymphocytes in the initiation of a T cell response. However, none of these papers addressed the role of circulating antibodies in the induction of T cell immunity.
In addition to the role B cells themselves play in T cell biology, an increasing body of evidence suggests that antibodies modulate the cellular immune response (4). Enhanced specific T cell responses have been observed after treatment with MUC 1 (5) and HER2/neu (6) specific antibodies in humans and after injection of neu-specific antibodies in mice that received a neu- expressing whole cell vaccine (7). This enhancement was shown to be FcgR-dependent and mediated by dendritic cells (DC).
We and others have previously demonstrated that specific antibodies can enhance uptake and presentation of soluble antigen by DCs both in vivo and in vitro through formation of immune complexes (8-10). The potency of immune complexes to induce T cell responses has been attributed, not only to their capacity to effectively target the antigen to DCs, but also to induce DC maturation. Both processes are facilitated by the IgG Fc receptor (FcgR).
These earlier studies were performed with optimized immune complexes pre-formed in vitro.
Whether actual circulating antigen-specific antibodies can be effective for T cell priming needs to be investigated. The information gained from such an experiment is of importance for the understanding of the therapeutic potential of tumor-associated antigens (TAA)-specific antibodies, intensively used in the clinic nowadays. Thus, we developed an experimental model that enabled us to dissect the role of antibodies in the induction of T cell immunity. We induced a classical hapten-specific antibody response in mice that were naïve for the T cell antigen (OVA).
In these mice, we were able to demonstrate that hapten-specific circulating antibodies markedly enhanced the priming of OVA-specific CD4+ and CD8+ T lymphocytes after vaccination with haptenated OVA. By specific conditional depletion of CD11c positive DCs we were also able to show that CD11c+ DCs were crucial for the antibody enhanced priming of CD8+ T cells. In conclusion, our data indicate that enhanced T cell immunity can be achieved by in vivo formation of antigen-antibody complexes that target the antigen to CD11c+ dendritic cells.
Materials and Methods
Mice and reagents
All mouse studies were approved by the Leiden University Medical Center Institutional Review Board. C57BL/6 mice were purchased from Charles Rivers laboratories. CD90.1 congenic mice on C57BL/6 background, OT-1 (CD90.1+) mice (CD8+ T cell transgenic mice expressing a TCR recognizing OVA257-264 SIINFEKL in H2-Kb) and OT-2 (CD90.2+) mice (CD4+ T cell transgenic mice expressing a TCR recognizing OVA323-339 in I-Ab) were all bred in our institute.
CD11c Cre/iDTR mice were generated by crossing CD11c Cre mice (11) with iDTR mice (12). 2,4,6-trinitrophenol (TNP) conjugated to bovine serum albumin (TNP(15)-BSA) and TNP conjugated to ovalbumin (TNP(12)-OVA) were purchased from Biosearch Technologies.
Complete Freund’s adjuvant (CFA) and Incomplete Freunds’s adjuvant (IFA) were purchased from Difco.
Induction of hapten-specific antibody responses
C57BL/6 mice or CD90.1 mice were subcutaneously injected with 50µg TNP-BSA in 100 µl of an emulsion of saline and CFA (50/50 v/v). After 4 and 6 weeks, mice were boosted with 50µg TNP-BSA in 100 µl emulsion of saline and IFA (50/50 v/v) subcutaneously. Seven to ten days after the boost, blood samples were collected from the tail vein of the individual mice in order to evaluate antibody titres. Serum was collected and stored at -20 degrees.
Measurement of antibody titers
Antibody titers in the serum of mice were assessed with enzyme-linked immune absorbent assay (Elisa). Nunc 96-wells microtiter plates were coated with 50 µl/well TNP-BSA, TNP-OVA, OVA (Worthington) or BSA (Sigma) at 4µg/ml. Plates were blocked 1h at 37 ºC and washed with 100 µl/well PBS-0.05% Tween. Plates were incubated 2h at 37 ºC with 50 µl/well serum diluted in PBS-0.05% Tween. Serum dilutions started at 1:500. Subsequently, plates were incubated for 2h with 50 µl/well goatαmouse horseradish peroxidase (HRP) diluted 1:1000 in PBS-0.05% Tween at room temperature in the dark. Subsequently 50 µl/well ABTS (0.4 µL H2O2/mL ABTS) was added as a substrate. Absorption was measured at 415 nm.
In vivo antigen presentation assay
Recipient mice received CFSE-labelled whole splenocyte/lymph node preparations iv from OT-1 or OT-2 mice containing 3x106 Va2+/CD8+ or 3x106 Vβ5+/CD4+ cells respectively. For OT-2 cells, CD90.1 congenic mice were used as recipients. One day later, TNP-OVA or TNP- BSA was injected in 200 µl saline iv. Four days after the injection of the antigen, spleen and lymph nodes were collected and proliferation of CFSE-labelled T lymphocytes was analyzed by flow cytometry. OT-1 cells were gated on CD8+/CD90.1+ cells. OT-2 cells were gated on CD4+/ CD90.2+ cells.
Serum/antibody transfer experiments
Either CFSE-labelled whole splenocyte/lymph node preparations from OT-1 mice (Fig. 4A, as described above) or enriched CD8+ T lymphocytes from OT-1 mice (BD Biosciences Mouse
CD8 T Lymphocyte Enrichment set) from OT-1 mice (Fig. 4B/6) were injected into naïve recipient mice at day -1. After enrichment, the cell suspension contained approximately 93%
Va2+/CD8+ cells. Day 0, pooled sera from Ab+ mice or 100 μl of rabbit anti-OVA (200 μg) or control rabbit sera was injected iv into mice and a few hours later 10 μg of TNP-OVA or 5μg of OVA was injected (iv). Day 4, proliferation in from spleen and lymph nodes was analyzed by flow cytometry. OT-1 cells were gated on CD8+/CD90.1+ cells.
Depletion of CD11c+ cells
In experiments were CD11c Cre/iDTR mice were used, diphtheria toxin (DT, calbiochem) was injected i.p. on day -3, -1 and 1 at a dose of 25 ng/g body weight. CD11c depletion on day 0 was evaluated: both CD11c high and intermediate cells were effectively eradicated in mice receiving DT.
In vivo uptake experiments
TNP-BSA was conjugated with the DyLight 649 Antibody Labelling Kit (Pierce) according to the manufacturer’s guide-lines. TNP seropositive or seronegative mice were iv injected with 10μg of DyLight 649 labeled TNP-BSA. After 4 or 24 hour, mice were sacrificed. Spleen and pooled lymph nodes of each mouse were isolated and incubated with 4 WU/mL of Liberase R1 (Roche) for 30 minutes at 37 ºC. The different populations of APCs were gated as indicated (Figure 3A). Subsequently, the percentage of Dylight 649+ cells within each gated population was analyzed. Conventional DCs were gated as CD11chigh and further as CD8α+ CD11blow or CD8α- CD11b+ (13). Plasmacytoid DCs (pDCs) were gated as CD11cinterm and further as Gr-1+, B220+. Macrophages were gated as CD11b+ and CD11c-. B lymphocytes were gated as B220+ CD11c- CD8α- (Fig 3A). Background was defined by analyzing a spleen of a naive mouse that did not receive the labelled antigen.
Statistics
Means of the groups were compared with the student’s t-test and in the figures *** equals p<0.001, ** equals p<0.01 and * equals p<0.05. ns indicates not significant.
Figure 1: Mice vaccinated with TNP-BSA have circulating antibodies specific for TNP and BSA but not for the model T cell antigen OVA. (A) Serum of mice vaccinated two times with 50 μg TNP-BSA was diluted 8000 times and tested in ELISA to bind OVA, BSA, TNP-OVA or TNP-BSA. (B) Serum dilutions of 9 mice vaccinated with TNP-BSA was tested by ELISA for specificity to TNP and compared to dilutions of pooled serum from naïve mice.
Data are shown as mean+/-SD.
Results
Mice with circulating hapten-specific antibodies, but naïve for the model-antigen OVA
To study the role of specific antibodies in the induction phase of a T cell immune response, we used a classical haptenated protein antigen. The antigen is composed of a B cell epitope, the hapten 2,4,6-trinitrophenol (TNP), conjugated to ovalbumin (OVA). To obtain TNP seropositive mice, naive mice were vaccinated with 50 mg TNP-BSA in Freund’s adjuvant. After a second and third vaccination, serum was analyzed by ELISA for antibodies against OVA, BSA, TNP-BSA or TNP-OVA (Figure 1A). All mice had circulating antibodies specific for TNP-OVA and TNP- BSA, indicating specificity for TNP. In addition, mice had circulating antibodies against BSA, but importantly, they were still naïve for our model T cell antigen OVA (Figure 1A). TNP-specific antibody titers are shown in Figure 1B, demonstrating a robust TNP-specific antibody response.
Screening for the different IgG subclasses revealed that TNP-specific antibodies were mostly IgG1 but all vaccinated mice had measurable titers of IgG2a and IgG2b while IgG3 was not demonstrable (not shown). All of these isotypes are known to cross-link mouse FcγR when they are complexed (14,15).
Figure 2: Enhanced OVA-specific CD4+ T cell induction in mice with circulating hapten-specific antibodies after injection with haptenated OVA
TNP seronegative (Ab-) or seropositive (Ab+) mice received 3x106 CFSE-labelled OVA-specific OT-2 CD4+ T cells and were subsequently intravenously injected with antigen as indicated. After four days, mice were sacrificed and spleen and inguinal lymph nodes were isolated and analyzed by flow cytometry. (A) CFSE dilution of CD90.2+/CD4+ cells from the spleen of one representative mouse per group. The graphs represent % of proliferating cells in Ab- mice not injected with antigen (-), Ab- mice iv injected with 5 μg TNP-OVA, Ab+ mice iv injected with 5 μg TNP-BSA or Ab+ mice iv injected with 5 μg TNP-OVA. (B/C) Mean and SD of % of proliferating CD4+ cells per group (n=6) measured in spleen (B) and lymph nodes (C). This experiment has been performed two times with similar results.
CD4+ T cell priming is enhanced in seropositive mice
TNP-OVA was used as antigenic construct to investigate whether, in these mice with circulating anti-TNP antibodies but naive for OVA, TNP-specific antibodies were able to enhance an OVA specific T cell response. Proliferation of CFSE labelled OVA-specific CD4+ T lymphocytes (OT-2) was used as read-out for T cell induction. Non-vaccinated (seronegative, Ab-) mice or mice vaccinated with TNP-BSA (seropositive, Ab+) received CFSE-labelled OVA-specific naive CD4+ T lymphocytes and were subsequently injected with 5 mg TNP-OVA intravenously. As a control, Ab+ mice were injected with 5 mg TNP-BSA. CD4+ T cell proliferation was significantly enhanced in both spleen (Fig. 2A/B) and lymph nodes of seropositive mice compared to seronegative mice (Fig. 2A/C). Proliferation of OVA-specific CD4+ T lymphocytes was not enhanced after injection of TNP-BSA (Fig. 2A/B/C) indicating that the enhanced proliferation of CD4+ T lymphocytes is antigen-specific and not caused by the circulating antibodies alone.
CD8+ T cell priming is enhanced in seropositive mice
In a similar experiment as above, the role of hapten-specific circulating antibodies on the induction of CD8+ T lymphocytes was studied by using CFSE-labelled OVA-specific CD8+ T lymphocytes (OT-1). OT-1 cell proliferation was significantly enhanced in both spleen and lymph nodes of seropositive compared to seronegative mice after injection of TNP-OVA (Fig 3A/B). In contrast, after injection of TNP-BSA, proliferation of OT-1 cells in Ab+ mice was comparable to the proliferation observed in naïve control mice (Fig 3A/B) signifying that the enhanced CD8+ T cell induction is antigen specific.
Systemic CD8+ T cell priming is enhanced in seropositive mice after sc injection of the haptenated antigen In the previously described experiments, the antigen was applied systemically through intravenous injection. Next, we studied the role of circulating antibodies in the cross-presentation of a subcutaneously administered antigen. In the draining inguinal lymph node, a high percentage of proliferating CD8+ T lymphocytes was observed in both the Ab+ and the Ab- mice (Fig. 3C).
In contrast, antigen-specific CD8+ T cell proliferation was only significantly enhanced in the spleens and mesenteric lymph nodes of Ab+ mice (Fig. 3C). These results suggest that circulating antibodies enhance T cell induction only when the antigen reaches the circulation but not when it is exclusively present in the skin close to the draining lymph node.
Passive transfer of antigen-specific antibodies or sera from seropositive mice increase T cell priming in response to an antigen challenge
We reasoned that if the enhanced T cell immunity in seropositive mice is mediated by circulating antibodies specific for the injected Ag, the effect is transferable to naive mice by transfer of serum. To study this, serum derived from Ab+ mice was transferred into naïve recipient mice. Proliferation of OVA-specific CD8+ T lymphocytes was significantly enhanced in mice that received Ab+ serum compared to mice that did not receive serum (Figure 4A). Similarly, enhanced OVA-specific CD8+ T cell proliferation was observed in mice that received polyclonal OVA-specific rabbit IgG compared to mice that were injected with rabbit control serum (Figure 4B). The data indicate that the enhanced antigen-specific CD8+ T cell proliferation is mediated by antigen-specific antibodies and are not attributed to other vaccine-induced effects.
Figure 3: Enhanced OVA-specific CD8+ T cell induction in mice with circulating hapten-specific antibodies after injection with haptenated OVA Non-immunized mice (Ab-) or mice with high antibody titers to TNP (Ab+) received 3x106 CFSE-labelled OVA-specific OT-1 CD8+ T cells and were subsequently intravenously injected with TNP- OVA or TNP-BSA as indicated. After four days, mice were sacrificed and spleen and inguinal lymph nodes were isolated and analyzed by flow cytometry. (A) CFSE dilution of CD90.1+/CD8+ cells from the spleen of one representative mouse per group. The graphs represent % of proliferating cells in Ab- mice not injected with antigen (-), Ab- mice iv injected with 10 μg TNP-OVA, Ab+ mice iv injected with 10 μg TNP-BSA or Ab+ mice iv injected with 10 μg TNP- OVA. (B) Mean and SD of percentage of proliferating CD8+ cells per group (n=4) measured in spleen and lymph nodes.
Means of the groups were compared by the student’s t-test. This experiment has been performed three times with similar results. (C) Seronegative (Ab-) or seropositive (Ab+) mice received 3x106 CFSE-labelled OVA-specific OT-1 CD8+ T cells and were subcutaneously injected with 10 μg TNP-OVA. After four days, mice were sacrificed and spleens, inguinal draining lymph nodes (DLN) and mesenteric lymph nodes (NDLN) were isolated and analyzed by flow cytometry. Mean and SD of percentage of proliferating CD8+ T cells per group (n=4) is shown.
Antigen uptake is improved by circulating antibodies
To further address the mechanism of enhanced T cell proliferation, we investigated which cells captured the antigen in the Ab+ and Ab- mice by using Dylight 647 labelled haptenated antigen. When injecting the Ag iv, enhanced uptake was observed by conventional DCs and plasmacytoid DCs in Ab+ compared to Ab- mice (Fig 5B). Macrophages and B cells also engulfed the antigen, but only macrophages demonstrated enhanced antibody mediated uptake (4 hours) which was lost after 24 hours. The percent of macrophages that had acquired the antigen was higher than the percent of CD11c+ DCs that had acquired the antigen in the Ab+ animals.
Moreover, macrophages outnumber the CD11c+ DCs in the spleen. Thus, although more antigen was routed to professional APCs when complexed in vivo, antigen was also “lost” after being captured by the macrophage/B cell pool. Uptake of Dylight 647-labelled haptenated antigen was also investigated after sc injection. No significant differences in uptake by DCs in the draining lymph node were observed between Ab+ and Ab- mice (data not shown). This correlates with comparable percentages of proliferating T cells in the draining lymph nodes of Ab+ and Ab- mice (Fig 3C). Although enhanced proliferation of CD8+ T lymphocytes was observed in the spleen of Ab+ mice after sc antigen injection, we did not observe significant antigen uptake in the spleen after sc injection, probably due to the detection limit of the experimental procedure.
Figure 4: Transfer of antibodies enhances CD8 T cell induction through in vivo formation of antigen- antibody immune complexes Naive mice that had received 3x106 CFSE-labelled OVA-specific OT-1 CD8 T cells were intravenously injected with 200 ml of pooled serum from TNP seropositive mice (A) or 100 ml of OVA-specific rabbit IgG or 100 ml control rabbit sera (B). Hours after sera/antibody injection they were intravenously injected with 10 mg of TNP-OVA (A) or 5 mg of OVA (B). After four days, mice were sacrificed and spleens were isolated and CD8 T cell proliferation was analyzed by flow cytometry. Mean (SD) percentage of proliferating cells per group is shown. This experiment was performed at least three times with similar results.
CD11c+ cells are crucial for antigen-presentation of an in vivo complexed antigen
Since various cell types take up complexed antigen we aimed to elucidate the role of CD11c+
cells in the antibody-mediated T cell priming. CD11c-Cre mice (11) were crossed to iDTR (12) mice. In the CD11c Cre/iDTR offspring, CD11c+ cells were depleted by repetitive injections of diphtheria toxin (DT). Passive transfer of anti-OVA antibodies was used to ensure circulating pre-existing antibodies at the moment of antigen injection. Upon selective depletion of CD11c+ dendritic cells in the mice, proliferation of OVA-specific T lymphocytes was completely abolished, while in control mice (CD11c Cre/iDTR) that did not receive DT, proliferation was comparable to wt mice (Figure 6A). The experiment indicates that CD11c+ cells are crucially involved in antigen-presentation of an in vivo complexed antigen. Cell depletion was also controlled for by staining splenocytes with anti-CD11c. The data confirmed successful depletion and established that no transfer of CD11c+ CFSE+ cells had taken place (data not shown).
Figure 5: Enhanced uptake of intravenously injected antigen by splenic APC in seropositive mice compared to seronegative mice. (A) Gating of the different populations of APCs. (B) Mean+/-SD of percentage of Dylight positive cells per gated population of APCs in spleens of seropositive (Ab+) or seronegative (Ab-) mice at 4 hrs or 24 hrs after iv injection of Dylight 649 conjugated TNP-BSA. This experiment has been performed twice. The statistical comparison was made between the seronegative and the seropositive group of each subset of cells.
Figure 6: CD11c+ cells are crucial for CD8+ T cell priming in a host with circulating antigen-specific antibodies.
Wild type or conditional knock-out mice (CD11c Cre/iDTR) were adoptively transferred with CD8+ enriched CFSE labelled OT-1 cells and 1 day later they were iv injected with anti-OVA or control serum. A few hours later the mice were injected with OVA (iv). As indicated mice were also given DT at specific time-points to remove CD11c+ cells. After four days, proliferation of OT-1 cells was analyzed in spleen and lymph node of the mice. Proliferating OT-1 cells from spleen (A) and the inguinal lymph nodes (B). Cumulative data from the experiment are shown as histograms (C). This experiment was performed twice with similar results.
Discussion
As therapeutic antibodies are entering the clinic as therapy for malignant diseases there has been a growing number of reports arguing that antibodies improve or enhance T cell activation.
However, to date, no report has applied a model in which antigen-specific antibodies are present, while T cells are still naïve for their antigen. This model system is crucial to establish that antibodies aid T cell priming. Our model system, using hapten-specific antibodies induced by TNP-BSA, and naïve T cells that recognize the OVA peptide SIINFEKL bound to Kb makes a distinction between B and T cell immunity that is necessary to be able to answer the question posed above.
Our data demonstrate that circulating antibodies improve T cell priming, both for the CD4+
and the CD8+ T cell arm. The antigen can be delivered either iv or sc to the mice. The increase in T cell proliferation correlates with enhanced antigen uptake in DCs and macrophages. This is likely mediated by in vivo formation of immune complexes, since these are much more efficiently captured by splenic APCs than non-complexed protein antigen in the circulation (10,16).
Together with previous work showing that intravenously injected pre-formed antigen-antibody complexes are cross-presented more efficient to CD8+ T lymphocytes than soluble Ag (10), we conclude that the enhancement of T cell immunity is mediated by in vivo formation of Ag- Ab immune complexes that are efficiently cross-presented by APCs. Others have also made attempts to study this, however, by using mice with a pre-existing T cell immunity against the antigen (7). Thus the decisive role of antibodies on T cell priming was not conclusive.
When studying the antigen uptake after in vivo complex formation it is evident that all three subsets of DCs (spleen resident DCs are commonly divided in three subtypes: CD8+ and CD8- conventional DCs (cDCs) and plasmacytoid DCs (13,17) capture more antigen in Ab+ mice than Ab- mice after iv injection of the antigen. This correlates with the expression of the different classes of FcgRs in cDCs (18) as well as pDCs (19). CD8+ cDCs can present soluble or cell- bound antigen (17,20) to CD8+ T lymphocytes while cross-presentation of immune complexes from the blood is mediated by both subsets of cDCs (18). In contrast, pDCs have a poor capacity to present Ab-bound antigen to T lymphocytes (19).
The results indicate that circulating antibodies enhance uptake exclusively when the specific antigen reaches the circulation. Immune complex formation in the skin may be hampered by limited access of antibodies to the skin, but also by the low efficiency of drainage of immune complexes to the lymph node (21). Subcutaneously injected soluble antigen is transported very fast and efficiently through the conduit system into the B cell (22) and T cell follicles (23) of the draining lymph node. As the conduit is a tubular transport system only accessible to small antigens with a size exclusion of approximately 70kDa, antigen-antibody complexes are excluded from this system (22).
It was previously demonstrated that both DCs and macrophages can capture pre-formed antigen-antibody immune complexes in vivo after intravenous injection (16), however, priming of antigen specific CD4+ T lymphocytes is restricted to DCs. Similarly, it has been shown that DCs are crucial for cross-presentation to CD8+ T lymphocytes (24). Both CD8a+ and CD8a- dendritic cells can cross-present pre-formed immune complexes after iv injection (18). To test whether CD11c+ dendritic cells are the major players in presenting the antigen when the antigen has been complexed in vivo we analysed T cell priming in the absence of CD11c+ DCs.
Using CD11c CRE/iDTR mice we demonstrate that cross-presentation of in vivo formed immune complexes to CD8+ T lymphocytes is completely dependent on CD11c+ cells. Although B lymphocytes and macrophages initially ingest a relative large part of the injected antigen (data not shown) and they also outnumber DCs in the spleen and lymphoid organs, our data demonstrate that CD11c+ cells are key players in cross-presentation of in vivo formed antigen- antibody complexes. Thus, DCs are extremely efficient antigen presenting cells compared to B lymphocytes and macrophages. The later cell types possibly act as an antigen sink in our model.
The efficiency of antigen presentation of DCs compared to other APCs might be explained by the antigen storage compartments that we recently described to facilitate prolonged cross- presentation capacity (25). The percentage of antigen-positive DCs over time in our current study was relatively stable compared to the percentage of antigen-positive macrophages that rapidly declined between 4 and 24 hours, suggestive of antigen degradation (data not shown).
This is in line with previous work showing that DCs have a lower degradative capacity compared to macrophages (26).
In conclusion, by making use of a classical haptenated protein antigen we unequivocally show that Ag-specific circulating antibodies markedly enhance the activation of naive CD4+ and CD8+ T lymphocytes to a soluble protein antigen. Thus, antibodies can link the B and T lymphocyte compartments. The relevance of this link is further illustrated by several studies from the vaccine field. In a study in which patients were vaccinated with adenovirus vectors it was shown that high serum titers of adenovirus specific antibodies enhanced the CD4+ and CD8+ T cell response through maturation of dendritic cells (27). Moreover, in a recent study it was shown that combination of a gp75 DNA vaccine with a monoclonal antibody to gp75 enhances the CD8+ T cell response (28).
Our study illustrates the role of antibodies in T cell priming in a host naïve for the antigen.
Together with the literature mentioned above, our work suggests that T and B lymphocytes act synergistically in the response to vaccine antigen. Using antibodies as natural adjuvant to enhance T cell induction might be favorable for the improvement of cancer immunotherapy and treatment of persistent infections.
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