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.     

Noncovalent association with stress protein facilitates cross-priming of CD8+ T cells to tumor cell antigens by dendritic cells.

A viral oncogene carrying well-defined K(b)/D(b)-restricted epitopes was expressed in a heat shock protein (hsp)-associated or nonassociated form in the murine tumor cells P815 and Meth-A. Wild-type SV40 large T-Ag (wtT-Ag) is expressed without stable hsp association; mutant (cytoplasmic cT-Ag) or chimeric (cT272-green fluorescent fusion protein) T-Ag is expressed in stable association with the constitutively expressed, cytosolic hsp73 (hsc70) protein. In vitro, remnants from apoptotic wtT-Ag- or cT-Ag-expressing tumor cells are taken up and processed by immature dendritic cells (DC), and the K(b)/D(b)-binding epitopes T1, T2/3, and T4 of the T-Ag are cross-presented to CTL in a TAP-independent way. DC pulsed with remnants of transfected, apoptotic tumor cells cross-presented the three T-Ag epitopes more efficiently when they processed ATP-sensitive hsp73/cT-Ag complexes than when they processed hsp-nonassociated (native) T-Ag. In vivo, more IFN-gamma-producing CD8+ T cells were elicited by a DNA vaccine that encoded hsp73-binding mutant T-Ag than by a DNA vaccine that encoded native, non-hsp-binding T-Ag. Three- to 5-fold higher numbers of T-Ag (T1-, T2/3-, or T4-) specific, D(b)/K(b)-restricted IFN-gamma-producing CD8+ T cells were primed during the growth of transfected H-2(d) Meth-A/cT tumors than during the growth of transfected Meth-A/T tumors in F(1)(b x d) hosts. Hence, the association of an oncogene with constitutively expressed, cytosolic hsp73 facilitates cross-priming in vitro and in vivo of CTL by DC that process material from apoptotic cells.     

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.     

Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo

Flagellin is secreted by many enteric bacteria and, upon reaching the basolateral membrane of the intestinal epithelium, activates Toll-like receptor 5-mediated innate immune signaling pathways. We hypothesized that any flagellin that gets beyond the epithelium might also regulate cells of the adaptive immune system. Here we demonstrate that the clonal expansion of naive DO11.10 CD4 T cells in response to OVA peptide (323-339) was enhanced 3- to 10-fold in the presence of purified bacterial flagellin in vivo. OVA-specific CD4 T cells were also shown to have undergone more cell division in vivo if flagellin was coinjected with OVA. Flagellin administration increased the expression of B7-1 on splenic dendritic cells, and coinjection of CTLA4-Ig, which is known to block B7 function in vivo, completely ablated the adjuvant effect on CD4 T cells. Therefore, a conserved bacterial protein produced by many intestinal microbes can modulate CD4 T cell activation in vivo. Such an adjuvant effect for flagellin has important implications for vaccine development and the generation of CD4 T cell responses to enteric bacteria.     

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.     

Uptake of apoptotic antigen-coupled cells by lymphoid dendritic cells and cross-priming of CD8(+) T cells produce active immune unresponsiveness

The induction of immunologic unresponsiveness by i.v. administration of Ag-coupled lymphoid cells has been studied extensively, but the mechanisms remain unclear. We have further explored this model by examining the role of Fas/Fas ligand (FasL)-mediated apoptosis. Using i.v. injection of trinitrophenyl-coupled splenocytes (TNP-spl) as tolerogen, we found that Fas signaling for apoptosis in the spleen cells delivered by FasL in the recipient is the critical event. The requirement for Fas and FasL was overcome by prior induction of apoptosis in TNP-spl, making the tolerogen 100 times more potent. Prevention of apoptosis by a caspase inhibitor blocks tolerance. Interestingly, while blocking CD40/CD40 ligand interaction does not prevent tolerance induction, an agonist anti-CD40 Ab turns tolerogenic TNP-spl into an immunizing Ag. Studies further showed that tolerance is induced through cross-presentation of Ag in a class I MHC-dependent manner by CD8(+)CD11c(+) lymphoid-derived dendritic cells to regulatory T cells. The results provide a mechanism for a well-established method of inducing immunologic unresponsiveness.     

Antigen persistence is required for dendritic cell licensing and CD8+ T cell cross-priming

It has been demonstrated that CD4(+) T cells require Ag persistence to achieve effective priming, whereas CD8(+) T cells are on "autopilot" after only a brief exposure. This finding presents a disturbing conundrum as it does not account for situations in which CD8(+) T cells require CD4(+) T cell help. We used a physiologic in vivo model to study the requirement of Ag persistence for the cross-priming of minor histocompatibility Ag-specific CD8(+) T cells. We report inefficient cross-priming in situations in which male cells are rapidly cleared. Strikingly, the failure to achieve robust CD8(+) T cell activation is not due to a problem with cross-presentation. In fact, by providing "extra help" in the form of dendritic cells (DCs) loaded with MHC class II peptide, it was possible to achieve robust activation of CD8(+) T cells. Our data suggest that the "licensing" of cross-presenting DCs does not occur during their initial encounter with CD4(+) T cells, thus accounting for the requirement for Ag persistence and suggesting that DCs make multiple interactions with CD8(+) T cells during the priming phase. These findings imply that long-lived Ag is critical for efficient vaccination protocols in which the CD8(+) T cell response is helper-dependent.     

Efficient delivery of Antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8+ T cells.

The in vivo induction of a CTL response using Antennapedia homeodomain (AntpHD) fused to a poorly immunogenic CTL epitope requires that the Ag is given in presence of SDS, an unacceptable adjuvant for human use. In the present report, we developed a hybrid CTL epitope delivery system consisting of AntpHD peptide vector formulated in liposomes as an alternative approach to bypass the need for SDS. It is proposed that liposomes will prevent degradation of the Ag in vivo and will deliver AntpHD recombinant peptide to the cytosol of APCs. We show in this work that dendritic cells incubated with AntpHD-fused peptide in liposomes can present MHC class I-restricted peptide and induce CTL response with a minimal amount of Ag. Intracellular processing studies have shown that encapsulated AntpHD recombinant peptide is endocytized before entering the cytosol, where it is processed by the proteasome complex. The processed liposomal peptides are then transported to the endoplasmic reticulum. The increase of the CTL response induced by AntpHD-fused peptide in liposomes correlates with this active transport to the class I-processing pathway. In vivo studies demonstrated that positively charged liposomes increase the immunogenicity of AntpHD-Cw3 when injected s.c. in mice in comparison to SDS. Moreover, addition of CpG oligodeoxynucleotide immunostimulatory sequences further increase the CD8+ T cell response. This strategy combining lipid-based carriers with AntpHD peptide to target poorly immunogenic Ags into the MHC class I processing pathway represents a novel approach for CTL vaccines that may have important applications for development of cancer vaccines.     

Radiotherapy promotes tumor-specific effector CD8+ T cells via dendritic cell activation

Radiotherapy is an important treatment for cancer. The main mode of action is thought to be the irreversible damage to tumor cell DNA, but there is evidence that irradiation mobilizes tumor-specific immunity, and recent studies showed that the efficacy of high-dose radiotherapy depends on the presence of CD8(+) T cells. We show in this study that the efficacy of radiotherapy given as a single, high dose (10 Gy) crucially depends on dendritic cells and CD8(+) T cells, whereas CD4(+) T cells or macrophages are dispensable. We show that local high-dose irradiation results in activation of tumor-associated dendritic cells that in turn support tumor-specific effector CD8(+) T cells, thus identifying the mechanism that underlies radiotherapy-induced mobilization of tumor-specific immunity. We propose that in the absence of irradiation, the activation status of dendritic cells rather than the amount of tumor-derived Ag is the bottleneck, which precludes efficient anti-tumor immunity.     

ICAM-1 contributes to but is not essential for tumor antigen cross-priming and CD8+ T cell-mediated tumor rejection in vivo

ICAM-1 has been described to provide both adhesion and costimulatory functions during T cell activation. In the setting of antitumor immunity, ICAM-1/LFA-1 interactions could be important at the level of T cell priming by APCs in draining lymph nodes as well as for transendothelial migration and tumor cell recognition at the tumor site. To determine the contribution of ICAM-1 to tumor rejection in vivo, we performed adoptive transfer of 2C TCR-transgenic/RAG2(-/-) T cells into TCRalpha(-/-) vs ICAM(-/-)/TCRalpha(-/-) recipient animals. ICAM-1-deficient mice successfully rejected HTR.C tumors expressing Ld recognized by the 2C TCR, albeit with a kinetic delay. Inasmuch as HTR.C tumor cells themselves express ICAM-1, a second model was pursued using B16-F10 melanoma cells that lack ICAM-1 expression. These cells were transduced to express the SIYRYYGL peptide recognized by the 2C TCR in the context of Kb, which is cross-presented by APCs in H-2b mice in vivo. These tumors also grew more slowly but were eventually rejected by the majority of ICAM-1(-/-)/TCRalpha(-/-) recipients. Delayed rejection in ICAM-1(-/-) mice was associated with diminished T cell priming as assessed by ELISPOT. In contrast, T cell penetration into the tumor was comparable in wild-type and ICAM-1(-/-) hosts, and adoptively transferred primed effector 2C cells rejected normally in ICAM-1(-/-) recipients. Our results suggest that ICAM-1 contributes to but is not absolutely required for CD8+ T cell-mediated tumor rejection in vivo and dominantly acts at the level of priming rather than the effector phase of the antitumor immune response.     

Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo

Dendritic cells (DCs) require a maturation signal to acquire efficient CTL-priming capacity. In vitro FcgammaR-mediated internalization of Ag-Ab immune complexes (ICs) can induce maturation of DCs. In this study, we show that IC-induced DC maturation in vitro enables DCs to prime peptide-specific CD8+ CTLs in vivo, independently of CD4+ Th cells. Importantly, OVA/anti-OVA IC-treated DCs not only primed CD8+ CTLs to an exogenously loaded peptide nonrelated to OVA, but also efficiently primed CTLs against the dominant CTL epitope derived from the OVA Ag present in the ICs. Our studies show that ICs fulfill a dual role in priming of CD8+ CTL responses to exogenous Ags: enhancement of Ag uptake by DCs and activation of DCs, resulting in "license to kill." These findings indicate that the presence of specific Abs can crucially affect the induction of cytotoxic cellular responses.     

Neutrophil involvement in cross-priming CD8+ T cell responses to bacterial antigens

Substantial CD8(+) T cell responses are generated after infection of mice with recombinant Listeria monocytogenes strains expressing a model epitope (lymphocytic choriomeningitis virus NP(118-126)) in secreted and nonsecreted forms. L. monocytogenes gains access to the cytosol of infected cells, where secreted Ags can be accessed by the endogenous MHC class I presentation pathway. However, the route of presentation of the nonsecreted Ag in vivo remains undefined. In this study we show that neutrophil-enriched peritoneal exudate cells from L. monocytogenes-infected mice can serve as substrates for in vitro cross-presentation of both nonsecreted and secreted Ag by dendritic cells as well as for in vivo cross-priming of CD8(+) T cells. In addition, specific neutrophil depletion in vivo by low dose treatment with either of two Ly6G-specific mAb substantially decreased the relative CD8(+) T cell response against the nonsecreted, but not the secreted, Ag compared with control Ab-treated mice. Thus, neutrophils not only provide rapid innate defense against infection, but also contribute to shaping the specificity and breadth of the CD8(+) T cell response. In addition, cross-presentation of bacterial Ags from neutrophils may explain how CD8(+) T cell responses are generated against Ags from extracellular bacterial pathogens.     

The role of dendritic cells in selection of classical and nonclassical CD8+ T cells in vivo

T cell development is determined by positive and negative selection events. An intriguing question is how signals through the TCR can induce thymocyte survival and maturation in some and programmed cell death in other thymocytes. This paradox can be explained by the hypothesis that different thymic cell types expressing self-MHC/peptide ligands mediate either positive or negative selection events. Using transgenic mice that express MHC class I (MHC-I) selectively on DC, we demonstrate a compartmentalization of thymic functions and reveal that DC induce CTL tolerance to MHC-I-positive hemopoietic targets in vivo. However, in normal and bone marrow chimeric mice, MHC-I+ DC are sufficient to positively select neither MHC-Ib (H2-M3)- nor MHC-Ia (H2-K)-restricted CD8+ T cells. Thus, thymic DC are specialized in tolerance induction, but cannot positively select the vast majority of MHC-I-restricted CD8+ T cells.     

CD103- and CD103+ bronchial lymph node dendritic cells are specialized in presenting and cross-presenting innocuous antigen to CD4+ and CD8+ T cells

Dendritic cells (DC) are able to capture, process, and present exogenous Ag to CD8(+) T lymphocytes through MHC class I, a process referred to as cross-presentation. In this study, we demonstrate that CD103(+) (CD11c(high)CD11b(low)) and CD103(-) (CD11c(int)CD11b(high)) DC residing in the lung-draining bronchial lymph node (brLN) have evolved to acquire opposing functions in presenting innocuous inhaled Ag. Thus, under tolerogenic conditions, CD103(-) DC are specialized in presenting innocuous Ag to CD4(+) T cells, whereas CD103(+) DC, which do not express CD8alpha, are specialized in presenting Ag exclusively to CD8(+) T cells. In CCR7-deficient but not in plt/plt mice, Ag-carrying CD103(+) DC are largely absent in the brLN, although CD103(+) DC are present in the lung of CCR7-deficient mice. As a consequence, adoptively transferred CD8(+) T cells can be activated under tolerizing conditions in plt/plt but not in CCR7-deficient mice. These data reveal that CD103(+) brLN DC are specialized in cross-presenting innocuous inhaled Ag in vivo. Because these cells are largely absent in CCR7(-/-) mice, our findings strongly suggest that brLN CD103(+) DC are lung-derived and that expression of CCR7 is required for their migration from the lung into its draining lymph node.     

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.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Autoimmune CD4+ T cells interfere with immune tolerance to a thymic-independent antigen

The ability of autoimmune T cell subsets to interfere with tolerization of B cells can be studied by using thymic-independent Ag. We have defined an abnormality within the CD4+ T cell compartment in young NZB and MRL-lpr/lpr mice by studying tolerance of spleen and B cells to the thymic independent Ag, fluorescein-Brucella abortus. Tolerization of spleen cells is defective in MRL-lpr/lpr mice, but not MRL-+/+ or C3H.lpr mice, suggesting that the defect requires both the autosomal MRL background and the lpr gene to be present. T enriched cells from NZB mice and from MRL-lpr/lpr mice (but not MRL-+/+ or C3H.lpr mice) reverse tolerance in spleen cells from [NZB X DBA/2]F1 and C3H/HeJ mice, respectively. This interference is removed by treatment with anti-CD4 antibody and C. Supernatants from cultured T cells of NZB and MRL-lpr/lpr mice also prevent tolerance in spleen cells of [NZB X DBA/2]F1 and MRL-+/+ mice, respectively, unless CD4+ cells are removed prior to T cell culture. Removal of T cells from NZB and MRL-lpr/lpr spleen cells allows normal tolerization of B cells, which is abrogated by the addition of syngeneic T cells or cultured T cell supernatants. This effect also depends on the presence of CD4+ T cells. These studies show that in MRL-lpr/lpr mice, through interaction of the lpr and MRL background genes in a T cell subset, and in NZB mice, CD4+ T cells interfere with B cell tolerance to a thymic-independent Ag.     

Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation,cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag "sequestration," or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by approximately 80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.     

Peripheral deletion of mature CD8+ antigen-specific T cells after in vivo exposure to male antigen.

It has been well established that T cell tolerance to self Ag occurs primarily via clonal deletion of immature thymocytes in the thymus. Evidence also exists that there are additional mechanisms operative on mature T cells for establishing and maintaining tolerance in the periphery. To follow the fate of mature Ag-specific T cells in vivo, we used female transgenic mice, which contain a large population of male H-Y Ag-specific T cells that can be identified by immunostaining with mAb directed against CD8 and the transgenic TCR. H-Y Ag was introduced into these mice by injecting Ag-bearing male lymphocytes using conditions known to induce CTL precursor response reduction. The number of Ag-reactive CD8+ transgenic T cells in the periphery started to decrease after 2 days of in vivo exposure to male Ag. Decline was maximum (up to 80% of total) by 7 days, and stayed at this level for at least 6 wk. CD4+ cells and those CD8+ cells that did not carry the transgenic TCR were not affected. Most or all of the remaining Ag-reactive CD8+ cells in the periphery were fully responsive when stimulated by male Ag in vitro. Maturation of transgenic T cells in the thymus of injected mice remained the same as that of control animals. Our data provide direct evidence that mature Ag-reactive CD8+ cells are susceptible to clonal deletion in the periphery when exposed to the Ag in vivo. These findings suggest the presence of two types of APC in the periphery: stimulatory APC (e.g., macrophages and dendritic cells) required for initiating an active immune response; and functionally deleting APC (or veto cells) capable of deleting mature T lymphocytes that recognize Ag presented on their surface. Functionally deleting APC that present self Ag to peripheral T cells may provide a fail-safe mechanism against autoreactive cells that escaped deletion during differentiation in the thymus.