Viral infections induce abundant numbers of senescent CD8 T cells.

Viral infections are often accompanied by extensive proliferation of reactive CD8 T cells. After a defined number of divisions, normal somatic cells enter a nonreplicative stage termed senescence. In the present study we have identified the inhibitory killer cell lectin-like receptor G1 (KLRG1) as a unique marker for replicative senescence of murine CD8 T cells. KLRG1 expression was induced in a substantial portion (30-60%) of CD8 T cells in C57BL/6 mice infected with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus, or vaccinia virus. Similarly, KLRG1 was found on a large fraction of LCMV gp33 peptide-specific TCR-transgenic (tg) effector and memory cells activated in vivo using an adoptive transfer model. Transfer experiments with CFSE-labeled TCR-tg cells into LCMV-infected hosts further indicated that induction of KLRG1 expression required an extensive number of cell divisions. Most importantly, KLRG1(+) TCR-tg effector/memory cells could efficiently lyse target cells and secrete cytokines, but were severely impaired in their ability to proliferate after Ag stimulation. Thus, this study demonstrates that senescent CD8 T cells are induced in abundant numbers during viral infections in vivo.

Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection.

Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.

Genomic structure, alternative splicing, and physical mapping of the killer cell lectin-like receptor G1 gene (KLRG1), the mouse homologue of MAFA.

The mouse killer cell lectin-like receptor G1 (KLRG1), the mouse homologue of the mast cell function-associated antigen (MAFA), is an inhibitory C-type lectin expressed on natural killer (NK) cells and activated CD8 T cells. Here we report the complete nucleotide sequence, alternatively spliced variants, and the physical mapping of the KLRG1 gene in the mouse. The gene spans about 13 kb and consists of five exons. Short interspersed repeats of the B1 and B2 family, a LINE-1-like element, and a (CTT)170 triplet repeat were found in intron sequences. In contrast to human KLRG1 and to the murine KLR family members, mouse KLRG1 locates outside the NK complex on Chromosome 6 between the genes encoding CD9 and CD4.

Identification of sequences in the human peptide transporter subunit TAP1 required for transporter associated with antigen processing (TAP) function.

The heterodimeric peptide transporter associated with antigen processing (TAP) consisting of the subunits TAP1 and TAP2 mediates the transport of cytosolic peptides into the lumen of the endoplasmic reticulum (ER). In order to accurately define domains required for peptide transporter function, a molecular approach based on the construction of a panel of human TAP1 mutants and their expression in TAP1(-/-) cells was employed. The characteristics and biological activity of the various TAP1 mutants were determined, and compared to that of wild-type TAP1 and TAP1(-/-) control cells. All mutant TAP1 proteins were localized in the ER and were capable of forming complexes with the TAP2 subunit. However, the TAP1 mutants analyzed transported peptides with different efficiencies and displayed a heterogeneous MHC class I surface expression pattern which was directly associated with their susceptibility to cytotoxic T lymphocyte-mediated lysis. Based on this study, the TAP1 mutants can be divided into three categories: those expressing a similar phenotype compared to TAP1(-/-) or wild-type TAP1 cells respectively, and those representing an intermediate phenotype in terms of peptide transport rate, MHC class I surface expression and immune recognition. Thus, the results provide evidence that specific regions in the TAP1 subunit are crucial for the proper processing and presentation of cytosolic antigens to MHC class I-restricted T cells, whereas others may play a minor role in this process.

Partial impairment of cytokine responses in Tyk2-deficient mice.

To assess the role of the Janus kinase (Jak) family member Tyk2, we have generated Tyk2-/- mice. In contrast to other Jaks, where inactivation leads to a complete loss of the respective cytokine receptor signal, Tyk2-/- mice display reduced responses to IFNalpha/beta and IL-12 and a selective deficiency in Stat3 activation in these pathways. Unexpectedly, IFNgamma signaling is also impaired in Tyk2-/- mice. Tyk2-/- macrophages fail to produce nitric oxide upon lipopolysaccharide induction. Tyk2-/- mice are unable to clear vaccinia virus and show a reduced T cell response after LCMV challenge. These data imply a selective contribution of Tyk2 to the signals triggered by various biological stimuli and cytokine receptors.

Differential requirement of perforin and IFN-gamma in CD8 T cell-mediated immune responses against B16.F10 melanoma cells expressing a viral antigen.

Perforin-mediated lysis and secretion of IFN-gamma belong to the key effector functions of CD8 T cells. To compare the anti-tumor activity of these two mechanisms, we used B16.F10 melanoma cells (B16GP33) expressing the cytotoxic T cell epitope GP33 and T cell receptor transgenic (TCR-tg) mice specific for GP33 and deficient in perforin or IFN-gamma. B16GP33 tumor cells, injected either i.v. to induce experimental metastases or s.c., were similarly controlled in both wild-type and perforin-deficient, but not in IFN-gamma-deficient TCR-tg mice. A similar result was obtained when the therapeutic efficacy of adoptively transferred TCR-tg effector cells from these mice was examined in the corresponding perforin- or IFN-gamma-deficient C57BL/6 hosts. Criss-cross experiments further revealed that IFN-gamma production by the host strongly influenced the efficiency of the adoptively transferred effector cells. In contrast to the potent ability of GP33-specific effector cells to mediate B16GP33 tumor regression without perforin, GP33-specific memory cells, induced with recombinant vaccinia virus expressing GP33, failed to control B16GP33 tumor growth in the absence of perforin. In conclusion, our data demonstrate a crucial role for IFN-gamma in B16GP33 tumor cell elimination in vivo and indicate a differential requirement of perforin by effector versus memory CD8 T cells in anti-tumor immunity.

Immunotherapy with dendritic cells and tumor major histocompatibility complex class I-derived peptides requires a high density of antigen on tumor cells.

Immunization with dendritic cells and unfractionated MHC class I-binding peptides derived from autologous tumor cells has been shown to induce effective antitumor immunity. However, the importance of the relative abundance of tumor peptides on the surface of tumor cells is not known. We have addressed this question using peptides isolated from three tumor cell lines transfected with a minigene encoding amino acids 33-41 of the lymphocytic choriomeningitis virus glycoprotein (LCMV(33-41)). The three cell lines expressed different levels of MHC class I molecules and had different abilities to stimulate proliferation of LCMV(33-41)-specific T cells in vitro. We found that antitumor immune responses were best elicited by immunizing mice with dendritic cells and synthetic LCMV(33-41) peptide. Peptide preparations from a given tumor cell line conferred protection against challenge with the same tumor cell line. However, protective immunity to a different tumor could be induced only if the cell line used for peptide preparation presented a high relative proportion of LCMV(33-41) in association with MHC class I. Our results suggest that multiple peptide epitopes are required for the induction of an effective antitumor immune response using MHC class I-binding peptides from tumor cells. Also, the ability to induce an antitumor immune response appears to correlate with the proportion, rather than the absolute amount, of tumor-specific peptide in the mixture used for immunization.

Break of T cell ignorance to a viral antigen in the liver induces hepatitis.

To study peripheral tolerance of CD8 T cells to a classically MHC-restricted peptide Ag expressed in hepatocytes, ALB1 transgenic (tg) mice expressing the CTL epitope GP33 of the lymphocytic choriomeningitis virus glycoprotein under control of the mouse albumin promoter were generated. ALB1 mice exclusively expressed the GP33 transgene in the liver and, at a 100- to 1000-fold lower level, in the thymus. TCR-tg mice specific for the GP33 epitope were used to directly follow GP33-specific T cells in vivo. These experiments revealed that 1) thymic expression of the GP33 transgene led to incomplete central deletion of TCR-tg cells; and 2) peripheral TCR-tg cells in ALB1 mice ignored the GP33 transgene expressed in hepatocytes. Ignorance of adoptively transferred TCR-tg cells in ALB1 mice was broken by infection with lymphocytic choriomeningitis virus, leading to induction of hepatitis in ALB1, but not in control, mice. Taken together, we have established a novel model of virus-induced CD8 T cell-mediated autoimmune hepatitis in mice and demonstrate that naive CD8 T cells may ignore Ags expressed in the liver.

CD8(+) T cells secreting type 2 lymphokines are defective in protection against viral infection.

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8(+) T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-gamma secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules.

Crucial role of TNF-alpha in CD8 T cell-mediated elimination of 3LL-A9 Lewis lung carcinoma cells in vivo.

The role of perforin, IFN-gamma, and TNF-alpha in anti-tumor CD8 T cell immunity was examined in a new tumor model using a CD8 T cell epitope (GP33) derived from lymphocytic choriomeningitis virus as a tumor-associated Ag. In contrast with parental 3LL-A9 (A9) Lewis lung carcinoma cells that progressively grow in C57BL/6 mice, s.c. injection of GP33-transfected A9GP33 tumor cells induced a protective GP33-specific CD8 T cell response that led to complete tumor cell elimination. Tumor regression was dependent on perforin, IFN-gamma, or TNF-alpha, because A9GP33 tumors developed in mice deficient in one of these genes. A9GP33 tumors arising in perforin- and IFN-gamma-deficient mice represented GP33 Ag-loss variants, demonstrating that GP33-specific CD8 T cells from these mice were able to exert an Ag selection pressure. In contrast, tumor cells growing in TNF-alpha knock-out mice still expressed the tumor-associated GP33 peptide despite the presence of activated GP33-specific CD8 T cells. These findings provide evidence for a crucial role of TNF-alpha in A9 tumor cell elimination by CD8 T cells in vivo.

Immune down-regulation and peripheral deletion of CD8 T cells does not require TNF receptor-ligand interactions nor CD95 (Fas, APO-1).

TNF receptor-ligand interactions and CD95 (Fas / APO-1) have been demonstrated to be involved in activation-induced death of mature T cells. Here, we examined the role of these molecules in the murine model of lymphocytic choriomeningitis virus (LCMV) infection using LCMV TCR transgenic (tg) mice lacking TNF, TNF receptor I (TNFR1), CD95 or both TNFR1 and CD95. This report demonstrates that neither TNF receptor-ligand interactions nor CD95 was required for down-regulation of LCMV-specific CD8 T cells following acute LCMV infection in vivo. Even LCMV-specific CD8 T cells lacking both TNFR1 and CD95 molecules declined after the acute phase of the infection with normal kinetics. Furthermore, peripheral deletion of LCMV-specific CD8 T cells induced by LCMV peptide injection or by adoptive transfer of tg spleen cells expressing the corresponding LCMV epitope was not impaired in mice lacking TNF, TNFR1 and / or CD95. Our data speak against an indispensable role of these molecules in antigen-induced apoptosis of CD8 T cells in vivo and suggest that T cell homeostasis after antigen challenge is controlled by additional mechanisms.

CpG-DNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses.

MHC class I-restricted T cell epitopes lack immunogenicity unless aided by IFA or CFA. In an attempt to circumvent the known inflammatory side effects of IFA and CFA, we analyzed the ability of immunostimulatory CpG-DNA to act as an adjuvant for MHC class I-restricted peptide epitopes. Using the immunodominant CD8 T cell epitopes, SIINFEKL from OVA or KAVYNFATM (gp33) from lymphocytic choriomeningitis virus glycoprotein, we observed that CpG-DNA conveyed immunogenicity to these epitopes leading to primary induction of peptide-specific CTL. Furthermore, vaccination with the lymphocytic choriomeningitis virus gp33 peptide triggered not only CTL but also protective antiviral defense. We also showed that MHC class I-restricted peptides are constitutively presented by immature dendritic cells (DC) within the draining lymph nodes but failed to induce CTL responses. The use of CpG-DNA as an adjuvant, however, initiated peptide presenting immature DC progression to professional licensed APC. Activated DC induced cytolytic CD8 T cells in wild-type mice and also mice deficient of Th cells or CD40 ligand. CpG-DNA thus incites CTL responses toward MHC class I-restricted T cell epitopes in a Th cell-independent manner. Overall, these results provide new insights into CpG-DNA-mediated adjuvanticity and may influence future vaccination strategies for infectious and perhaps tumor diseases.

Distinct migration patterns of naive and effector CD8 T cells in the spleen: correlation with CCR7 receptor expression and chemokine reactivity.

Changes in the migration pattern of lymphocytes represent a key event in the evolution of an immune response since they enable lymphocytes to gain access to infected tissues. We studied the location of virus-specific CD8 T cells in various splenic compartments in response to infection with lymphocytic choriomeningitis virus (LCMV), either in situ or by adoptive cell transfers using T cells from transgenic (tg) mice expressing an LCMV-specific TCR. Naive tg T cells were predominantly localized in the periarteriolar lymphoid sheath, where they proliferated extensively after virus infection. In contrast, in vivo activated effector T cells failed to enter white pulp areas and accumulated in the red pulp. The different homing patterns of naive and effector CD8 T cells in vivo correlated well with their CCR7 chemokine receptor expression and their reactivity to the secondary lymphoid tissue chemokine (SLC). Thus, down-regulation of CCR7 expression on CD8 effector T cells rendered them unre sponsive to SLC, which controls T cell homing into white pulp of spleen and lymph nodes. Exclusion of CD8 effector T cells from these sites may represent an important mechanism to protect professional antigen-presenting cells from cytotoxic T cell attack and thus to prevent a prematuredecline of the immune response.

T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein.

Infection of neonates with Borna disease virus (BDV) induces severe meningoencephalitis and neurological disorder in wild-type but not in beta(2)-microglobulin-deficient mice of strain MRL (H-2(k)). Temporary in vivo depletion of CD8(+) T cells delayed BDV-induced disease for several weeks. Depletion of CD4(+) T cells had a similar beneficial effect, indicating that the BDV-induced neurological disorder in mice is a CD4(+) T cell-dependent immunopathological process that is mediated by CD8(+) T cells. Lymphocytes prepared from brains of diseased mice were mainly from the CD8(+) T cell subset. They showed up-regulation of activation markers and exerted strong MHC I-restricted cytotoxic activity against target cells expressing the BDV nucleoprotein p40. Infection of B10.BR (H-2(k)) or congenic C57BL/10 (H-2(b)) mice resulted in symptomless, lifelong persistence of BDV in the brain. Superinfection with a recombinant vaccinia virus expressing BDV p40 but not with other vaccinia viruses induced severe neurological disease and encephalitis in persistently infected B10.BR mice but not in persistently infected C57BL/10 mice, indicating that the disease-inducing T cell response is restricted to the nucleoprotein of BDV in H-2(k) mice. Our results demonstrate that the cellular arm of the immune system may ignore the presence of a replicating virus in the central nervous system until proper antigenic stimulation at a peripheral site triggers the antiviral response.

Intrathymic deletion of MHC class I-restricted cytotoxic T cell precursors by constitutive cross-presentation of exogenous antigen.

Cross-priming of cytotoxic T lymphocytes by professional antigen-presenting cells (APC) is a potential hazard to self tolerance because it exposes naive T cells to tissue-specific self antigens in the context of co-stimulatory signals. Here we show that cross-presentation of exogenous material occurs constitutively within the thymus. Although efficient cross-presentation is a property of relatively few APC it results in thymocyte deletion both in vitro and in vivo, suggesting that intrathymic cross-presentation can operate as an effective component of tolerance to circulating self antigens. The capacity of minor cell populations to mediate thymocyte deletion but not positive selection reflects an underlying difference in the biology of these two processes.

A novel approach to visualize polyclonal virus-specific CD8 T cells in vivo.

Recent technical breakthroughs in generating soluble MHC class I-peptide tetramers now allow the direct visualization of virus-specific CD8 T cells after infection in vivo. However, this technique requires the knowledge of the immunodominant viral epitopes recognized by T cells. Here, we describe an alternative approach to visualize polyclonal virus-specific CD8 T cells in vivo using a simple adoptive transfer system. In our approach, C57BL/6 (Thy1.2) mice were infected with lymphocytic choriomeningitis virus, vesicular stomatitis virus, or vaccinia virus to induce virus-specific memory T cells. Tracer T cells (2 x 106) from these virus-immune mice were adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 mice. After infection of the F1-recipient mice with the appropriate virus, the transferred cells expanded vigorously, and on day 8 postinfection 60-80% of total CD8 T cells were of donor T cell origin. Under the same conditions memory CD4 T cells gave rise to at least 10 times less cell numbers than memory CD8 T cells. The transfer system described here not only allows to visualize effector and memory CD8 T cells in vivo but also to isolate them for further in vitro characterization without knowing the epitopes recognized by these Ag-specific CD8 T cells.

Identification of an alpha2,6-sialyltransferase induced early after lymphocyte activation.

We have used mRNA differential display PCR to search for genes induced in activated T cells and we identified a gene encoding an alpha2,6-sialyltransferase (ST6GalNAc IV) that is rapidly induced in lymphocytes after antigen or mitogen stimulation. The 3.6 kb full-length cDNA clone (MK45) obtained contained a single open reading frame encoding a 302 amino acid protein and a 2.5 kb 3' untranslated region. MK45 expression in in vivo-activated CD8 T cells reached the highest level 4 h after antigen triggering and then declined rapidly to nearly base levels within 45 h. Northern blot analysis further revealed that MK45 expression was also induced in LPS-activated B cells and antigen-triggered CD4 T cells in vitro. MK45 expression was low or undetectable in most other mouse tissues examined, when compared to activated lymphocytes. Importantly, the mRNA expression level of other sialyltransferases remained largely unchanged during the early stage of lymphocyte activation. Finally, increased ecto-sialyltransferase activity and an altered sialylation pattern were demonstrated on the cell surface of early activated CD8 T cells. Our report identifies a candidate sialyltransferase gene that is involved in the early alteration of the sialylation pattern of cell surface molecules in activated lymphocytes.

Molecular cloning and functional expression of two members of mouse NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2,6-sialyltransferase family, ST6GalNAc III and IV.

Two cDNA clones encoding NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2, 6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAcalpha2, 3Galbeta1, 3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Galbeta1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[14C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAcalpha2,6-sialyltransferase, which requires sialic acid residues linked to Galbeta1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.

Qualitative and quantitative requirements for CD4+ T cell-mediated antiviral protection.

CD4+ Th cells deliver the cognate and cytokine signals that promote the production of protective virus-neutralizing IgG by specific B cells and are also able to mediate direct antiviral effector functions. To quantitatively and qualitatively analyze the antiviral functions of CD4+ Th cells, we generated transgenic mice (tg7) expressing an MHC class II (I-Ab)-restricted TCR specific for a peptide derived from the glycoprotein (G) of vesicular stomatitis virus (VSV). The elevated precursor frequency of naive VSV-specific Th cells in tg7 mice led to a markedly accelerated and enhanced class switching to virus-neutralizing IgG after immunization with inactivated VSV. Furthermore, in contrast to nontransgenic controls, tg7 mice rapidly cleared a recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G) from peripheral organs. By adoptive transfer of naive tg7 CD4+ T cells into T cell-deficient recipients, we found that 105 transferred CD4+ T cells were sufficient to induce isotype switching after challenge with a suboptimal dose of inactivated VSV. In contrast, naive transgenic CD4+ T cells were unable to adoptively confer protection against peripheral infection with Vacc-IND-G. However, tg7 CD4+ T cells that had been primed in vitro with VSV-G peptide were able to adoptively transfer protection against Vacc-IND-G. These results demonstrate that the antiviral properties of CD4+ T cells are governed by the differentiation status of the CD4+ T cell and by the type of effector response required for virus elimination.

Kinetics of the response of naive and memory CD8 T cells to antigen: similarities and differences.

We have studied the kinetics of the antigen induced response of naive and memory CD8 T cells expressing a transgenic T cell receptor (TCR) specific for the glycoprotein peptide amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV). Memory T cells were generated in vivo by adoptive transfer of LCMV TCR transgenic T cells into normal recipient mice, followed by LCMV infection. The results demonstrated that the cell cycle progression and kinetics of TCR down-modulation, CD25 and CD69 up-regulation were identical in naive and memory T cells after antigen recognition. Moreover, the two T cell populations did not differ in respect of activation thresholds and in their proliferative capacities neither in vitro nor in vivo. However, memory CD8 T cells could be more rapidly induced to become cytolytic and to secrete high levels of interleukin-2 and interferon-gamma than naive T cells. LCMV GP33-specific CD8 memory T cells were only slightly more efficient in reducing LCMV titers in the spleen but were far more effective than naive LCMV GP33-specific T cells in controlling subcutaneous tumor growth of B16.F10 melanoma cells which expressed the LCMV GP33 epitope as tumor-associated antigen. Thus, in our experiments the main difference between CD8 memory T cells and naive cells is the ability of the former to rapidly acquire effector cell functions.