Plasmacytoid dendritic cells take up opsonized antigen leading to CD4+ and CD8+ T cell activation in vivo

Plasmacytoid dendritic cells (pDC) are the body's main source of IFN-alpha, but, unlike classical myeloid DC (myDC), they lack phagocytic activity and are generally perceived as playing only a minor role in Ag processing and presentation. We show that murine pDC, as well as myDC, express Fcgamma receptors (CD16/CD32) and can use these receptors to acquire Ag from immune complexes (IC), resulting in the induction of robust Ag-specific CD4(+) and CD8(+) T cell responses. IC-loaded pDC stimulate CD4(+) T cells to proliferate and secrete a mixture of IL-4 and IFN-gamma, and they induce CD8(+) T cells to secrete IL-10 as well as IFN-gamma. In contrast, IC-loaded myDC induce both CD4(+) and CD8(+) T cells to secrete mainly IFN-gamma. These results indicate that pDC can shape an immune response by acquiring and processing opsonized Ag, leading to a predominantly Th2 response.     

NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells

Modification in the function of dendritic cells (DC), such as that achieved by microbial stimuli or T cell help, plays a critical role in determining the quality and size of adaptive responses to Ag. NKT cells bearing an invariant TCR (iNKT cells) restricted by nonpolymorphic CD1d molecules may constitute a readily available source of help for DC. We therefore examined T cell responses to i.v. injection of soluble Ag in the presence or the absence of iNKT cell stimulation with the CD1d-binding glycolipid alpha-galactosylceramide (alpha-GalCer). Considerably enhanced CD4(+) and CD8(+) T cell responses were observed when alpha-GalCer was administered at the same time as or close to OVA injection. This enhancement was dependent on the involvement of iNKT cells and CD1d molecules and required CD40 signaling. Studies in IFN-gammaR(-/-) mice indicated that IFN-gamma was not required for the adjuvant effect of alpha-GalCer. Consistent with this result, enhanced T cell responses were observed using OCH, an analog of alpha-GalCer with a truncated sphingosine chain and a reduced capacity to induce IFN-gamma. Splenic DC from alpha-GalCer-treated animals expressed high levels of costimulatory molecules, suggesting maturation in response to iNKT cell activation. Furthermore, studies with cultured DC indicated that potentiation of T cell responses required presentation of specific peptide and alpha-GalCer by the same DC, implying conditioning of DC by iNKT cells. The iNKT-enhanced T cell responses resisted challenge with OVA-expressing tumors, whereas responses induced in the absence of iNKT stimulation did not. Thus, iNKT cells exert a significant influence on the efficacy of immune responses to soluble Ag by modulating DC function.     

CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity

The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response. DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers detectable approximately 40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the lymph node with kinetics that parallel the known kinetics of activation of CD8+ T cells to effector function. In the presence of high numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection. The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in "memory" mice in which memory CD8+ cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8+ T cells failed to divide in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback mechanism to limit the activity of DC within the lymph node.     

Mycobacterium tuberculosis-reactive CD8+ T lymphocytes: the relative contribution of classical versus nonclassical HLA restriction

Previous studies in mice and humans models have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). In humans, CD8+ Mtb-reactive T cells have been described that are HLA-A2-, B52-, as well as CD1-restricted. Recently, we have described Mtb-specific CD8+ T cells that are neither HLA-A-, B-, or C- nor group 1 CD1-restricted. At present, little is known about the relative contribution of each of these restriction specificities to the overall CD8+ response to Mtb. An IFN-gamma enzyme-linked immunospot assay was used to determine the frequency of Mtb-reactive CD8+ T cells directly from PBMC. The effector cell frequency among five healthy purified protein derivative-positive subjects was 1/7,600 +/- 4,300 compared with 1/16,000 +/- 7,000 in six purified protein derivative-negative controls. To determine the frequencies of classically, CD1-, and nonclassically restricted cells, a limiting dilution analysis was performed. In one purified protein derivative-positive subject, 192 clones were generated using Mtb-infected dendritic cells (DC). Clones were assessed for reactivity against control autologous DC, Mtb-infected autologous DC, and HLA-mismatched CD1+ targets (DC), as well as HLA-mismatched CD1- targets (macrophages). Of the 96 Mtb-reactive CD8+ T cell clones, four (4%) were classically restricted and 92 (96%) were nonclassically restricted. CD1-restricted cells were not detected. Of the classically restricted cells, two were HLA-B44 restricted and one was HLA-B14 restricted. These results suggest that while classically restricted CD8+ lymphocytes can be detected, they comprise a relatively small component of the overall CD8+ T cell response to Mtb. Further definition of the nonclassical response may aid development of an effective vaccine against tuberculosis.     

A low affinity TCR ligand restores positive selection of CD8+ T cells in vivo

The T cell repertoire is shaped by the processes of positive and negative selection. During development, the TCR binds self peptide-MHC complexes in the thymus, and the kinetics of this interaction are thought to determine the thymocyte's fate. For development of CD8(+) T cells, the data supporting such a model have been obtained using fetal thymic organ culture. To confirm the fidelity of this model in vivo, we studied development of OT-I TCR-transgenic mice that expressed different individual K(b) binding peptides in thymic epithelial cells under the control of the human keratin 14 promoter. We used a system that allowed TAP-independent expression of the peptide-MHC complex, such that the ability of given peptides to restore positive selection in TAP(o) mice could be assessed. We found that transgenic expression of a TCR antagonist peptide (E1) in vivo efficiently restored positive selection of OT-I T cells in TAP(o) mice. An unrelated transgenic peptide (SIY) did not restore selection of OT-I T cells, nor did the E1-transgenic peptide restore selection of an unrelated receptor (2C), showing that positive selection is peptide specific in vivo, as observed in organ cultures. Neither E1 nor SIY transgenes increased the polyclonal CD8 T cell repertoire size in non-TCR-transgenic animals, arguing that single class I binding peptides do not detectably affect the size of the CD8 T cell repertoire when expressed at low levels. We also observed that OT-I T cells selected in TAP(o)-E1 mice were functional in their response to Ag; however, there was a lag in this response, suggesting that the affinity of the TCR interaction with MHC-self peptide can result in fine-tuning of the T cell response.     

The role of antiviral CD8+ T cells in cognitive impairment

The impact of the immune system on the etiopathogenesis of neurodegenerative diseases, including Alzheimer's disease, is a rapidly growing area of investigation. Evidence from human patients and animal models implicates neurotropic viral infections, and specifically the antiviral immune response of brain-infiltrating CD8⁺ T cells, as potential drivers of disease pathology. While infiltration and retention of CD8⁺ T cells within the brain following viral infection is associated with improved survival, CD8⁺ T cells also contribute to neuronal death and gliosis which underlie cognitive impairment in several disease models. Here we review the role of antiviral CD8⁺ T cells as potential mediators of cognitive impairment and highlight the mechanisms by which brain-resident CD8⁺ T cells may contribute to neurodegenerative disease pathology.     

Memory CD8+ T cells protect dendritic cells from CTL killing

CD8(+) T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8(+) T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8(+) T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4(+) and CD8(+) T cell populations. Moreover, memory CD8(+) T cells that release the DC-activating factor TNF-alpha before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4(+) Th cells. The currently identified DC-protective function of memory CD8(+) T cells helps to explain the phenomenon of CD8(+) T cell memory, reduced dependence of recall responses on CD4(+) T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization.     

Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4+ and CD8+ T cells

Due to their potent immunostimulatory capacity, dendritic cells (DC) have become the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4(+) lymphocytes, which reciprocally activate DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8(+) lymphocytes. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCimm results in blockade of DC maturation in response to all activation signals, including CD40L, monocyte-conditioned medium, LPS, TNF-alpha, and poly(I:C), and failure to induce a CD8(+) response. By contrast, DCmat can be infected with rVV and induce a CD8(+) response, but, having lost phagocytic activity, fail to process the Ag via the exogenous class II pathway. To overcome these limitations, we used the CMV protein pp65 as a model Ag and designed a gene containing the lysosomal-associated membrane protein 1 targeting sequence (Sig-pp65-LAMP1) to target pp65 to the class II compartment. DCmat infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4(+) clones and efficiently induced a pp65-specific CD4(+) response, suggesting that after DC maturation the intracellular processing machinery for class II remains intact for at least 16 h. Moreover, infection of DCmat with rVV-Sig-pp65-LAMP1 resulted in at least equivalent presentation to CD8(+) cells as infection with rVV-pp65. These results demonstrate that despite rVV interference with DCimm maturation, a single targeting vector can deliver Ags to DCmat for the effective simultaneous stimulation of both CD4(+) and CD8(+) cells.     

NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires "help". We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4(+) T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4(+) T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4(+) T cell-independent priming of CTL. DC copresenting a K(b)-binding antigenic peptide and the CD1d-binding glycolipid alpha-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.     

Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.     

Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8- dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells

Mouse spleen contains three distinct mature dendritic cell (DC) populations (CD4(+)8(-), CD4(-)8(-), and CD4(-)8(+)) which retain a capacity to take up particulate and soluble AGS: Although the three splenic DC subtypes showed similar uptake of injected soluble OVA, they differed markedly in their capacity to present this Ag and activate proliferation in OVA-specific CD4 or CD8 T cells. For class II MHC-restricted presentation to CD4 T cells, the CD8(-) DC subtypes were more efficient, but for class I MHC-restricted presentation to CD8 T cells, the CD8(+) DC subtype was far more effective. This differential persisted when the DC were activated with LPS. The CD8(+) DC are therefore specialized for in vivo cross-presentation of exogenous soluble Ags into the class I MHC presentation pathway.     

CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Foxp3(+)CD25(+)CD4(+) regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-beta- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8(+) DEC-205/CD205(+) DCs, but not the major fraction of CD8(-) DC inhibitory receptor-2 (DCIR2)(+) DCs, induce functional Foxp3(+) Treg from Foxp3(-) precursors in the presence of low doses of Ag but without added TGF-beta. CD8(+)CD205(+) DCs preferentially express TGF-beta, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-beta. In contrast, CD8(-)DCIR2(+) DCs better induce Foxp3(+) Treg when exogenous TGF-beta is supplied. In vivo, CD8(+)CD205(+) DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG(-/-) Foxp3(-)CD4(+) T cells, whereas the CD8(-)DCIR2(+) DCs better stimulate natural Foxp3(+) Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3(+) Treg, in part through the endogenous formation of TGF-beta. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3(+) Treg for treatment of autoimmune diseases, transplant rejection, and allergy.     

Systemic control of plasmacytoid dendritic cells by CD8+ T cells and commensal microbiota

The composition of the intestinal microbial community is a distinctive individual trait that may divergently influence host biology. Because dendritic cells (DC) regulate the quality of the host response to microbiota, we evaluated DC in mice bearing distinct enteric microbial communities divergent for colitis susceptibility. Surprisingly, a selective, systemic reduction of plasmacytoid dendritic cells (pDC) was observed in isogenic mice with different microbiota: restricted flora (RF) vs specific pathogen free (SPF). This reduction was not observed in germfree mice, suggesting that the pDC deficiency was not simply due to a lack of intestinal microbial products. The microbial action was linked to cytotoxic CD8(+) T cells, since pDC in RF mice were preserved in the CD8(-/-) and perforin(-/-) genotypes, partially restored by anti-CD8beta Ab, and augmented in SPF mice bearing the TAP(-/-) genotype. Direct evidence for pDC cytolysis was obtained by rapid and selective pDC depletion in SPF mice transferred with RF CD8(+) T cells. These data indicate that commensal microbiota, via CTL activation, functionally shape systemic immune regulation that may modify risk of inflammatory disease.     

Negative selection of Tcra-V8+CD8+ T cells by MHC class I molecules

Tcrb-V-specific positive and negative selection of T cells has been well documented. In contrast, nothing is known about Tcra-V-specific selection. Using Tcra-V8-specific KT50 antibody Tcra-V8-specific selection of T cells has been examined. The CD8⁺ T cell subpopulation bearing Tcra-V8 are shown to be negatively selected by major histocompatibility complex (MHC) class I H-2K^d and H-2D^d/L^d molecules. Furthermore, percentages of these T cells are also influenced by Tcra-V haplotypes. Involvement of non-H-2 self (super)antigens in this MHC class I restricted negative selection, however, remains to be determined.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

Anergic CD8+ T cells can persist and function in vivo.

Using a mouse model system, we demonstrate that anergic CD8+ T cells can persist and retain some functional capabilities in vivo, even after the induction of tolerance. In TCR Vbeta5 transgenic mice, mature CD8+Vbeta5+ T cells transit through a CD8lowVbeta5low deletional intermediate during tolerance induction. CD8low cells are characterized by an activated phenotype, are functionally compromised in vitro, and are slated for deletion in vivo. We now demonstrate that CD8low cells derive from a proliferative compartment, but do not divide in vivo. CD8low cells persist in vivo with a t1/2 of 3-5 days, in contrast to their in vitro t1/2 of 0.5-1 day. During this unexpectedly long in vivo life span, CD8low cells are capable of producing IFN-gamma in vivo despite their inability to proliferate or to kill target cells in vitro. CD8low cells also accumulate at sites of inflammation, where they produce IFN-gamma. Therefore, rather than withdrawing from the pool of functional CD8+ T cells, anergic CD8low cells retain a potential regulatory role despite losing their capacity to proliferate. The ability of anergic cells to persist and function in vivo adds another level of complexity to the process of tolerance induction in the lymphoid periphery.     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

CD40-ligated dendritic cells effectively expand melanoma-specific CD8+ CTLs and CD4+ IFN-gamma-producing T cells from tumor-infiltrating lymphocytes

Professional APC, notably dendritic cells (DC), are necessary for stimulation and expansion of naive T cells. By means of murine models, the interaction between CD40 on DC and its ligand CD154 has been recognized as an important element for conditioning of DC to prime and expand CTL. We translated these findings into the human system, scrutinizing the ability of DC to initiate clonal expansion of single T cells. DC generated under completely autologous conditions from peripheral blood monocytes were cocultured at a rate of 0.3 cell/well with melanoma-infiltrating T cells; this procedure guaranteed that either a CD4+ or a CD8+ cell interacted with the DC, thus avoiding the contact of more than one T cell to the DC. In the absence of further stimulation, this cloning protocol yielded almost exclusively CD4+ T cell clones that predominantly exhibited a Th2 phenotype. However, cross-linking of CD40 on DC resulted in the induction of IFN-gamma-producing Th1 CD4+ T cell clones. In addition, CD40-activated DC were capable of expanding CD8+ CTL clones. The ratio of CD4 to CD8 T cell clones corresponded to the ratio present in the initial tumor-infiltrating lymphocyte preparation. The CTL clones efficiently lysed autologous tumor cells whereas autologous fibroblasts or MHC-mismatched melanoma cells were not killed. Our findings support the critical role of CD40/CD154 interactions for the induction of cellular immune responses.     

The fate of effector T cells in vivo is determined during activation and differs for CD4+ and CD8+ cells

Effector T cells generated in the mesenteric lymph nodes (mLN) are known to accumulate in mLN and the tissue drained by them after circulating in the blood. Their accumulation is due less to preferential entry into mLN but more to preferential proliferation within mLN. The factors regulating the proliferation of effector T cells in vivo are unclear, and it is unknown whether they are different for CD4(+) and CD8(+) effector T cells. Rat T cells from mLN or peripheral lymph nodes (pLN) were stimulated polyclonally via the TCR and CD28 and injected i.v. into congenic recipients. Using three-color flow cytometry and immunohistochemistry, they were identified in mLN, pLN, and blood over time, and proliferation was determined by measuring bromodeoxyuridine incorporation. Only effector mLN T cells showed a significantly increased proliferation rate after entry into mLN compared with that in pLN (2.4 +/- 1.8% vs 0.8 +/- 0.4%). Proliferation among the injected cells was higher when they had contact with dendritic cells within mLN (9.0 +/- 4.3%) than when they did not (4.1 +/- 2.1%). Furthermore, effector mLN T cells which were observed 56 days after injection maintained the capacity for preferential proliferation within mLN. Interestingly, CD4(+) effector mLN T cells proliferated at a higher rate (4.8 +/- 0.7%), remaining in mLN, whereas CD8(+) effector mLN T cells proliferated at a lower rate (3.3 +/- 1.0%) and were able to leave the mLN into the blood. Elucidating the factors regulating the proliferation of effector T cells in vivo will help to modify their distribution for therapeutic purposes.     

Antibody-independent antiviral function of memory CD4+ T cells in vivo requires regulatory signals from CD8+ effector T cells

Previous studies have shown that vaccine-primed CD4(+) T cells can mediate accelerated clearance of respiratory virus infection. However, the relative contributions of Ab and CD8(+) T cells, and the mechanism of viral clearance, are poorly understood. Here we show that control of a Sendai virus infection by primed CD4(+) T cells is mediated through the production of IFN-gamma and does not depend on Ab. This effect is critically dependent on CD8(+) cells for the expansion of CD4(+) T cells in the lymph nodes and the recruitment of memory CD4(+) T cells to the lungs. Passive transfer of a CD8(+) T cell supernatant into CD8(+) T cell-depleted, hemagglutinin-neuraminidase (HN)(421-436)-immune muMT mice substantially restored the virus-specific memory CD4(+) response and enhanced viral control in the lung. Together, the data demonstrate for the first time that in vivo primed CD4(+) T cells have the capacity to control a respiratory virus infection in the lung by an Ab-independent mechanism, provided that CD8(+) T cell "help" in the form of soluble factor(s) is available during the virus infection. These studies highlight the importance of synergistic interactions between CD4(+) and CD8(+) T cell subsets in the generation of optimal antiviral immunity.