Antigen-specific Interactions Research Articles

Regulatory B10 cell development and function.

B cells are known to instigate and promulgate immune responses by producing antibodies and presenting antigens to T cells. However, a rare but potent B-cell subset in both humans and mice is capable of inhibiting immune responses through the production of the anti-inflammatory cytokine IL-10. Regulatory B cells do not express any unique combination of surface markers but instead represent a small population of B cells that have acquired the unique ability to produce IL-10. This numerically rare B-cell subset is therefore functionally referred to as 'B10 cells' to reflect both their molecular program and the fact that their anti-inflammatory effects in models of autoimmunity, infection and cancer are solely attributable to IL-10 production. As with most B cells, B10 cell development and function appear to be predominantly, if not exclusively, driven by antigen-receptor signals. Once generated, B10 cells respond to both innate and adaptive immune signals, with a requirement for antigen-specific local interactions with T cells to induce IL-10 production and to provide optimal immune suppression in mouse models of autoimmune disease. B10 cells therefore provide an antigen-specific mechanism for delivering IL-10 locally to sites of immune activation and inflammation. The ability of B10 cells to regulate innate and adaptive immune responses makes them an ideal therapeutic target for the treatment of many immune-related disorders.

Beyond vascular inflammation--recent advances in understanding atherosclerosis.

Atherosclerosis is the most life-threatening pathology worldwide. Its major clinical complications, stroke, myocardial infarction, and heart failure, are on the rise in many regions of the world--despite considerable progress in understanding cause, progression, and consequences of atherosclerosis. Originally perceived as a lipid-storage disease of the arterial wall (Die cellularpathologie in ihrer begründung auf physiologische und pathologische gewebelehre. August Hirschwald Verlag Berlin, [1871]), atherosclerosis was recognized as a chronic inflammatory disease in 1986 (New Engl J Med 314:488-500, 1986). The presence of lymphocytes in atherosclerotic lesions suggested autoimmune processes in the vessel wall (Clin Exp Immunol 64:261-268, 1986). Since the advent of suitable mouse models of atherosclerosis (Science 258:468-471, 1992; Cell 71:343-353, 1992; J Clin Invest 92:883-893, 1993) and the development of flow cytometry to define the cellular infiltrate in atherosclerotic lesions (J Exp Med 203:1273-1282, 2006), the origin, lineage, phenotype, and function of distinct inflammatory cells that trigger or inhibit the inflammatory response in the atherosclerotic plaque have been studied. Multiphoton microscopy recently enabled direct visualization of antigen-specific interactions between T cells and antigen-presenting cells in the vessel wall (J Clin Invest 122:3114-3126, 2012). Vascular immunology is now emerging as a new field, providing evidence for protective as well as damaging autoimmune responses (Int Immunol 25:615-622, 2013). Manipulating inflammation and autoimmunity both hold promise for new therapeutic strategies in cardiovascular disease. Ongoing work (J Clin Invest 123:27-36, 2013; Front Immunol 2013; Semin Immunol 31:95-101, 2009) suggests that it may be possible to develop antigen-specific immunomodulatory prevention and therapy-a vaccine against atherosclerosis.

The active metabolite of spleen tyrosine kinase inhibitor fostamatinib abrogates the CD4⁺ T cell-priming capacity of dendritic cells.

Spleen tyrosine kinase (SYK) is a core signalling protein that drives inflammatory responses and is fundamental to the propagation of signals via numerous immune receptors, including the B cell receptor and Fc receptors (FcRs). Fostamatinib, a small molecule SYK inhibitor, has shown evidence of ameliorating inflammation in RA patients. We sought to understand how the active metabolite of fostamatinib, R406, affects the inflammatory response at the cellular level. Antigen-specific in vivo systems and in vitro fluorescence microscopy were combined to investigate the effects of fostamatinib on antigen-specific interactions between dendritic cells (DCs) and CD4(+) T cells. Although it has previously been shown that R406 reduces the response of DCs to immune complexes (ICs), we found that fostamatinib failed to reduce specific CD4(+) T cell proliferation in mice after immunization with ICs. However, we observed in vitro that R406 reduces both the area and duration of cellular interactions between IC-activated DCs and specific CD4(+) T cells during the initial phase of cellular crosstalk. This led to diminished proliferation of antigen-specific CD4(+) T cells after R406 treatment compared with vehicle controls. This decreased proliferative capacity of CD4(+) T cells was accompanied by reduced expression of the co-stimulatory molecules, inducible T cell co-stimulator (ICOS) and PD-1, and abrogation of the production of inflammatory cytokines such as IFN-γ and IL-17. Our findings indicate a potential mechanism by which this compound may be effective in inhibiting FcR-driven CD4(+) T cell responses.

Defining and Refining Tfh Cells in Antibody Responses and The Role of OX40 Costimulation.

Follicular helper T cells (Tfh) specifically provide help to the maturation and development of B cells in secondary lymphoid tissue follicle. They assist the antigen experienced B cells to form antibody producing cells, and they also provide help to the class switch of antibodies. As the master transcriptional factor, Bcl6 expression is instrumental to Tfh development. However, memory Tfh are defined as Bcl6-CXCR5+ CD4+ T cells, suggesting complexity of Bcl6 in Tfh subsets. In the follicle there is another CXCR5+ CD4 T cell subset which also expresses Foxp3. These follicular regulatory T cells (Tfr) cells inhibit the germinal center formation and antibody production mediated by Tfh. Besides antigen specific TCR interaction, the priming of Tfh also requires assistant from co-stimulation signal from antigen presenting cells. OX40, a member of the TNFR-superfamily, is a co-stimulation receptor presenting on the activated CD4 T cells surface. It is known to be related to the development of several CD4 subsets. Here we demonstrated decreased Tfh development and antibody production in OX40-/- mice after immunization. When given antigen and excess OX40 ligand (OX40L) as stimulation signal, there are more Tfh and Tfr cells both developed in OTII mice. In OX40L transgenic mice, there are more memory Tfh cells developed and enhanced secondary response with antigen re-boost, but the excess OX40 signal can not increase Tfh development in P52-/- mouse as in wild type mice. So it indicates the OX40 signaling mediated Tfh development is activated through non-canonical NFkB pathway. In the allograft transplantation model, donor graft provides antigen and triggers strong immune responses. In both the skin and heart transplantation model, we observed increased Tfh development and donor specific antibody production. In the skin transplantation model, the secondary transplantation will recall both Tfh and antibody production to a higher level. The allograft tissue was rejected faster than the first transplantation. All of these indicate the Tfh might be related to the antibody mediated allograft rejection. We uncovered the relationship of OX40 signaling with Tfh development, further studies are ongoing to investigate its function in regulating allograft rejection in transplantation model. These studies will be important in developing better therapies to prevent antibody-mediated graft damage.

Multiplex and functional detection of antigen-specific human T cells by ITRA—Indirect T cell recognition assay

The identification and functional characterization of pathogen-specific T cells plays a critical role in immunological research and diagnostics. In addition to the present standard technologies such as intracellular cytokine staining (ICS), enzyme-linked immunospot (ELISPOT) and peptide-major-histocompatibility-complex (MHC) multimer staining, we aimed to develop a multiplex detection assay, which provides fast in vitro functional data for both human CD4 and CD8 T cells with different antigen specificities in one sample. In this study, we have exploited the expression of CD83 on B cells to develop the cell array-based indirect T cell recognition assay (ITRA). In detail, B cells are pulsed with different pathogen peptide pools and fluorescently barcoded. Thereafter the B cells are pooled and co-cultured with autologous T cells. Subsequently each B cell population is analyzed via flow cytometry for CD83 expression, which indicates antigen-specific interaction with CD4 T cells. Moreover, we revealed donor dependent variations of cytotoxic activity of pathogen-specific CD4 T cells and CD8 T cells, evidenced by specific lysis of peptide-pulsed B cells. Taken together, ITRA is a novel antigen presenting cell (APC) array based method to analyze the presence and function of various antigen-specific T cells in one sample. It has the potential to be used in the future for epitope/antigen screening in research and for analysis of anti-tumor, anti-pathogen or autoimmune T cell responses in patient samples.

Differentiation of germinal center B cells and follicular helper T cells as viewed by tracking Bcl6 expression dynamics

Development of germinal center (GC) B cells and follicular helper T (Tfh) cells requires the transcription factor B-cell lymphoma 6 (Bcl6). Expression of Bcl6 in B cells and helper T cells is regulated by complex signals including those generated through their antigen-specific interactions, which take place in various microenvironments depending on their activation/differentiation states. In the last several years, it has become possible to detect Bcl6 protein in individual B cells and T cells by intracellular staining with the newly developed antibodies or by using the reporter mice. Experiments using these reagents have started to clarify microanatomical location of early Bcl6 upregulation in B cells and T cells, and contributed to reveal the dispensability and indispensability of B cells in the early and late phase of Tfh differentiation. They also started to reveal the diversity, plasticity, and/or instability of Tfh cells. We summarize the recent findings made by tracking Bcl6 expression together with the updated knowledge about dynamics of antigen-engaged B cells and helper T cells and discuss them in relation to possible signaling requirements for the development of GC B cells and Tfh cells.

Endosomal clathrin drives actin accumulation at the immunological synapse

Antigen-specific cognate interaction of T lymphocytes with antigen-presenting cells (APCs) drives major morphological and functional changes in T cells, including actin rearrangements at the immune synapse (IS) formed at the cell-cell contact area. Here we show, using cell lines as well as primary cells, that clathrin, a protein involved in endocytic processes, drives actin accumulation at the IS. Clathrin is recruited towards the IS with parallel kinetics to that of actin. Knockdown of clathrin prevents accumulation of actin and proteins involved in actin polymerization, such as dynamin-2, the Arp2/3 complex and CD2AP at the IS. The clathrin pool involved in actin accumulation at the IS is linked to multivesicular bodies that polarize to the cell-cell contact zone, but not to plasma membrane or Golgi complex. These data underscore the role of clathrin as a platform for the recruitment of proteins that promote actin polymerization at the interface of T cells and APCs.

B cells require "nurturing" by CD4 T cells during development in order to respond in chronic graft-versus-host model of systemic lupus erythematosus.

The murine chronic GVH (cGVH) model of SLE is induced by allo-recognition of foreign major histocompatibility complex (MHC) class II determinants. Previous studies have shown that syngeneic CD4(+) T cells are needed during B cell development in order to induce cGVH response in CD4KO mice. Our present studies show that B cells require "nurturing" by CD4 T cells through much of their ontogeny in order to respond to allo-signaling and become autoreactive. The nurturing process does not require antigen-specific cognate interactions between CD4 T cells and B cells. It is mediated by IL-4, but not IL-10, IL-6 and IFN-gamma. The CD4 T cell nurturing may be supplanted by large doses of IL-4 and/or by agonistic anti-CD40 mAb. Understanding the mechanism of this "nurturing" process may yield clues to the role of CD4 T cells in lupus and in host defense in general.

Active Stochastic Microrheology using Optical Tweezers

Cells are dynamic structures capable of generating and reacting to physical cues in their environment. Measuring mechanical properties is thus essential for elucidating cell or other material structure-function in particular during dynamic rearrangement of the cytoskeleton. Although a variety of rheological techniques have been developed using video microscopy, AFM, and magnetic traps, the measurable frequency range is limited by the time to obtain the measurement, and forcing conditions such as amplitude, direction, contact geometry, and probe location. Here, we developed active stochastic microrheology using optical tweezers to enhance the temporal resolution and precision of detection. A stochastic force is generated by moving the trap relative to the sample. Both bead displacement and trap position are monitored simultaneously by separate position sensitive devices. With this method, both storage and loss shear moduli of the extracellular matrices can be extracted over a wide frequency range of 10−2 – 103 Hz within a few minutes. Also, this method was used to probe the local mechanical environment of B-cell receptor using antigen specific interaction. We showed that the local mechanical properties are strengthened in response to antigen binding and repeated external excitation in a physiological range of 1–100pN. The mechanical responses can also be measured with respect to direction such as force applied normal and perpendicular to the cell membrane. This technique is useful in characterizing the mechanical properties at a user-defined location and magnitude, over a wide frequency spectrum, in a short time, and with a small deformation < 100nm. With these advantages, the method can also be applied to other cell processes, studies of complex fluids, fibril growth, and polymer solutions. Support from the NIGMS (GM-076689), an NSF Career Award (0643745), and the Singapore-MIT Alliance for Research and Technology (SMART-BioSyM) are gratefully acknowledged.

Tunable Chemokine Production by Antigen Presenting Dendritic Cells in Response to Changes in Regulatory T Cell Frequency in Mouse Reactive Lymph Nodes

BackgroundAlthough evidence exists that regulatory T cells (Tregs) can suppress the effector phase of immune responses, it is clear that their major role is in suppressing T cell priming in secondary lymphoid organs. Recent experiments using two photon laser microscopy indicate that dendritic cells (DCs) are central to Treg cell function and that the in vivo mechanisms of T cell regulation are more complex than those described in vitro.Principal FindingsHere we have sought to determine whether and how modulation of Treg numbers modifies the lymph node (LN) microenvironment. We found that pro-inflammatory chemokines—CCL2 (MCP-1) and CCL3 (MIP-la)—are secreted in the LN early (24 h) after T cell activation, that this secretion is dependent on antigen-specific DC–T cell interactions, and that it was inversely related to the frequency of Tregs specific for the same antigen. Furthermore, we demonstrate that Tregs modify the chemoattractant properties of antigen-presenting DCs, which, as the frequency of Tregs increases, fail to produce CCL2 and CCL3 and to attract antigen-specific T cells.ConclusionsThese results substantiate a major role of Tregs in LN patterning during antigen-specific immune responses.

Antigen-specific recognition is critical for the function of regulatory CD8 +CD28 − T cells

The immunomodulatory properties of CD8 T cells with regulatory phenotype have become evident. It remains unclear whether the immunomodulatory function of CD8 +CD28 − T cells requires antigen-specific TCR interaction with major histocompatibility complex class I (MHC I). We have isolated naïve CD8 +CD28 − T suppressor cells (Tsup) from H2-Kk Des-TCR mice that express a transgenic, MHC class I-restricted, clonotypic TCR against an allogeneic MHC class I molecule (H2-Kb) plus self-peptide. These cells were compared to B10.BR wild type (w/t) CD8 +CD28 − T cells and to naïve CD4 +CD25 + regulatory T cells (Treg) of the same strains. Des CD8 effector T cells proliferated more readily when stimulated by H2-Kb splenocytes than w/t controls, whereas Des CD4 T cells showed the same alloresponse as w/t cells. Activation and proliferation of B10.BR CD4 T cells stimulated by H2-Kb APC were suppressed more effectively by Des CD8 + CD28 − T cells than by w/t CD8 +CD28 − T cells. On the contrary, Des CD4 +CD25 + T cells inhibited T cell proliferation less effectively than w/t CD4 +CD25 + T cells. In conclusion, we demonstrate that the function of naive Tsup is strongly enhanced by antigen recognition. Therefore we expect that Tsup are possible candidates for antigen-specific immunosuppressive therapy.

Dopamine released by dendritic cells polarizes Th2 differentiation

A major neurotransmitter dopamine transmits signals via five different seven transmembrane G protein-coupled receptors termed D1-D5. It is now evident that dopamine is released from leukocytes and acts as autocrine or paracrine immune modulator. However, the role of dopamine for dendritic cells (DCs) and T(h) differentiation remains unclear. We herein demonstrate that human monocyte-derived dendritic cells (Mo-DCs) stored dopamine in the secretary vesicles. The storage of dopamine in Mo-DCs was enhanced by forskolin and dopamine D2-like receptor antagonists via increasing cyclic adenosine 3',5'-monophosphate (cAMP) formation. Antigen-specific interaction with naive CD4(+) T cells induced releasing dopamine-including vesicles from Mo-DCs. In naive CD4(+) T cells, dopamine dose dependently increased cAMP levels via D1-like receptors and shifts T-cell differentiation to T(h)2, in response to anti-CD3 plus anti-CD28 mAb. Furthermore, we demonstrated that dopamine D2-like receptor antagonists, such as sulpiride and nemonapride, induced a significant DC-mediated T(h)2 differentiation, using mixed lymphocyte reaction between human Mo-DCs and allogeneic naive CD4(+) T cells. When dopamine release from Mo-DCs is inhibited by colchicines (a microtubule depolymerizer), T-cell differentiation shifts toward T(h)1. These findings identify DCs as a new source of dopamine, which functions as a T(h)2-polarizing factor in DC-naive T-cell interface.

PKC-θ selectively controls the adhesion-stimulating molecule Rap1

AbstractThe antigen-specific interaction of a T cell with an antigen-presenting cell (APC) results in the formation of an immunologic synapse (IS) between the membranes of the 2 cells. β2 integrins on the T cell, namely, leukocyte function-associated antigen 1 (LFA-1) and its counter ligand, namely, immunoglobulin-like cell adhesion molecule 1 (ICAM-1) on the APC, critically stabilize this intercellular interaction. The small GTPase Rap1 controls T-cell adhesion through modulating the affinity and/or spatial organization of LFA-1; however, the upstream regulatory components triggered by the T-cell receptor (TCR) have not been resolved. In the present study, we identified a previously unknown function of a protein kinase C-θ (PKC-θ)/RapGEF2 complex in LFA-1 avidity regulation in T lymphocytes. After T-cell activation, the direct phosphorylation of RapGEF2 at Ser960 by PKC-θ regulates Rap1 activation as well as LFA-1 adhesiveness to ICAM-1. In OT-II TCR-transgenic CD4+ T cells, clustering of LFA-1 after antigen activation was impaired in the absence of PKC-θ. These data define that, among other pathways acting on LFA-1 regulation, PKC-θ and its effector RapGEF2 are critical factors in TCR signaling to Rap1. Taken together, PKC-θ sets the threshold for T-cell activation by positively regulating both the cytokine responses and the adhesive capacities of T lymphocytes.

Deletional tolerance mediated by extrathymic Aire-expressing cells.

The prevention of autoimmunity requires the elimination of self-reactive T cells during their development and maturation. The expression of diverse self-antigens by stromal cells in the thymus is essential to this process and depends, in part, on the activity of the autoimmune regulator (Aire) gene. Here we report the identification of extrathymic Aire-expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally derived eTACs express a diverse array of distinct self-antigens and are capable of interacting with and deleting naïve autoreactive T cells. Using two-photon microscopy, we observed stable antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen-expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection.

CD40L‐expressing CD8 T cells prime CD8α+ DC for IL‐12p70 production

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Intercellular Adhesion Molecule-1-Dependent Stable Interactions between T Cells and Dendritic Cells Determine CD8+ T Cell Memory

The initiation of cytotoxic immune responses requires the direct interaction between naive CD8+ T lymphocytes and dendritic cells (DCs). Multiphoton imaging in intact lymph nodes (LNs) showed that during priming, naive T cells and DCs establish sequentially brief (i.e., minutes) and long (hours) antigen-specific contacts. We show here that the expression of the Intercellular Adhesion Molecule-1 (ICAM-1) by mature DCs is critical for long-lasting contacts with CD8+ T cells but dispensable for short-lived antigen-specific interactions. Serial brief DC-T cell contacts induced early CD8+ T cell activation, proliferation, and differentiation into effector cytotoxic T lymphocytes in the first few days after immunization. ICAM-1-deficient mature DCs, however, failed to induce fully effective priming, because CD8+ T cells produced reduced amounts of interferon gamma and were clonally depleted after 2 weeks. In addition, Icam1(-/-) mice failed to respond to rechallenge. We conclude that ICAM-1-dependent long-lasting interactions between mature DCs and naive CD8+ T cells determine the survival of activated CD8+ T cells and the establishment of effective memory.

T cell-induced secretion of MHC class II–peptide complexes on B cell exosomes

Antigen-specific interactions between B cells and T cells are essential for the generation of an efficient immune response. Since this requires peptide-MHC class II complexes (pMHC-II) on the B cell to interact with TCR on antigen-specific T cells, we have examined the mechanisms regulating the persistence, loss, and secretion of specific pMHC-II complexes on activated B cells. Using a mAb that recognizes specific pMHC-II, we found that activated B cells degrade approximately 50% of pMHC-II every day and release 12% of these pMHC-II from the cell on small membrane vesicles termed exosomes. These exosomes directly stimulate primed, but not naïve, CD4 T cells. Interestingly, engagement of antigen-loaded B cells with specific CD4 T cells stimulates exosome release in a manner that can be mimicked by pMHC-II crosslinking. Biochemical studies revealed that the pMHC-II released on exosomes was previously expressed on the plasma membrane of the B cells, suggesting that regulated exosome release from activated B cells is a mechanism to allow pMHC-II to escape intracellular degradation and decorate secondary lymphoid organs with membrane-associated pMHC-II complexes.

Influenza specific CD8 T cells require interactions with pulmonary DC in the lungs in order to mount an effective immune response (42.8)

Abstract Our studies show that respiratory dendritic cells (rDC) transiently migrate (i.e. within the first 48 hours post infection) from the lungs to the lymph nodes (LN) following influenza virus infections. However, DC are continually recruited into the lungs even after DC migration to the LN has ceased. Since CD8 T cell activation and programming occurs in the LN, the role of these lung-recruited DC in influenza immunity was unclear. To better understand their role, we depleted rDC after the conclusion of normal rDC trafficking to the LN and assessed the immune response. Our results show decreased CD8 T cell responses, resulting in increased viral titers, morbidity and mortality. Initial T cell expansion and activation in the LN, and migration to the lungs appears equivalent in both wildtype and rAPC depleted mice (rAPCneg). However, in rAPCneg mice, these activated T cells fail to continue their expansion once arriving in the lungs. Examination of the DC subtypes present in the lungs revealed decreased numbers of both pulmonary pDC and CD8+ DC in rAPCneg mice through 6 days p.i. Reconstitution of rAPCneg mice with purified pulmonary pDC or CD8+ DC restores influenza-specific CD8 T cell immunity in a MHC I dependent manner. These results suggest that activated influenza-specific CD8 T cells may require additional antigen-specific interactions with recruited DC in the lungs in order to protect against influenza virus infections.