An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.     

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.     

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.     

CD8+ T cells accumulate in the lungs of Mycobacterium tuberculosis-infected Kb-/-Db-/- mice,but provide minimal protection

Recent studies have shown that MHC class I molecules play an important role in the protective immune response to Mycobacterium tuberculosis infection. Here we showed that mice deficient in MHC class Ia, but possessing MHC class Ib (K(b-/-)D(b-/-) mice), were more susceptible to aerosol infection with M. tuberculosis than control mice, but less susceptible than mice that lack both MHC class Ia and Ib (beta(2)m(-/-) mice). The susceptibility of K(b-/-)D(b-/-) mice cannot be explained by the failure of CD8(+) T cells (presumably MHC class Ib-restricted) to respond to the infection. Although CD8(+) T cells were a relatively small population in uninfected K(b-/-)D(b-/-) mice, most already expressed an activated phenotype. During infection, a large percentage of these cells further changed their cell surface phenotype, accumulated in the lungs at the site of infection, and were capable of rapidly producing IFN-gamma following TCR stimulation. Histopathologic analysis showed widespread inflammation in the lungs of K(b-/-)D(b-/-) mice, with a paucity of lymphocytic aggregates within poorly organized areas of granulomatous inflammation. A similar pattern of granuloma formation has previously been observed in other types of MHC class I-deficient mice, but not CD8alpha(-/-) mice. Thus, neither the presence of MHC class Ib molecules themselves, nor the activity of a population of nonclassical CD8(+) effector cells, fully restored the deficit caused by the absence of MHC class Ia molecules, suggesting a unique role for MHC class Ia molecules in protective immunity against M. tuberculosis.     

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help

The unique ether glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

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.     

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.     

Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice

Classical CD4(+) and CD8(+) T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4(-/-) mice mount a strong, readily detectable CD8(+) T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8(+)CD4(-) T cells are more similar to classical CD8(+) T cells than to CD4(+) T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8(+) T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4(-/-) mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4(-/-) mice consists mainly of CD8(-)CD4(-) T cells. In contrast to CD8(+) T cells, MHCII-restricted CD8(-)CD4(-) T cells are capable of producing IL-2 in addition to IFN-gamma and thus appear to have attributes characteristic of CD4(+) T cells rather than CD8(+) T cells. Therefore, MHCII-restricted T cells in CD4(-/-) mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8(+) T cells or with MHCII-restricted CD4(+) T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.     

Quantitation of CD8+ T cell expansion,memory, and protective immunity after immunization with peptide-coated dendritic cells

Dendritic cells (DCs) are potent APCs for naive CD8(+) T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8(+) T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 x 10(5) mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8(+) T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8(+) T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91-99-coated BMDCs are protected against high dose challenge with virulent LM. This protection was enhanced by diversifying the memory CD8(+) T cell compartment, even in the absence of a large increase in Ag-specific CD8(+) memory T cells.     

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.     

H2-M3-restricted memory T cells: persistence and activation without expansion

H2-M3-restricted T cells respond more rapidly to primary Listeria monocytogenes infection than conventional MHC class Ia-restricted T cells. Reinfection with L. monocytogenes, while inducing explosive proliferation of H2-K(d)-restricted T cells, does not stimulate significant expansion of H2-M3-restricted CTL. These disparate responses to reinfection are apparent within 5 days of primary L. monocytogenes infection. However, H2-M3-restricted memory T cells are generated, and are indistinguishable from classically restricted T cells in terms of cell surface memory markers and longevity. Early responses of H2-M3- and H2-K(d)-restricted memory T cells to reinfection are similar, with increases in size and expression of activation markers. Interestingly, priming of H2-M3-restricted T cells with an L. monocytogenes-derived N-formyl peptide plus anti-CD40 generates memory T cells that expand upon re-exposure to Ag during L. monocytogenes infection. Our data indicate that disparate H2-M3- and MHC class Ia-restricted memory T cell responses reflect intrinsic differences between these T cell populations. Although distinct proliferative programs appear to be hardwired in these populations during primary L. monocytogenes infection, under different inflammatory circumstances M3-restricted T cell populations can maintain the ability to expand upon re-exposure to Ag.     

Functional characterization of class Ia- and non-class Ia-restricted Chlamydia-reactive CD8+ T cell responses in humans

CD8(+) T cells are a key immune component for the eradication of many intracellular pathogens. This study aims to characterize the human CD8(+) T cell response to naturally processed chlamydial Ags in individuals exposed to the intracellular pathogen Chlamydia trachomatis. By using C. trachomatis-infected autologous dendritic cells (DCs) as stimulators, Chlamydia-reactive CD8(+) T cell responses were detected in all 10 individuals tested. The majority of the Chlamydia-reactive CD8(+) T cells were non-MHC class Ia restricted in all three of the individuals tested. From one donor, three non-class Ia-restricted and two class Ia-restricted Chlamydia-specific CD8(+) T cells were cloned and characterized further. All five T cell clones secreted IFN-gamma in response to autologous DCs infected with viable Chlamydia, but not with DCs pulsed with inactivated chlamydial elementary bodies. MHC class Ia-restricted and non-class Ia-restricted responses were inhibited by DC treatment with a proteasomal inhibitor and an endoplasmic reticulum-Golgi transport inhibitor, suggesting that these T cells recognize a peptide Ag translocated to the host cell cytosol during infection that is processed via the classical class Ia Ag-processing pathway. Even though both restricted and nonrestricted CD8(+) T cells produced IFN-gamma in response to Chlamydia-infected fibroblasts, only the non-class Ia-restricted cells were lytic for these targets. The class Ia-restricted CTLs, however, were capable of cytolysis as measured by redirected killing. Collectively, these data demonstrate that both class Ia-restricted and non-classically restricted CD8(+) T cells are elicited in C. trachomatis-exposed individuals. Their role in host immunity remains to be elucidated.     

Polyclonal MHC Ib-restricted CD8+ T cells undergo homeostatic expansion in the absence of conventional MHC-restricted T cells

Naive T cells have the capacity to expand in a lymphopenic environment in a process called homeostatic expansion, where they gain a memory-like phenotype. Homeostatic expansion is dependent on competition for a number of factors, including growth factors and interactions with their selecting self-MHC molecules. In contrast to conventional T cells, it is unclear whether class Ib-restricted CD8+ T cells have a capacity to undergo homeostatic expansion. In this study, we demonstrate that polyclonal MHC Ib-restricted CD8+ T cells can undergo homeostatic expansion and that their peripheral expansion is suppressed by conventional MHC-restricted T cells. The acute depletion of CD4+ T cells in MHC class Ia-deficient Kb-/-Db-/- mice led to the substantial expansion of class Ib-restricted CD8+ T cells. Adoptive transfer of class Ib-restricted CD8+ T cells to congenic lymphopenic recipients revealed their ability to undergo homeostatic expansion in a MHC Ib-dependent manner. To further study the homeostatic expansion of MHC Ib-restricted T cells in the absence of all conventional MHC-restricted T cells, we generated mice that express only MHC Ib molecules by crossing H-2Kb-/-Db-/- with CIITA-/- mice. CD8+ T cells in these mice exhibit all of the hallmarks of naive T cells actively undergoing homeostatic expansion with constitutive memory-like surface and functional phenotype. These findings provide direct evidence that MHC Ib-restricted CD8+ T cells have the capacity to undergo homeostatic expansion. Their peripheral expansion is suppressed under normal conditions by a numerical excess of conventional MHC class Ia- and class II-restricted T cells.     

Cutting edge: selective impairment of CD8+ T cell function in mice lacking the TNF superfamily member LIGHT

Interactions of LIGHT and its receptors, herpesvirus entry mediator on T cells and lymphotoxin beta receptor on stromal cells, are implicated in the regulation of lymphoid organogenesis, costimulation of T cells, and activation of dendritic cells. In this work we report that LIGHT-deficient mice had normal lymphoid organs with T cells and APCs that normally responded to Ag stimulation and normally stimulated T cells. Although the number of Vbeta8(+) T cells in naive LIGHT(+/+) and LIGHT(-/-) mice was identical, Vbeta8(+)CD8(+) T cell proliferation in response to staphylococcal enterotoxin B was significantly lower in LIGHT(-/-) mice. Consistently, induction and cytokine secretion of CD8(+) CTL to MHC class I-restricted peptide was also reduced in LIGHT(-/-) mice. However, the proliferative response of Vbeta8(+)CD4(+) T cells to staphylococcal enterotoxin B was comparable in LIGHT(-/-) and LIGHT(+/+) mice. Our results suggest that LIGHT is required for activation of normal CD8(+) T cells but not CD4(+) T cells.     

A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae

An intact T cell compartment and IFN-gamma signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8(+) T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8(+) Tc1 response against various Cpn Ags, and that CD8(+) CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2(b)-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-gamma-producing CD8(+) CTL against all seven CTL epitopes. CD8(+) T cell lines generated to minigene-encoded CTL epitopes secreted IFN-gamma and TNF-alpha and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with beta(2)-microglobulin(-/-) mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8(+) T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4(+) T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8(+) T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.     

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.     

Cross-reactive antigen is required to prevent erosion of established T cell memory and tumor immunity: a heterologous bacterial model of attrition

Induction and maintenance of T cell memory is critical for the control of intracellular pathogens and tumors. Memory T cells seem to require few "maintenance signals," though often such studies are done in the absence of competing immune challenges. Conversely, although attrition of CD8(+) T cell memory has been characterized in heterologous viral models, this is not the case for bacterial infections. In this study, we demonstrate attrition of T cell responses to the intracellular pathogen Listeria monocytogenes (LM) following an immune challenge with a second intracellular bacterium, Mycobacterium bovis (bacillus Calmette-Guérin, BCG). Mice immunized with either LM or recombinant LM (expressing OVA; LM-OVA), develop a potent T cell memory response. This is reflected by peptide-specific CTL, IFN-gamma production, and frequency of IFN-gamma-secreting T cells to native or recombinant LM Ags. However, when the LM-infected mice are subsequently challenged with BCG, there is a marked reduction in the LM-specific T cell responses. These reductions are directly attributable to the effects on CD4(+) and CD8(+) T cells and the data are consistent with a loss of LM-specific T cells, not anergy. Attrition of the Ag (OVA)-specific T cell response is prevented when LM-OVA-immunized mice are challenged with a subsequent heterologous pathogen (BCG) expressing OVA, demonstrating memory T cell dependence on Ag. Although the reduction of the LM-specific T cell response did not impair protection against a subsequent LM rechallenge, for the first time, we show that T cell attrition can result in the reduction of Ag-specific antitumor (B16-OVA) immunity previously established with LM-OVA immunization.     

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.     

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded β(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.     

Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection

CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response. A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.