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.     

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.     

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.     

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.     

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.     

An MHC class Ib-restricted TCR that cross-reacts with an MHC class Ia molecule

TCR transgenic 6C5 T cells recognize an insulin B chain epitope presented by the nonclassical class I MHC molecule, Qa-1(b). Positive selection of these T cells was shown previously to require Qa-1(b). Despite dedicated specificity for Qa-1(b), evidence presented in the current study indicates that 6C5 T cells can cross-recognize a classical class I molecule. Clonal deletion was observed unexpectedly in 6C5.H-2(bxq) mice, which do not express I-E MHC class II molecules and thus should not be subject to superantigen-mediated negative selection. 6C5 T cells were observed to respond in vivo and in vitro to spleen cells from allogeneic H-2(q) mice, and specificity was mapped to D(q). Evidence was obtained for direct recognition of D(q), rather than indirect presentation of a D(q)-derived peptide presented by Qa-1(b). Polyclonal CD8(+) T cells from class Ia-deficient K(b)D(b-/-) mice reacted in vitro to allogeneic spleen cells with an apparent frequency comparable to conventional class Ia-restricted T cells. Our results provide a clear example of a Qa-1-specific TCR that can cross-react with a class Ia molecule and evidence supporting the idea that this may be a common property of T cells selected by class Ib molecules.     

Memory phenotype of CD8+ T cells in MHC class Ia-deficient mice

B6.K(b-)D(b-) mice are devoid of class Ia but express normal levels of class Ib molecules. They have low levels of CD8 T cells in both the thymus as well as peripheral T cell compartments. Although the percentage of splenic CD8 alpha alpha T cells is increased in these animals, approximately 90% of CD8 T cells are CD8 alpha beta. In contrast to B6 animals, most of the CD8 T cells from these mice have a memory phenotype (CD44(high)CD122(high) CD62L(low)) including both CD8 alpha beta and CD8 alpha alpha subsets. In the thymus of B6.K(b-)D(b-) animals, there is a decrease in the percentage of SP CD8 T cells, although most are CD44(low), similar to that seen in B6 mice. The spleens from day 1-old B6 and B6.K(b-)D(b-) mice have a relatively high proportion of CD44(high)CD62L(low) CD8 T cells. However, by day 28 most CD8 T cells in B6 mice have a naive phenotype while in B6.K(b-)D(b-) mice the memory phenotype remains. Unlike CD44(high) cells that are found in B6 animals, most CD44(high) cells from B6.K(b-)D(b-) mice do not secrete IFN-gamma rapidly upon activation. The paucity of CD8 T cells in B6.K(b-)D(b-) mice might be due in part to their inability to undergo homeostatic expansion. Consistent with this, we found that CD8 T cells from these animals expand poorly in X-irradiated syngeneic hosts compared with B6 CD8 T cells that respond to class Ia Ags. We examined homeostatic expansion of B6 CD8 T cells in single as well as double class Ia knockout mice and were able to estimate the fraction of cells reactive against class Ia vs class Ib molecules.     

Cutting edge: a single MHC class Ia is sufficient for CD8 memory T cell differentiation

Recent studies have suggested a role for MHC class Ib molecules in providing signals for memory T cell differentiation during the early phases of acute infection. To test this hypothesis, we assessed the development of effector and memory CD8 T cells in transgenic mice expressing a single chain H-2D(d)/beta2-microglobulin (beta2M) fusion protein on a beta2M-deficient background. These mice thus express a single MHC class Ia in the absence of all other beta2M-dependent class Ia and Ib molecules. Following infection with a recombinant vaccinia virus expressing a known D(d)-restricted epitope from HIV-1 gp160, the development of effector and memory cells CD8 T cells was comparable to control mice. Furthermore, these memory cells responded rapidly and robustly to antigenic restimulation. Therefore, we conclude that full CD8 memory differentiation requires only a single MHC class Ia chain, ruling out a requirement for MHC class Ib molecules in this process.     

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.     

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.     

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.     

Adaptive immunity and enhanced CD8+ T cell response to Listeria monocytogenes in the absence of perforin and IFN-gamma

Single Ag-specific CD8+ T cells from IFN-gamma-deficient (GKO) or perforin-deficient (PKO) mice provide substantial immunity against murine infection with Listeria monocytogenes. To address the potential for redundancy between perforin and IFN-gamma as CD8+ T cell effector mechanisms, we generated perforin/IFN-gamma (PKO/GKO) double-deficient mice. PKO/GKO-derived CD8+ T cells specific for the immunodominant listeriolysin O (LLO91-99) epitope provide immunity to LM infection similar to that provided by Ag-matched wild-type (WT) CD8+ T cells in the liver but reduced in the spleen. Strikingly, polyclonal CD8+ T cells from immunized PKO/GKO mice were approximately 100-fold more potent in reducing bacterial numbers than the same number of polyclonal CD8+ T cells from immunized WT mice. This result is probably quantitative, because the frequency of the CD8+ T cell response against the immunodominant LLO91-99 epitope is >4.5-fold higher in PKO/GKO mice than WT mice at 7 days after identical immunizations. Moreover, PKO/GKO mice can be immunized by a single infection with attenuated Listeria to resist >80,000-fold higher challenges with virulent organisms than naive PKO/GKO mice. These data demonstrate that neither perforin nor IFN-gamma is required for the development or expression of adaptive immunity to LM. In addition, the results suggest the potential for perforin and IFN-gamma to regulate the magnitude of the CD8+ T cell response to infection.     

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 CD8+ T cells induced by peptide-pulsed dendritic cells confer protection against Mycobacterium tuberculosis

One of the oligopolymorphic MHC class Ib molecules, H2-M3, presents N-formylated peptides derived from bacteria. In this study, we tested the ability of an H2-M3-binding peptide, TB2, to induce protection in C57BL/6 mice against Mycobacterium tuberculosis. Immunization with bone marrow-derived dendritic cell (BMDC) pulsed with TB2 or a MHC class Ia-binding peptide, MPT64(190-198) elicited an expansion of Ag-specific CD8+ T cells in the spleen and the lung. The number of TB2-specific CD8+ T cells reached a peak on day 6, contracted with kinetics similar to MPT64(190-198)-specific CD8+ T cells and was maintained at an appreciable level for at least 60 days. The TB2-specific CD8+ T cells produced less effector cytokines but have stronger cytotoxic activity than MPT64(190-198)-specific CD8+ T cells. Mice immunized with TB2-pulsed BMDC as well as those with MPT64(190-198)-pulsed BMDC showed significant protection against an intratracheal challenge with M. tuberculosis H37Rv. However, histopathology of the lung in mice immunized with TB2-pulsed BMDC was different from mice immunized with MPT64(190-198)-pulsed BMDC. Our results suggest that immunization with BMDC pulsed with MHC class Ib-restricted peptides would be a useful vaccination strategy against M. tuberculosis.     

Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity.

This study shows that removal of a T cell subpopulation can evoke effective tumor immunity in otherwise nonresponding animals. Elimination of CD25-expressing T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, elicited potent immune responses to syngeneic tumors in vivo and eradicated them. The responses were mediated by tumor-specific CD8+ CTLs and tumor-nonspecific CD4-8- cytotoxic cells akin to NK cells. Furthermore, in vitro culture of CD25+4+ T cell-depleted splenic cell suspensions prepared from tumor-unsensitized normal mice led to spontaneous generation of similar CD4-8- cytotoxic cells capable of killing a broad spectrum of tumors; reconstitution of CD25+4+ T cells inhibited the generation. In this culture, self-reactive CD25-4+ T cells responding to self peptides/class II MHC complexes on APCs spontaneously proliferated upon removal of CD25+4+ T cells, secreting large amounts of IL-2. The IL-2 thus produced appeared to be responsible for the generation of CD4-8- NK cells as lymphokine-activated killer cells, because direct addition of an equivalent amount of IL-2 to the culture of CD4-8- cells generated similar lymphokine-activated killer/NK cells, whereas coculture of normal CD4-8- cells with CD25-4+ T cells from IL-2-deficient mice did not. Thus, removal of immunoregulatory CD25+4+ T cells can abrogate immunological unresponsiveness to syngeneic tumors in vivo and in vitro, leading to spontaneous development of tumor-specific effector cells as well as tumor-nonspecific ones. This novel way of evoking tumor immunity would help to devise effective immunotherapy for cancer in humans.     

A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including beta(2)-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8(+) T cell populations with an activated/memory CD44(high) phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.     

V beta T cell repertoire of CD8+ splenocytes selected on nonpolymorphic MHC class I molecules

In this work, we have studied the role of the MHC class Ib molecules in the selection and maintenance of CD8(+) T splenocytes. We have compared the CD8(+) T cell repertoires of wild-type, H-2K-deficient, H-2D-deficient, or double knockout C57BL/6 mice. We show that the different CD8(+) repertoires, selected either by class Ia and class Ib or by class Ib molecules only, use the various V alpha (AV) and V beta (BV) rearrangements in the same proportion and without biases in the CDR3 size distribution. Furthermore, we have estimated the size of the BV repertoire in the four different strains of mice. Interestingly, we have found that the BV repertoire size is proportional to the overall number of CD8(+) splenocytes. This observation implies that BV diversity is positively correlated with the number of CD8(+) cells, even when the number of CD8(+) splenocytes is dramatically reduced (90% in the double knockout mice).     

T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules

Despite being a major group of intracellular pathogens, the role of class I-restricted T cells in the clearance of Gram-negative bacteria is not resolved. Using a murine typhoid model, a role for class I-restricted T cells in the immune response to the Gram-negative pathogen Salmonella typhimurium is revealed. Class I-deficient beta2-microglobulin-/- mice show increased susceptibility to infection with S. typhimurium. Following infection, CD8+ CTLs specific for Salmonella-infected targets can be readily detected. The Salmonella-specific CTLs recognize infected H-2-mismatched targets, suggesting the involvement of shared class Ib molecules. Studies using transfectants expressing defined class Ia and class Ib molecules indicate the involvement of the class Ib molecule, Qa-1. Ab-blocking studies and the measurement of bacteria-specific CTL frequencies identified Qa-1 as a dominant restricting element. The Qa-1-restricted CTL recognition depends on TAP and proteasome functions. Surprisingly, Qa-1-restricted CTLs recognized cells infected with other closely related Gram-negative bacteria. Taken together, these observations indicate that Salmonella-specific CTLs recognize a cross-reactive epitope presented by Qa-1 molecules and, as such, may be novel targets for vaccine development.     

Priming for in vitro and in vivo anti-human T lymphotropic virus type 1 cellular immunity by virus-related protein reconstituted into liposome

In vitro and in vivo anti-human T lymphotropic virus type 1 (HTLV-1) cellular immunity was examined by immunizing rats with a truncated hybrid protein (228 amino acids) of gag and env of HTLV-1 produced by Escherichia coli. Animals were immunized with the hybrid protein reconstituted into mannan-derivative-coated liposomes (gag-env-lipo). In vitro sensitization with a HTLV-1-positive cell line, TARS-1, of spleen cells obtained from these animals generated killer cells specific for syngeneic HTLV-1-positive cells. No killer activity was generated when spleen cells were obtained from animals immunized with the hybrid protein alone, the liposome alone, or the hybrid protein reconstituted into conventional liposomes with no polysaccharide coating. Killer cells were CD8+ CTL restricted to MHC class I. Analysis of CD8+ and CD4+ subsets in spleens showed the existence of primed CD8+ T cells in animals immunized with gag-env-lipo. Rats immunized with gag-env-lipo displayed accelerated rejection of TARS-1 but not of two other HTLV-1-negative tumor lines. Injection of carrageenan into animals strongly inhibited generation of killer cells, which indicates the necessity of macrophages for priming of CD8+ T cells with gag-env-lipo. Injection of carrageenan also cancelled in vivo immunity against HTLV-1+ cells induced with gag-env-lipo. These results, taken together, indicate that exogenous protein reconstituted into appropriate liposomes can effectively prime MHC class I restricted CD8+ T cells in vivo with macrophage dependency.     

Antigen form influences induction and frequency of influenza-specific class I and class II MHC-restricted cytolytic T lymphocytes

The induction of class I and class II MHC-restricted CTL in response to different forms of A/JAP/57 influenza virus was compared. Splenocytes removed from influenza-immune BALB/c mice and stimulated in vitro with infected syngeneic splenocytes are mainly CD8+ (Lyt-2+) and specifically lyse infected Ia- and Ia+ target cells. To a lesser extent they also lyse non-infectious virus-pulsed Ia+ but not Ia- target cells. In contrast, syngeneic stimulators pulsed with non-infectious virus (exogenous Ag) induce effector T cells that specifically lyse both infected and non-infectious virus-pulsed Ia+ target cells. The cells present in this heterogeneous culture predominantly express the CD4 (L3T4) cell surface marker. Frequency analysis by limiting dilution of splenocytes derived directly from influenza-immune mice revealed a similar pattern of precursor induction: In vitro stimulation with infected splenocytes yielded primarily class I MHC-restricted CTL, whereas stimulation with non-infectious virus reciprocally induced primarily class II MHC-restricted CTL. Thus, the Ag form and consequently the intracellular route of viral Ag presentation profoundly influence the MHC restriction of CTL precursors induced.