Distinct regulation of H2-M3-restricted memory T cell responses in lymph node and spleen

CD8 T cell populations restricted by H2-M3 MHC class Ib molecules expand rapidly during primary Listeria monocytogenes infection but only minimally upon reinfection. In contrast, CD8 T cells restricted by MHC class Ia molecules undergo more delayed expansion during primary infection but rapid and robust expansion following reinfection. In this study we demonstrate that primary H2-M3-restricted CD8 T cell responses are unaffected by the frequency of naive MHC class Ia-restricted T cells during L. monocytogenes infection. The magnitude of H2-M3-restricted memory responses, in contrast, is down-modulated by increasing frequencies of MHC class Ia-restricted effector T cells following secondary systemic infection. Suppression by MHC class Ia-restricted T cells, however, is not a universal feature of MHC class Ib-restricted memory responses. Primary systemic L. monocytogenes infection followed by secondary tissue infection, for example, results in robust expansion of H2-M3-restricted memory T cells in draining lymph nodes, despite the activation of MHC class Ia-restricted memory T cell responses. Thus, whereas MHC class Ia-restricted memory T cell populations predominate in spleens following systemic reinfection, H2-M3-restricted memory T cell responses remain prominent in lymph nodes draining localized infections. Our studies demonstrate that interactions between CD8 T cell populations can differ, depending on the status of the responding T cells (naive vs memory) and the route of reinfection. These results may have important implications for prime-boost vaccination strategies.     

Variable immunodominance hierarchies for H2-M3-restricted N-formyl peptides following bacterial infection

H2-M3-restricted presentation of N-formyl methionine (f-Met) peptides to CD8(+) T cells provides a mechanism for selective recognition of bacterial infection. In this report we demonstrate that Listeria monocytogenes infection induces distinct CD8(+) T cell populations specific for each of the known Listeria-derived formyl methionine peptides presented by M3. The sum H2-M3-restricted, Listeria-specific T cell response constitutes a major fraction of the total CD8(+) T cell response to primary infection. H2-M3-restricted T cell populations expand synchronously in vivo and achieve peak frequencies approximately 2 days earlier than MHC class Ia-restricted T cell populations. Although cross-recognition of different f-Met peptides by M3-restricted T cells was previously described, costaining of CD8(+) T cells ex vivo with H2-M3 tetramers complexed with different f-Met peptides shows that the majority of Listeria-specific, M3-restricted CD8(+) T cells are peptide specific. In contrast to the highly predictable size and immunodominance hierarchies of MHC class Ia-restricted T cell responses, the magnitudes of T cell responses specific for H2-M3-restricted peptides are remarkably variable between genetically identical mice. Our findings demonstrate that H2-M3-restricted T cell responses are distinct from classically restricted T cell responses to bacterial infection.     

Selective dependence of H2-M3-restricted CD8 responses on IL-15

We studied whether CD8 T cell responses that are mediated by unconventional MHC class Ib molecules are IL-15 dependent in mice. CD8(+) T cell responses to Listeria monocytogenes infection that are restricted by the MHC class Ib molecule H2-M3 decreased in the absence of IL-15, whereas other primary MHC class Ib- and MHC class Ia-restricted responses were IL-15 independent. This result was confirmed in MHC class Ia-deficient mice in which IL-15 deficiency also reduced H2-M3-restricted but not all CD8 T cell responses to L. monocytogenes. IL-15 deficiency did not affect proliferation or survival of responding H2-M3-restricted CD8(+) T cells, but IL-15 was necessary to detect H2-M3-restricted CD8(+) T cells in naive mice. This finding suggests that these CD8(+) T cells require IL-15 during development, but become IL-15 independent after activation. IL-15 was necessary for the survival of most class Ib-restricted CD8(+) T cells, starting at the mature thymocyte stage in naive mice, but does not affect a distinct CD44(low)/CD122(low) subpopulation. These data suggest that the nature of the selecting MHC class Ib molecule determines whether CD8(+) T cells acquire IL-15 dependence during thymic development.     

H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO(190-201) (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-gamma-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.     

Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes

Listeria monocytogenes is an intracellular bacterium that causes systemic infections after traversing the intestinal mucosa. Clearance of infection and long term protective immunity are mediated by L. monocytogenes-specific CD8 T lymphocytes. In this report, we characterize the murine CD8 T cell response in the lamina propria and intestinal epithelium after enteric L. monocytogenes infection. We find that the frequency of MHC class Ia-restricted, L. monocytogenes-specific T cells is approximately 4- to 5-fold greater in the lamina propria than in the spleen of mice after oral or i.v. infection. Although the kinetics of T cell expansion and contraction are similar in spleen, lamina propria, and intestinal epithelium, high frequencies of Ag-specific T cells are detected only in the lamina propria 1 mo after infection. In contrast to MHC class Ia-restricted T cells, the frequency of H2-M3-restricted, L. monocytogenes-specific T cells is decreased in the intestinal mucosa relative to that found in the spleen. In addition to this disparity, we find that MHC class Ia-restricted CD8 T cells specific for a dominant L. monocytogenes epitope have different TCR V beta repertoires in the spleen and intestinal mucosa of individual mice. These findings indicate that the intestinal mucosa is a depot where L. monocytogenes-specific effector CD8 T cells accumulate during and after infection irrespective of immunization route. Furthermore, our results demonstrate that CD8 T cell populations in these two sites, although overlapping in Ag specificity, are distinct in terms of their repertoire.     

MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection

Infection of B6 mice with the intracellular pathogen Listeria monocytogenes (LM) results in the activation of CD8(+) T cells that respond to Ag presented by both MHC class Ia and class Ib molecules. Enzyme-linked immunospot analysis reveals that these CTL populations expand and contract at different times following a primary sublethal LM infection. Between days 4 and 6 postinfection, class Ib-restricted CTL exhibit a rapid proliferative response that is primarily H2-M3 restricted. The peak response of class Ia-restricted CD8(+) T cells occurs a few days later, after the majority of bacteria have been cleared. Although class Ia-restricted CTL exhibit a vigorous recall response to secondary LM infection, we observe limited expansion of class Ib-restricted memory CTL, even in MHC class Ia-deficient mice (B6.K(b-/-)D(b-/-)). Despite this lack of enhanced expansion in vivo, class Ib-restricted memory CTL retain the ability to proliferate and expand when provided with Ag in vitro. Furthermore, we demonstrate that in vivo depletion of CD8(+) T cells in LM-immune B6.K(b-/-)D(b-/-) mice severely impairs memory protection. Together, these data demonstrate that class Ib-restricted CTL play an important role in clearing a primary LM infection and generate a memory population capable of providing significant protection against subsequent infection.     

Promiscuity of MHC class Ib-restricted T cell responses

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8(+) T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.     

Partially TAP-independent protection against Listeria monocytogenes by H2-M3-restricted CD8+ T cells

Effective protection against Listeria monocytogenes requires Ag-specific CD8(+) T cells. A substantial proportion of CD8(+) T cells activated during L. monocytogenes infection of C57BL/6 mice are restricted by the MHC class Ib molecule H2-M3. In this study, an H2-M3-restricted CD8(+) T cell clone specific for a known H2-M3 epitope (fMIGWII) was generated from L. monocytogenes-infected mice. The clone was cytotoxic, produced IFN-gamma, and could mediate strong protection against L. monocytogenes when transferred to infected mice. Macrophages pulsed with heat-killed LISTERIAE: presented Ag to the clone in a TAP-independent manner. Both TAP-independent and -dependent processing occurred in vivo, as TAP-deficient mice infected with L. monocytogenes were partially protected by adoptive transfer of the clone. This is the first example of CD8(+) T cell-mediated, TAP-independent protection against a pathogen in vivo, confirming the importance of alternative MHC class I processing pathways in the antibacterial immunity.     

CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell-independent

CD8+ T cells are important for immunity to the intracellular bacterial pathogen Chlamydia pneumoniae (Cpn). Recently, we reported that type 1 CD8+ (Tc1) from Cpn-infected B6 mice recognize peptides from multiple Cpn Ags in a classical MHC class Ia-restricted fashion. In this study, we show that Cpn infection also induces nonclassical MHC class Ib-(H2-M3)-restricted CD8+ T cell responses. H2-M3-binding peptides representing the N-terminal formylated sequences from five Cpn Ags sensitized target cells for lysis by cytolytic effectors from the spleens of infected B6 mice. Of these, only peptides fMFFAPL (P1) and fMLYWFL (P4) stimulated IFN-gamma production by infection-primed splenic and pulmonary CD8+ T cells. Studies with Cpn-infected Kb-/-/Db-/- mice confirmed the Tc1 cytokine profile of P1- and P4-specific CD8+ T cells and revealed the capacity of these effectors to exert in vitro H2-M3-restricted lysis of Cpn-infected macrophages and in vivo pulmonary killing of P1- and P4-coated splenocytes. Furthermore, adoptive transfer of P1- and P4-specific CD8+ T cells into naive Kb-/-/Db-/- mice reduced lung Cpn loads following challenge. Finally, we show that in the absence of MHC class Ia-restricted CD8+ T cell responses, CD4+ T cells are largely expendable for the control of Cpn growth, and for the generation, memory maintenance, and secondary expansion of P1- and P4-specific CD8+ T cells. These results suggest that H2-M3-restricted CD8+ T cells contribute to protective immunity against Cpn, and that chlamydial Ags presented by MHC class Ib molecules may represent novel targets for inclusion in anti-Cpn vaccines.     

Nonconventional CD8+ T cell responses to Listeria infection in mice lacking MHC class Ia and H2-M3

CD8(+) T cells restricted to MHC class Ib molecules other than H2-M3 have been shown to recognize bacterial Ags. However, the contribution of these T cells to immune responses against bacterial infection is not well defined. To investigate the immune potential of MHC class Ib-restricted CD8(+) T cells, we have generated mice that lack both MHC class Ia and H2-M3 molecules (K(b-/-)D (b-/-)M3(-/-)). The CD8(+) T cells present in K(b-/-)D (b-/-)M3(-/-) mice display an activated surface phenotype and are able to secrete IFN-γ rapidly upon anti-CD3 and anti-CD28 stimulation. Although the CD8(+) T cell population is reduced in K(b-/-)D (b-/-)M3(-/-) mice compared with that in K(b-/-)D (b-/-) mice, this population retains the capacity to expand significantly in response to primary infection with the bacteria Listeria monocytogenes. However, K(b-/-)D (b-/-)M3(-/-) CD8(+) T cells do not expand upon secondary infection, similar to what has been observed for H2-M3-restricted T cells. CD8(+) T cells isolated from Listeria-infected K(b-/-)D (b-/-)M3(-/-) mice exhibit cytotoxicity and secrete proinflammatory cytokines in response to Listeria-infected APCs. These T cells are protective against primary Listeria infection, as Listeria-infected K(b-/-)D (b-/-)M3(-/-) mice exhibit reduced bacterial burden compared with that of infected β(2)-microglobulin-deficient mice that lack MHC class Ib-restricted CD8(+) T cells altogether. In addition, adoptive transfer of Listeria-experienced K(b-/-)D (b-/-)M3(-/-) splenocytes protects recipient mice against subsequent Listeria infection in a CD8(+) T cell-dependent manner. These data demonstrate that other MHC class Ib-restricted CD8(+) T cells, in addition to H2-M3-restricted T cells, contribute to antilisterial immunity and may contribute to immune responses against other intracellular bacteria.     

Differing roles of inflammation and antigen in T cell proliferation and memory generation

Recent studies have demonstrated that viral and bacterial infections can induce dramatic in vivo expansion of Ag-specific T lymphocytes. Although presentation of Ag is critical for activation of naive T cells, it is less clear how dependent subsequent in vivo T cell proliferation and memory generation are upon Ag. We investigated T cell expansion and memory generation in mice infected alternately with strains of Listeria monocytogenes that contained or lacked an immunodominant, MHC class I-restricted T cell epitope. We found substantial differences in the responses of effector and memory T cells to inflammatory stimuli. Although effector T cells undergo in vivo expansion in response to bacterial infection in the absence of Ag, memory T cells show no evidence for such bystander activation. However, Ag-independent expansion of effector T cells does not result in increased memory T cell frequencies, indicating that Ag presentation is critical for effective memory T cell generation. Early reinfection of mice with L. monocytogenes before the maximal primary T cell response induces typical memory expansion, suggesting that the capacity for a memory T cell response exists within the primary effector population. Our findings demonstrate that T cell effector proliferation and memory generation are temporally overlapping processes with differing requirements for Ag.     

Nonsecreted bacterial proteins induce recall CD8 T cell responses but do not serve as protective antigens

Secreted or nonsecreted Ag expressed by recombinant Listeria monocytogenes can prime CD8 T cells. However, Ag-specific memory CD8 T cells confer protection against bacteria secreting Ag, but not against bacteria expressing the nonsecreted form of the same Ag. This dichotomy may be explained by a long-standing hypothesis that nonsecreted Ags are less effective than secreted Ags at inducing a protective immune response at the onset of infection. We tested this hypothesis by examining whether these two different forms of Ag induce different primary and secondary CD8 T cell responses. The primary responses to secreted and nonsecreted Ags expanded and contracted almost synchronously, although the responses to nonsecreted Ags were of lower magnitude. These results demonstrate that the kinetics of the CD8 T cell response are similar regardless of whether Ag is accessible to the endogenous MHC class I pathway or can only be presented through cross-presentation. No differences were detected in the CD8 T cell recall response to L. monocytogenes expressing secreted or nonsecreted Ags. Nonsecreted Ags are as effective as secreted Ags at the induction of a rapid recall response by memory CD8 T cells. Thus, the inability of nonsecreted bacterial proteins to serve as protective Ags cannot be attributed to a defective CD8 T cell response.     

Immunization with gp96 from Listeria monocytogenes-infected mice is due to N-formylated listerial peptides.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.     

Enhancement of the Listeria monocytogenes p60-specific CD4 and CD8 T cell memory by nonpathogenic Listeria innocua

The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory. This hypothesis was evaluated in the model of murine Listeria monocytogenes infection. The influence of nonpathogenic L. innocua on the L. monocytogenes p60-specific T cell response was analyzed. We show that some CD4 T cell clones raised against purified p60 from L. monocytogenes cross-react with p60 purified from L. innocua. The L. monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L. innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue. In vitro experiments revealed that after L. monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited. L. innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II. In contrast to these in vitro experiments the infection of mice with L. monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L. innocua infection failed to induce p60-specific T cells. L. innocua booster infection, however, expanded p60-specific memory T cells induced by previous L. monocytogenes infection. In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L. innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen.     

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.     

Serum antibodies critically affect virus-specific CD4+/CD8+ T cell balance during respiratory syncytial virus infections

Following infection with respiratory syncytial virus (RSV), reinfection in healthy individuals is common and presumably due to ineffective memory T cell responses. In peripheral blood of healthy adults, a higher CD4(+)/CD8(+) memory T cell ratio was observed compared with the ratio of virus-specific effector CD4(+)/CD8(+) T cells that we had found in earlier work during primary RSV infections. In mice, we show that an enhanced ratio of RSV-specific neutralizing to nonneutralizing Abs profoundly enhanced the CD4(+) T cell response during RSV infection. Moreover, FcγRs and complement factor C1q contributed to this Ab-mediated enhancement. Therefore, the increase in CD4(+) memory T cell response likely occurs through enhanced endosomal Ag processing dependent on FcγRs. The resulting shift in memory T cell response was likely amplified by suppressed T cell proliferation caused by RSV infection of APCs, a route important for Ag presentation via MHC class I molecules leading to CD8(+) T cell activation. Decreasing memory CD8(+) T cell numbers could explain the inadequate immunity during repeated RSV infections. Understanding this interplay of Ab-mediated CD4(+) memory T cell response enhancement and infection mediated CD8(+) memory T cell suppression is likely critical for development of effective RSV vaccines.     

Th1 T cell responses to HIV-1 Gag protein delivered by a Listeria monocytogenes vaccine are similar to those induced by endogenous listerial antigens.

Listeria monocytogenes is a facultative intracellular bacterium that lives and grows in the cytoplasm of the host cell. The hallmark of a listerial infection is a cell-mediated immune response to its own secreted virulence factors. Thus, L. monocytogenes vaccines engineered to secrete HIV proteins may be ideal vectors for boosting cellular immune responses against HIV. Using strains of L. monocytogenes that stably express and secrete HIV Gag (Lm-Gag) to deliver this Ag to the immune system, we have previously shown strong MHC class I-restricted cytotoxic T cell responses to this protein. In this study, we examine MHC class II-restricted T cell responses to HIV-Gag delivered by Lm-Gag. We demonstrate the induction of CD4+ T cells that are HIV-Gag specific and identify three epitopes in two strains of mice, BALB/c (H-2d) and C57BL/6 (H-2b), two of which are both H-2d and H-2b restricted, but are not immunodominant for both haplotypes. In addition, we show that the CD4+ T cells induced are of the Th1 phenotype that produce IFN-gamma at levels similar to CD4+ T cells induced to endogenous listerial Ags. These studies suggest that chromosomally modified strains of L. monocytogenes may be useful as vaccine vectors for the induction of Th1 T cell responses against HIV.     

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.     

Antigen secreted from noncytosolic Listeria monocytogenes is processed by the classical MHC class I processing pathway.

Intracellular bacteria can reside in a vacuolar compartment, or they can escape the vacuole and become free living in the cytoplasm. The presentation of Ag by class I MHC molecules has been defined primarily for Ag present in the cytoplasm. It was therefore thought that Ags from bacteria that remain in a vacuole would not be presented by MHC class I molecules. Although some studies have provided data to support this idea, it is not necessarily true for all intracellular bacteria. For example, we have previously demonstrated that an epitope from the p60 protein secreted by LLO- Listeria monocytogenes, which does not reside in the cytoplasm, can be presented by MHC class I molecules to a T cell clone specific for the epitope, p60217-225. We have further examined the route by which Ag secreted by LLO- L. monocytogenes is presented by MHC class I molecules. Using pharmacological inhibitors, we demonstrate that MHC class I presentation of the p60 epitope derived from by LLO- L. monocytogenes requires phagolysosome fusion and processing by the proteasome. Lysosomal cathepsins, however, are not required for processing of the p60 epitope. Similarly, processing of the AttM epitope, secreted by LLO- L. monocytogenes and presented by H2-M3, also requires phagolysosome fusion and cleavage by the proteasome. Thus, p60 and AttM secreted by LLO- L. monocytogenes are processed via the classical class I pathway for presentation by MHC class I molecules.     

CD8 T cell recall responses are regulated by the tissue tropism of the memory cell and pathogen

Whether memory CD8 T cells can be reactivated in nonlymphoid tissues is unclear. Using mice lacking the spleen, lymph nodes, or both, we show that the secondary T cell response, but not homeostatic maintenance of memory cells, required lymphoid tissue. Whereas primary and secondary CD8 T cell responses to vesicular stomatitis virus infection were lymph node dependent, responses to Listeria monocytogenes infection were driven primarily in the spleen. Memory cell subset reactivation was also regulated by location of the responding population and the pathogen. Thus, CD62Llow effector memory T cells (TEM) cells responded nearly as well as CD62Lhigh central memory T cells (TCM) and TCM cells after L. monocytogenes infection, and both subsets generated equivalent populations of secondary memory cells. In contrast, TCM cells, but not TEM cells, mounted a robust response to vesicular stomatitis virus infection. TCM and TEM cells also required lymphoid tissue to mount recall responses, and the bone marrow did not contribute significantly to the response of either subset. Our findings indicated that characteristics of the infectious agent and the migratory preferences of memory cells dictated the secondary lymphoid tissue requirement for the recall response to infection.