Induction of CD70 on dendritic cells through CD40 or TLR stimulation contributes to the development of CD8+ T cell responses in the absence of CD4+ T cells

The expansion of CD8(+) T cells in response to Ag can be characterized as either dependent or independent of CD4(+) T cells. The factors that influence this dichotomy are poorly understood but may be dependent upon the degree of inflammation associated with the Ag. Using dendritic cells derived from MHC class II-deficient mice to avoid interaction with CD4(+) T cells in vivo, we have compared the immunogenicity of peptide-pulsed dendritic cells stimulated with molecules associated with infection to those stimulated via CD40. In the absence of CD4(+) T cell help, the expansion of primary CD8(+) T cells after immunization with TNF-alpha- or poly(I:C)-stimulated dendritic cells was minimal. In comparison, LPS- or CpG-stimulated dendritic cells elicited substantial primary CD8(+) T cell responses, though not to the same magnitude generated by immunization with CD40L-stimulated dendritic cells. Remarkably, mice immunized with any stimulated dendritic cell population generated fully functional recall CD8(+) T cells without the aid of CD4(+) T cell help. The observed hierarchy of immunogenicity was closely correlated with the expression of CD70 (CD27L) on the stimulated dendritic cells, and Ab-mediated blockade of CD70 substantially prevented the CD4(+) T cell-independent expansion of primary CD8(+) T cells. These results indicate that the expression of CD70 on dendritic cells is an important determinant for helper-dependence of primary CD8(+) T cell expansion and provide an explanation for the ability of a variety of pathogens to stimulate primary CD8(+) T cell responses in the absence of CD4(+) T cells.     

Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8α(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8α(+) DCs are sufficient to subsequent CD8(+) T cell responses.     

Langerhans cells derived from genetically modified human CD34+ hemopoietic progenitors are more potent than peptide-pulsed Langerhans cells for inducing antigen-specific CD8+ cytolytic T lymphocyte responses

Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34(+) hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34(+) hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20-30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100-150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-gamma secretion (100-123 spot-forming cells/10(5) CD8(+) T cells) than did fluMP-pulsed LCs (10-91 spot-forming cells/10(5) CD8(+) T cells). CD8(+) T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8(+) T cell responses than are peptide-pulsed LCs.     

Dendritic cells in distinct oral mucosal tissues engage different mechanisms to prime CD8+ T cells

Although oral dendritic cells (DCs) were shown to induce cell-mediated immunity, the identity and function of the various oral DC subsets involved in this process is unclear. In this study, we examined the mechanisms used by DCs of the buccal mucosa and of the lining mucosa to elicit immunity. After plasmid DNA immunization, buccally immunized mice generated robust local and systemic CD8(+) T cell responses, whereas lower responses were seen by lining immunization. A delayed Ag presentation was monitored in vivo in both groups; yet, a more efficient presentation was mediated by buccal-derived DCs. Restricting transgene expression to CD11c(+) cells resulted in diminished CD8(+) T cell responses in both oral tissues, suggesting that immune induction is mediated mainly by cross-presentation. We then identified, in addition to the previously characterized Langerhans cells (LCs) and interstitial dendritic cells (iDCs), a third DC subset expressing the CD103(+) molecule, which represents an uncharacterized subset of oral iDCs expressing the langerin receptor (Ln(+)iDCs). Using Langerin-DTR mice, we demonstrated that whereas LCs and Ln(+)iDCs were dispensable for T cell induction in lining-immunized mice, LCs were essential for optimal CD8(+) T cell priming in the buccal mucosa. Buccal LCs, however, failed to directly present Ag to CD8(+) T cells, an activity that was mediated by buccal iDCs and Ln(+)iDCs. Taken together, our findings suggest that the mechanisms engaged by oral DCs to prime T cells vary between oral mucosal tissues, thus emphasizing the complexity of the oral immune network. Furthermore, we found a novel regulatory role for buccal LCs in potentiating CD8(+) T cell responses.     

Loss of TLR2, TLR4, and TLR5 on Langerhans cells abolishes bacterial recognition

It is unknown whether closely related epidermal dendritic cells, Langerhans cells (LCs), and dermal dendritic cells (DDCs) have unique functions. In this study, we show that human DDCs have a broad TLR expression profile, whereas human LCs have a selective impaired expression of cell surface TLR2, TLR4, and TLR5, all involved in bacterial recognition. This distinct TLR expression profile is acquired during the TGF-beta1-driven development of LCs in vitro. Consequently, and in contrast to DDCs, LCs weakly respond to bacterial TLR2, TLR4, and TLR5 ligands in terms of cytokine production and maturation, as well as to whole Gram-positive and Gram-negative bacteria, whereas their responsiveness to viral TLR ligands and viruses is fully active and comparable to DDCs. Unresponsiveness of LCs to bacteria may be a mechanism that contributes to tolerance to bacterial commensals that colonize the skin.     

Differential Capacity of Human Skin Dendritic Cells to Polarize CD4+T Cells into IL-17, IL-21 and IL-22 Producing Cells

Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c⁺CD14⁻ and CD14⁺ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4⁺ or CD8⁺T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c⁺CD14⁻DDCs were able to differentiate naïve CD4⁺T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4⁺T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4⁺ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.     

CD27 stimulation promotes the frequency of IL-7 receptor-expressing memory precursors and prevents IL-12-mediated loss of CD8(+) T cell memory in the absence of CD4(+) T cell help

Fully functional CD8(+) T cell memory is highly dependent upon CD4(+) T cell support. CD4(+) T cells play a critical role in inducing the expression of CD70, the ligand for CD27, on dendritic cells. In this study, we demonstrate that CD27 stimulation during primary CD8(+) T cell responses regulates the ability to mount secondary CD8(+) T cell responses. CD27 stimulation during vaccinia and dendritic cell immunization controls the expression of the IL-7R (CD127), which has been shown to be necessary for memory CD8(+) T cell survival. Furthermore, CD27 stimulation during primary CD8(+) T cell responses to vaccinia virus restrained the late expression on memory precursor cells of cytokine receptors that support terminal differentiation. The formation of CD8(+) T cell memory precursors and secondary CD8(+) T cell responses was restored in the absence of CD27 costimulation when endogenous IL-12 was not available. Similarly, the lesion in CD8(+) T cell memory that occurs in the absence of CD4(+) T cells did not occur in mice lacking IL-12. These data indicate that CD4(+) T cell help and, by extension, CD27 stimulation support CD8(+) T cell memory by modulating the expression of cytokine receptors that influence the differentiation and survival of memory CD8(+) T cells.     

Langerhans-type dendritic cells genetically modified to express full-length antigen optimally stimulate CTLs in a CD4-dependent manner

Oncoretroviral vectors encoding either full-length Ag or a corresponding immunodominant peptide were expressed in Langerhans-type dendritic cells (LCs) differentiated from CD34(+) progenitors. We used human CMV as a model Ag restricted by HLA-A*0201 to define parameters for eventual expression of cancer Ags by LCs for active immunization against tumors. Stimulation by CMVpp65(495-503)-pulsed LCs, CMVpp65(495-503)-transduced LCs, and full-length CMVpp65-transduced LCs respectively increased tetramer-reactive T cells with an effector memory phenotype by 10 +/- 11, 34 +/- 21, and 51 +/- 24-fold (p < 0.05) from CMV-seropositive donors. CMV-specific CD8(+) CTLs achieved respective frequencies of 231 +/- 102, 583 +/- 219, and 714 +/- 281 spot-forming cells per 10(5) input cells (p < 0.01) in ELISPOT assays for IFN-gamma secretion. LCs expressing full-length Ag stimulated greater lytic activity than either peptide-transduced or peptide-pulsed LCs (p < 0.05), all in the absence of exogenous cytokines. pp65-transduced LCs presenting class I and II MHC-restricted epitopes expanded IFN-gamma-secreting CD4(+) T cells, whereas pp65(495-503)-transduced LCs did not. CD4(+) T cell numbers even declined after stimulation by pp65(495-503) peptide-pulsed LCs. CD4(+) T cell depletion confirmed their contribution to the more robust CTL responses. LCs, transduced with a retroviral vector encoding full-length Ag, stimulate potent CTLs directed against multiple epitopes in a CD4(+) Th cell-dependent manner.     

Th cells act via two synergistic pathways to promote antiviral CD8+ T cell responses

The mechanisms of how Th cells promote CD8(+) T cell responses during viral infections are largely unknown. In this study, we unraveled the mechanisms of T cell help for CD8(+) T cell responses during vaccinia virus infection. Our results demonstrate that Th cells promote vaccinia virus-specific CD8(+) T cell responses via two interconnected synergistic pathways: First, CD40L expressed by activated CD4(+) T cells instructs dendritic cells to produce bioactive IL-12p70, which is directly sensed by Ag-specific CD8(+) T cells, resulting in increased IL-2Rα expression. Second, Th cells provide CD8(+) T cells with IL-2, thereby enhancing their survival. Thus, Th cells are at the center of an important communication loop with a central role for IL-2/IL-2R and bioactive IL-12.     

Mature human Langerhans cells derived from CD34+ hematopoietic progenitors stimulate greater cytolytic T lymphocyte activity in the absence of bioactive IL-12p70, by either single peptide presentation or cross-priming, than do dermal-interstitial or monocyte-derived dendritic cells

The emerging heterogeneity of dendritic cells (DCs) mirrors their increasingly recognized division of labor at myriad control points in innate and acquired cellular immunity. We separately generated blood monocyte-derived DCs (moDCs), as well as Langerhans cells (LCs) and dermal-interstitial DCs (DDC-IDCs) from CD34(+) hematopoietic progenitor cells. Differential expression of CD11b, CD52, CD91, and the CD1 isoforms proved useful in distinguishing these three DC types. All mature DCs uniformly expressed comparable levels of HLA-DR, CD83, CD80, and CD86, and were potent stimulators of allogeneic T cells after exposure either to recombinant human CD40L trimer or a combination of inflammatory cytokines with PGE(2). moDCs, however, required 0.5-1 log greater numbers than LCs or DDC-IDCs to stimulate comparable T cell proliferation. Only moDCs secreted the bioactive heterodimer IL-12p70, and moDCs phagocytosed significantly more dying tumor cells than did either LCs or DDC-IDCs. LCs nevertheless proved superior to moDCs and DDC-IDCs in stimulating CTL against a recall viral Ag by presenting passively loaded peptide or against tumor Ag by cross-priming autologous CD8(+) T cells. LCs also secreted significantly more IL-15 than did either moDCs or DDC-IDCs, which is especially important to the generation of CTL. These findings merit further comparisons in clinical trials designed to determine the physiologic relevance of these distinctions in activity between LCs and other DCs.     

Invariant NKT cells promote CD8+ cytotoxic T cell responses by inducing CD70 expression on dendritic cells

Activation of invariant NK T (iNKT) cells with the glycolipid alpha-galactosylceramide promotes CD8(+) cytotoxic T cell responses, a property that has been used to enhance the efficacy of antitumor vaccines. Using chimeric mice, we now show that the adjuvant properties of iNKT cells require that CD40 triggering and Ag presentation to CD8(+) T cells occur on the same APCs. We demonstrate that injection of alpha-galactosylceramide triggers CD70 expression on splenic T cell zone dendritic cells and that this is dependent on CD40 signaling. Importantly, we show that blocking the interaction between CD70 and CD27, its costimulatory receptor on T cells, abrogates the ability of iNKT cells to promote a CD8(+) T cell response and abolishes the efficacy of alpha-GalCer as an adjuvant for antitumor vaccines. These results define a key role for CD70 in linking the innate response of iNKT cells to the activation of CD8(+) T cells.     

Polyomavirus-infected dendritic cells induce antiviral CD8(+) T lymphocytes

CD8(+) T cells are critical for the clearance of acute polyomavirus infection and the prevention of polyomavirus-induced tumors, but the antigen-presenting cell(s) involved in generating polyomavirus-specific CD8(+) T cells have not been defined. We investigated whether dendritic cells and macrophages are permissive for polyomavirus infection and examined their potential for inducing antiviral CD8(+) T cells. Although dendritic cells and macrophages both supported productive polyomavirus infection, dendritic cells were markedly more efficient at presenting the immunodominant viral epitope to CD8(+) T cells. Additionally, infected dendritic cells, but not infected macrophages, primed anti-polyomavirus CD8(+) T cells in vivo. Treatment with Flt3 ligand, a hematopoietic growth factor that dramatically expands the number of dendritic cells, markedly enhanced the magnitude of virus-specific CD8(+) T-cell responses during acute infection and the pool of memory anti-polyomavirus CD8(+) T cells. These findings suggest that virus-infected dendritic cells induce polyomavirus-specific CD8(+) T cells in vivo and raise the potential for their use as cellular adjuvants to promote CD8(+) T cell surveillance against polyomavirus-induced tumors.     

Down-regulation of Toll-like receptor expression in monocyte-derived Langerhans cell-like cells: implications of low-responsiveness to bacterial components in the epidermal Langerhans cells

In the skin, there are unique dendritic cells called Langerhans cells, however, it remains unclear why this particular type of dendritic cell resides in the epidermis. Langerhans cell-like dendritic cells (LCs) can be generated from CD14(+) monocytes in the presence of GM-CSF, IL-4, and TGF-beta1. We compared LCs with monocyte-derived dendritic cells (DCs) generated from CD14(+) monocytes in the presence of GM-CSF and IL-4 and examined the effect of exposure to two distinct bacterial stimuli via Toll-like receptors (TLRs), such as peptidoglycan (PGN) and lipopolysaccharide (LPS) on LCs and DCs. Although stimulation with both ligands induced a marked up-regulation of CD83 expression on DCs, PGN but not LPS elicited up-regulation of expression CD83 on LCs. Consistent with these results, TLR2 and TLR4 were expressed on DCs, whereas only TLR2 was weakly detected on LCs. These findings suggest the actual feature of epidermal Langerhans cells with low-responsiveness to skin commensals.     

Timing and magnitude of type I interferon responses by distinct sensors impact CD8 T cell exhaustion and chronic viral infection

Type I interferon (IFN-I) promotes antiviral CD8(+)T cell responses, but the contribution of different IFN-I sources and signaling pathways are ill defined. While plasmacytoid dendritic cells (pDCs) produce IFN-I upon TLR stimulation, IFN-I is induced in most cells by helicases like MDA5. Using acute and chronic lymphocytic choriomeningitis virus (LCMV) infection models, we determined that pDCs transiently produce IFN-I that minimally impacts CD8(+)T cell responses and viral persistence. Rather, MDA5 is the key sensor that induces IFN-I required for CD8(+)T cell responses. In the absence of MDA5, CD8(+)T cell responses to acute infection rely on CD4(+)T cell help, and loss of both CD4(+)T cells and MDA5 results in CD8(+)T cell exhaustion and persistent infection. Chronic LCMV infection rapidly attenuates IFN-I responses, but early administration of exogenous IFN-I rescues CD8(+)T cells, promoting viral clearance. Thus, effective antiviral CD8(+)T cell responses depend on the timing and magnitude of IFN-I production.     

CD4+ T cells regulate CD8+ T cell-mediated cutaneous immune responses by restricting effector T cell development through a Fas ligand-dependent mechanism

The magnitude and duration of CD8(+) T cell-mediated responses in the skin to hapten sensitization and challenge, contact hypersensitivity (CHS), is negatively regulated by CD4(+) T cells through an unknown mechanism. In this study we show that CD4(+) T cells restrict the development and expansion of hapten-specific CD8(+) T cells mediating CHS responses to 2,4-dinitrofluorobenzene. In the absence of CD4(+) T cells, high numbers of hapten-specific CD8(+) T cells producing IFN-gamma were detected in the skin-draining lymph nodes on day 5 postsensitization, and these numbers decreased slightly, but were maintained through day 9, correlating with the increased magnitude and duration of CHS responses observed in these mice. In the presence of CD4(+) T cells, the number of hapten-specific CD8(+) T cells producing IFN-gamma detected on day 5 postsensitization was lower and quickly fell to background levels by day 7. The limited development of effector CD8(+) T cells was associated with decreased numbers of hapten-presenting dendritic cells in the lymphoid priming site. This form of immunoregulation was absent after sensitization of Fas ligand-defective gld mice. Transfer of wild-type CD4(+) T cells to gld mice restored the negative regulation of CD8(+) T cell priming and the immune response to hapten challenge in gld-recipient mice. These results indicate that CD4(+) T cells restrict hapten-specific CD8(+) T cell priming for CHS responses through a Fas ligand-dependent mechanism.     

Activated CD34-derived Langerhans cells mediate transinfection with human immunodeficiency virus

Langerhans cells (LCs) are a subset of dendritic cells (DCs) that reside within epidermal and mucosal tissue. Because of their location, LCs are potentially the first cells to encounter human immunodeficiency virus (HIV) during sexual transmission. We report that LCs purified from CD34(+)-derived DCs can facilitate the transinfection of target cells but only after activation. Virions were observed in an intracellular compartment that contains several tetraspanins, in addition to the unique LC markers langerin and CD1a. This reveals that the trafficking of HIV within LCs is reminiscent of that which occurs in mature monocyte-derived DCs and that it varies with the activation state of the cell. The observation that activated LCs can mediate transinfection suggests a potential role for these cells in the known increase in HIV transmission associated with sexually transmitted infections that would cause inflammation of the genital lining.     

Langerhans cells that have matured in vivo in the absence of T cells are fully capable of inducing a helper CD4 as well as a cytotoxic CD8 response

Langerhans cells (LCs) are immature dendritic cells (DCs) present in the skin epithelium. Upon Ag exposure, they migrate to the draining lymph nodes where they mature into potent stimulators of naive T cells. The aim of this study was to investigate the influence of T cells on LC migration and maturation. Therefore, the in vivo migration and maturation of LCs after sensitization with the hapten FITC was compared between C57BL/6 or BALB/c mice used as positive controls, and recombination activating gene (RAG) 1 knockout (-/-) mice or SCID mice used as T cell-deficient mice. Phenotypically, there was no difference between migrated LCs from RAG1-/- or SCID mice vs normal C57BL/6 or BALB/c mice: both populations of FITC+ cells had a dendritic morphology and a mature phenotype as they expressed high levels of MHC class II molecules and costimulatory molecules CD80, CD86, and CD54. Sorted migrated LCs of RAG1-/- or SCID mice were efficient stimulators of allogeneic T cells and Ag-specific CD4+ T cells. The same results were found if migrated LCs were fixed instead of irradiated, excluding the possibility that LCs derived from RAG1-/- or SCID mice would mature in the presence of T cells during the stimulation tests. Importantly, fixed migrated LCs of RAG1-/- mice were also efficient stimulators of cytotoxic CD8+ T cells. These data suggest that T cells are not required for full maturation of LCs.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.     

Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host

Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination. Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen. Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections. The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection. However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection. Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells. However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice. This situation occurred in response to lymphocytic choriomeningitis virus or L. monocytogenes infection and for CD8(+) T cell responses against multiple epitopes. The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells. Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response.