Search

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8 + T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR + PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR + LysoDCs) in PP and examine their role in CD8 + T cell immune induction. Stimulation of C5aR + LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8 + T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Legionella pneumophila , an intracellular bacterium, may cause life-threatening pneumonia in immunocompromised individuals. Mononuclear cells and antibodies have been reported to be associated with the host defense response against L. pneumophila. This study is to determine whether Legionella peptidoglycan-associated lipoprotein (PAL)-specific CD8 + T cells are directly associated with protection against L. pneumophila , with a focus on potential epitopes. Synthetic peptides derived from PAL of L. pneumophila were obtained and tested through in vitro and in vivo cytotoxic T lymphocyte (CTL) assays for immunogenicity. PAL DNA vaccines or a peptide epitope with or without CpG-oligodeoxynucleotides (ODN) was evaluated for protection against L. pneumophila infection in animal models. When mice were immunized with DNA vaccines expressing the PAL of L. pneumophila , they were significantly protected against a lethal challenge with L. pneumophila through induction of antigen-specific CD8 + CTLs. Of the 13 PAL peptides tested, PAL 92-100 (EYLKTHPGA) was the most immunogenic and induced the strongest CTL responses. When mice were immunized with the PAL 92-100 peptide plus CpG-ODN, they were protected against the lethal challenge, while control mice died within 3-6 days after the challenge. Consistent with lung tissue histological data, bacterial counts in the lungs of immunized mice were significantly lower than those in control mice. Also, the amino acid sequence of PAL 92-100 peptides is conserved among various Legionella species. To our knowledge, this study is the first to demonstrate that PAL 92-100 -specific CD8 + T cells play a central role in the host defense response against L. pneumophila ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Kim, Sin and Kim.)

Subversion of antigen-specific immune responses by intracellular pathogens is pivotal for successful colonisation. Bacterial pathogens, including Shigella, deliver effectors into host cells via the type III secretion system (T3SS) in order to manipulate host innate and adaptive immune responses, thereby promoting infection. However, the strategy for subverting antigen-specific immunity is not well understood. Here, we show that Shigella flexneri invasion plasmid antigen H (IpaH) 4.5, a member of the E3 ubiquitin ligase effector family, targets the proteasome regulatory particle non-ATPase 13 (RPN13) and induces its degradation via the ubiquitin-proteasome system (UPS). IpaH4.5-mediated RPN13 degradation causes dysfunction of the 19S regulatory particle (RP) in the 26S proteasome, inhibiting guidance of ubiquitinated proteins to the proteolytically active 20S core particle (CP) of 26S proteasome and thereby suppressing proteasome-catalysed peptide splicing. This, in turn, reduces antigen cross-presentation to CD8+ T cells via major histocompatibility complex (MHC) class I in vitro. In RPN13 knockout mouse embryonic fibroblasts (MEFs), loss of RPN13 suppressed CD8+ T cell priming during Shigella infection. Our results uncover the unique tactics employed by Shigella to dampen the antigen-specific cytotoxic T lymphocyte (CTL) response., (© 2018 John Wiley & Sons Ltd.)

Infection of specific pathogen-free mice with lymphocytic choriomeningitis virus (LCMV) is a widely used model to study antiviral T-cell immunity. Infections in the real world, however, are often accompanied by coinfections with unrelated pathogens. Here we show that in mice, systemic coinfection with E. coli suppresses the LCMV-specific cytotoxic T-lymphocyte (CTL) response and virus elimination in a NK cell- and TLR2/4-dependent manner. Soluble TLR4 ligand LPS also induces NK cell-mediated negative CTL regulation during LCMV infection. NK cells in LPS-treated mice suppress clonal expansion of LCMV-specific CTLs by a NKG2D- or NCR1-independent but perforin-dependent mechanism. These results suggest a TLR4-mediated immunoregulatory role of NK cells during viral-bacterial coinfections.

Commensal bacteria shape the colonic regulatory T (Treg) cell population required for intestinal tolerance. However, little is known about this process. Here, we use the transfer of naive commensal-reactive transgenic T cells expressing colonic Treg T cell receptors (TCRs) to study peripheral Treg (pTreg) cell development in normal hosts. We found that T cells were activated primarily in the distal mesenteric lymph node. Treg cell induction was rapid, generating >40% Foxp3(+) cells 1 week after transfer. Contrary to prior reports, Foxp3(+) cells underwent the most cell divisions, demonstrating that pTreg cell generation can be the dominant outcome from naive T cell activation. Moreover, Notch2-dependent, but not Batf3-dependent, dendritic cells were involved in Treg cell selection. Finally, neither deletion of the conserved nucleotide sequence 1 (CNS1) region in Foxp3 nor blockade of TGF-β (transforming growth factor-β)-receptor signaling completely abrogated Foxp3 induction. Thus, these data show that pTreg cell selection to commensal bacteria is rapid, is robust, and may be specified by TGF-β-independent signals., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Fungal infections have skyrocketed in immune-compromised patients lacking CD4+ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A+ CD8+ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4+ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ+ CD8+ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8+ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.

Natural killer cells play a crucial role in the initial defense against bacterial pathogens. The crosstalk between host cells infected with intracellular pathogens and NK cells has been studied intensively, but not much attention has been given to characterize the role of NK cells in the response to extracellular bacterial pathogens such as yersiniae. In this study we used antibody-mediated NK cell depletion to address the importance of this immune cell type in controlling a Y. pseudotuberculosis infection. Analysis of the bacterial counts was used to follow the infection and flow cytometry was performed to characterize the composition and dynamic of immune cells. Depletion of NK cells led to higher bacterial loads within the mesenteric lymph nodes. We further show that in particular CD11b+ CD27+ NK cells which express higher levels of the activation marker CD69 increase within the mesenteric lymph nodes during a Y. pseudotuberculosis infection. Moreover, in response to the activation NK cells secrete higher levels of IFNy, which in turn triggers the production of the proinflammatory cytokine TNFα. These results suggest, that NK cells aid in the clearance of Y. pseudotuberculosis infections mainly by triggering the expression of proinflammatory cytokines manipulating the host immune response.

Porcine ileum is populated with a high proportion of B cells but previous studies have shown that they are not developed there. While B cells prevail in the ileum even in germ-free animals, microbial colonization is a major factor that causes even a greater prevalence of B cells in the ileum and further differential representation of lymphoid cells throughout small intestine. Analysis of lymphoid subpopulations showed that the effector cells appear only after colonization. These include class-switched IgM(+)IgA(+) B cells, primed CD2(-)CD21(+) B cells, antibody-producing/memory CD2(+)CD21(-) B cells, and effector/memory CD4(+)CD8(+) αβ Th cells. While colonization resulted in a uniform distribution of effector cells throughout the gut, it caused a decrease in the frequency of cytotoxic αβ and CD2(+)CD8(+) γδ T cells. These results suggest that the ileum is a site where naive B cells expand presumably to increase antibody repertoire but the entire small intestine is immunofunctionally comparable., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To prevent the global spread of tuberculosis (TB), more effective vaccines and vaccination strategies are urgently needed. As a result of the success of bacillus Calmette-Guérin (BCG) in protecting children against miliary and meningeal TB, the majority of individuals will have been vaccinated with BCG; hence, boosting BCG-primed immunity will probably be a key component of future vaccine strategies. In this study, we compared the ability of DNA-, protein- and lentiviral vector-based vaccines that express the antigens Ag85B and Rv3425 to boost the effects of BCG in the context of immunity and protection against Mycobacterium tuberculosis in C57BL/6 mice. Our results demonstrated that prime-boost BCG vaccination with a lentiviral vector expressing the antigens Ag85B and Rv3425 significantly enhanced immune responses, including T helper type 1 and CD8(+) cytotoxic T lymphocyte responses, compared with DNA- and protein-based vaccines. However, lentivirus-vectored and DNA-based vaccines greatly improved the protective efficacy of BCG against M. tuberculosis, as indicated by a lack of weight loss and significantly reduced bacterial loads and histological damage in the lung. Our study suggests that the use of lentiviral or DNA vaccines containing the antigens Ag85B and Rv3425 to boost BCG is a good choice for the rational design of an efficient vaccination strategy against TB., (© 2014 John Wiley & Sons Ltd.)

Despite the importance and success of vaccine immunization against bacterial diseases in fish, little is known about the molecular mechanisms of vaccine-induced immune protection in teleost fish. In this study, the live attenuated Edwardsiella tarda vaccine strain WED, which has been shown to evoke efficacious protection against edwardsiellosis and ascites diseases in fish, was extensively evaluated for multiple parameters in a 5-week immunization and challenge experiment in zebrafish. The parameters evaluated included the immunologic potency (relative percent survival, RPS), the specific IgM antibody titers and the expression profiles of multiple immune-related gene markers at multiple time points following immunization and challenge. During the 4-week immunization phase, the toll-like receptor (TLR) 5 signaling pathway, the MHC-I antigen processing pathway and cytotoxic T lymphocyte (CTL) responses were activated in succession. In contrast, the MHC-II antigen processing pathway and the markers of CD4(+) T lymphocyte activation were down-regulated, and IgM transcription and specific IgM antibody titers were not significantly induced following immunization. During the 1-week challenge phase, the induction of MHC-I and CTL responses and the inhibition of MHC-II and CD4(+) T cell responses were similarly observed in immunized zebrafish following challenge with wild E. tarda. With the 5-week immunization and challenge model, our data suggest the basic mechanism that underlying the long-lasting protective immunity elicited by WED in zebrafish. This mechanism involved the induction of the TLR-5 signaling pathway, the MHC-I antigen processing pathway and CTL effector function, and CTL function seems play a major role in the protection against E. tarda infection in zebrafish., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Growing evidence has shown that regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) abnormally increase in cancer cachectic patients. Suppressions of Tregs and MDSCs may enhance anti-tumor immunity for cancer patients. Fish oil and selenium have been known to have many biological activities such as anti-inflammation and anti-oxidation. Whether fish oil and/or selenium have an additional effect on population of immunosuppressive cells in tumor-bearing hosts remained elusive and controversial. To gain insights into their roles on anti-tumor immunity, we studied the fish oil- and/or selenium-mediated tumor suppression and immunity on lung carcinoma, whereof cachexia develops. Advancement of cachexia in a murine lung cancer model manifested with such indicative symptoms as weight loss, chronic inflammation and disturbed immune functionality. The elevation of Tregs and MDSCs in spleens of tumor-bearing mice was positively correlated with tumor burdens. Consumption of either fish oil or selenium had little or no effect on the levels of Tregs and MDSCs. However, consumption of both fish oil and selenium together presented a synergistic effect--the population of Tregs and MDSCs decreased as opposed to increase of anti-tumor immunity when both fish oil and selenium were supplemented simultaneously, whereby losses of body weight and muscle/fat mass were alleviated significantly.

Brucella abortus is a Gram-negative, facultative intracellular pathogen for several mammals, including humans. Live attenuated B. abortus strain RB51 is currently the official vaccine used against bovine brucellosis in the United States and several other countries. Overexpression of protective B. abortus antigen Cu/Zn superoxide dismutase (SOD) in a recombinant strain of RB51 (strain RB51SOD) significantly increases its vaccine efficacy against virulent B. abortus challenge in a mouse model. An attempt has been made to better understand the mechanism of the enhanced protective immunity of RB51SOD compared to its parent strain RB51. We previously reported that RB51SOD stimulated enhanced Th1 immune response. In this study, we further found that T effector cells derived from RB51SOD-immunized mice exhibited significantly higher cytotoxic T lymphocyte activity than T effector cells derived from RB51-immunized mice against virulent B. abortus-infected target cells. Meanwhile, the macrophage responses to these two strains were also studied. Compared to RB51, RB51SOD cells had a lower survival rate in macrophages and induced lower levels of macrophage apoptosis and necrosis. The decreased survival of RB51SOD cells correlates with the higher sensitivity of RB51SOD, compared to RB51, to the bactericidal action of either Polymyxin B or sodium dodecyl sulfate (SDS). Furthermore, a physical damage to the outer membrane of RB51SOD was observed by electron microscopy. Possibly due to the physical damage, overexpressed Cu/Zn SOD in RB51SOD was found to be released into the bacterial cell culture medium. Therefore, the stronger adaptive immunity induced by RB51SOD did not correlate with the low level of innate immunity induced by RB51SOD compared to RB51. This unique and apparently contradictory profile is likely associated with the differences in outer membrane integrity and Cu/Zn SOD release.

Cytotoxic T-lymphocytes (CTL) are famous for their ability to kill tumor, allogeneic and virus-infected cells. However, an emerging literature has now demonstrated that CTL also possess the ability to directly recognize and kill bacteria, parasites, and fungi. Here, we review past and recent findings demonstrating the direct microbicidal activity of both CD4(+) and CD8(+) CTL against various microbial pathogens. Further, this review will outline what is known regarding the mechanisms of direct killing and their underlying signalling pathways.

Autoimmune diseases tend to be chronic and progressive, but how these responses are sustained is not clear. One cell type that might contribute to autoimmunity is the cytotoxic T lymphocyte (CTL), which, as a consequence of causing tissue destruction and production of cytokines, could provide a sustained supply of antigen and inflammatory signals for dendritic cells to maintain immune stimulation. Here we examined whether such CTL-mediated tissue damage alone could provide antigen in the right context to recruit immune effectors and sustain autoimmunity. We show that while CTL-mediated tissue damage caused the release of self-antigens that stimulated the proliferation of naive autoreactive CD8(+) T cells, such responses failed to precipitate disease and, instead, led to deletional tolerance. These findings indicate that despite the capacity of CTLs to produce inflammatory cytokines and to cause tissue damage, their responses are not sustaining, but instead favor induction of self-tolerance.

Previously, a peptide epitope derived from the Hsp 60 molecule of Salmonella that is presented by the major histocompatibility complex (MHC) class Ib molecule Qa-1 to CD8(+) cytotoxic T cells (CTLs) was described. In the present study we investigated the Salmonella-induced processing and presentation pathway for generating this Qa-1-restricted epitope. Live bacteria and, to a lesser extent, opsonized heat-killed bacteria are able to sensitize target cells for lysis by Salmonella-specific CTL. In contrast, heat-killed bacteria cannot sensitize target cells. Presentation of the Hsp 60 epitope appears independent of bacterial internalization, because cytochalasin D does not affect presentation. Moreover, Salmonella strains defective in the InvA or InvE operon, two critical components of the type III secretion pathway, are as efficient as wild-type Salmonella enterica serovar Typhimurium in sensitizing infected targets to lysis. Collectively, these results suggest the existence of a novel antigen-processing pathway in which exogenous antigens gain access to the cytosolic MHC class I processing machinery. Considering the abundant nature of bacterial Hsp 60 and the upregulation of this protein after Salmonella infection of eukaryotic cells, this mode of antigen presentation may be particularly relevant to understanding the host defense mechanisms against gram-negative bacteria.

The role of HLA-A*6802-restricted CD8+ T cells in chlamydial disease was investigated in human ocular infections. Peptides with predicted binding motifs for HLA-A*6802 were synthesized using sequences based on chlamydial antigens, major outer membrane protein (MOMP), macrophage infectivity potentiator (MIP) and heat shock protein (hsp70). Peptides were pooled according to Chlamydia trachomatis protein type and serovar, and were tested in 51Cr-release cytotoxic T lymphocyte (CTL) and enzyme-linked immunospot (ELISPOT) assays, using peripheral blood mononuclear cells (PBMC) isolated from subjects living in trachoma-endemic communities in The Gambia. Significant CTL activity or interferon-gamma release was not detected in any of the subjects, suggesting either that HLA-A*6802 CD8+ T cells may not be important in ocular infections or that the peptides chosen did not represent epitopes.

Infection of BALB/c mice with Listeria monocytogenes stimulates an antilisterial immune response evident by the appearance of H2-Kd-restricted CD8(+) cytotoxic T lymphocytes (CTLs) specific for the nanomer peptides amino acids (aa) 91 to 99 of listeriolysin O (LLO 91-99) and aa 217 to 225 of the p60 molecule (p60 217-225). We have introduced point mutations at anchor residues within LLO 91-99 (92F) or p60 217-225 (218F), and BALB/c mice infected with L. monocytogenes strains containing these point mutations do not develop CTLs specific for LLO 91-99 or p60 217-225, respectively. We have used these strains to test whether primary CTL responses against L. monocytogenes-derived determinants can be stimulated within an environment of existing antilisterial immunity. We found that the development of a primary L. monocytogenes-specific CTL response is not altered by existing immunity to L. monocytogenes. For example, primary immunization with the p60 218F strain of L. monocytogenes followed by a secondary immunization with wild-type L. monocytogenes results in stimulation of p60 217-225-specific CTLs at primary response levels and LLO 91-99-specific effectors at levels consistent with a memory CTL response. Similarly, primary immunization with the 92F strain of L. monocytogenes followed by a secondary immunization with wild-type L. monocytogenes results in stimulation of LLO 91-99-specific CTLs at primary response levels and p60 217-225-specific effectors at levels consistent with a memory CTL response. These results provide additional support for the use of L. monocytogenes as a recombinant vaccine vector and show that antivector immunity does not inhibit the development of a primary CTL response when the epitope is delivered by L. monocytogenes as the vaccine strain.

Protective immunity to infection by many intracellular pathogens requires recognition by cytotoxic T lymphocytes (CTLs) of antigens presented on major histocompatibility complex (MHC) class I molecules. To be presented for recognition by pathogen-specific CTLs, these antigens must gain access to the host cell class I processing pathway. In the case of intracellular bacterial pathogens, the majority of bacterial proteins are retained within the bacterial membrane and therefore remain inaccessible to the host cell for antigen processing. We have isolated a CTL clone from a C57BL/6 mouse infected with the intracellular gram-positive bacterium Listeria monocytogenes (LM) and have identified the source of the antigen. Using a genomic expression library, we determined that the clone recognizes an antigenic N-formyl peptide presented by the nonpolymorphic murine MHC class Ib molecule, H2-M3. Several lengths of this peptide were able to sensitize cells for lysis by this CTL clone. The source of this antigenic peptide is a 23-amino acid polypeptide encoded at the start of a polycistronic region. Analysis of mRNA secondary structure of this region suggests that this polypeptide may be a leader peptide encoded by a transcriptional attenuator.

In enterobacteria-induced reactive arthritis (ReA), different T cell subsets play a role in the induction and maintenance of the synovitic process. Synovial fluid-derived alphabeta CD4, alphabeta CD8, and gammadelta T lymphocyte clones (TLC) that recognize Yersinia or Salmonella antigens on professional antigen-presenting cells (APC) have been characterized, and T cells themselves can function as nonprofessional APC. T cells were infected with the facultatively intracellular, arthritogenic enterobacterium Yersinia enterocolitica O:3. A CD8 TLC isolated from a patient with Yersinia-induced ReA recognized and efficiently lysed autologous and allogeneic Yersinia-infected T cells. Infected cytotoxic T lymphocytes (CTL) had a reduced lytic capacity against syngeneic and allogeneic infected target cells, suggesting that the infection of CTL by bacteria may represent a mechanism of immune escape. In ReA, antigen presentation by T cells may modify the antibacterial immune response and may also contribute to network control mechanisms of T cell-mediated cytotoxicity.

CD8+ T cells are essential for protection against mycobacteria, as is clearly demonstrated by the fatal outcome of experimental infection of beta-2 microglobulin knockout mice. However, the mechanisms and antigens (Ags) leading to CD8+ T-cell activation and regulation have been poorly characterized. Here we show that, upon immunization of major histocompatibility complex (MHC)-congenic mice with Mycobacterium bovis bacillus Calmette-Guérin (BCG), a cytotoxic response against BCG culture filtrate (CF) Ags (CFAgs) is induced in H-2b and H-2bxd haplotypes but not in H-2d haplotype. This response is mediated by CD8+ T cells and absolutely requires the activation of CD4+ T cells and their secretion of interleukin 2. The lack of cytotoxic response in H-2d mice cannot be explained by impaired cytokine production or by a defect in Ag presentation by H-2d macrophages. Using the MHC class I mutant B6.C-H-2bm13 mouse strain, we demonstrate that cytotoxic T lymphocytes (CTLs) recognize CFAgs exclusively in association with D(b) molecules. These Ags are cross-reactive in mycobacteria, since BCG-induced CTLs also recognize macrophages pulsed with CF from Mycobacterium tuberculosis H37Rv and H37Ra and from two virulent strains of M. bovis. Moreover, immunization with Mycobacterium kansasii induces CTLs able to lyse macrophages pulsed with BCG CF. Finally, we have found that these Ags can be characterized as hydrophilic proteins, since they do not bind to phenyl-Sepharose CL-4B. Our results indicate that MHC-linked genes exert a profound influence on the generation of CD8+ CTLs following BCG vaccination.

The effect of herpes simplex virus type 1 (HSV-1) infection on human cytotoxic T-lymphocyte (CTL) lytic function was assessed. All HSV-infected CTL populations tested were significantly inhibited in lysing target cells. The inhibition of CTL lytic function by infection with HSV-1 was independent of T-cell receptor-mediated antigen recognition and did not involve virus-induced shutoff of host protein synthesis, the expression of the HSV-1 transactivation protein, ICP4, or replicating virus. Understanding the functional impairment of CTL following infection with HSV may have important implications for HSV-induced immunosuppression and the mechanism of HSV persistence in immunocompetent hosts.

During hepatitis B virus (HBV) infection, distinct host-virus interactions may establish the patterns of viral clearance and persistence and the extent of virus-associated pathology. It is generally thought that HBV-specific class I-restricted cytotoxic T lymphocytes (CTLs) play a critical role in this process by destroying infected hepatocytes. This cytopathic mechanism, however, could be lethal if most of the hepatocytes are infected. In the current study, we demonstrate that class I-restricted HBV-specific CTLs profoundly suppress hepatocellular HBV gene expression in HBV transgenic mice by a noncytolytic process, the strength of which greatly exceeds the cytopathic effect of the CTLs in magnitude and duration. We also show that the regulatory effect of the CTLs is initially mediated by interferon gamma and tumor necrosis factor alpha, is delayed in onset, and becomes independent of these cytokines shortly after it begins. The data indicate that the anti-viral CTL response activates a complex regulatory cascade that inhibits hepatocellular HBV gene expression without killing the cell. The extent to which this mechanism contributes to viral clearance or viral persistence during HBV infection remains to be determined.

We previously reported that the clone JK34 was cross-reactive for dengue virus types 1, 2, 3, and 4 and recognized NS3 (I. Kurane, M. A. Brinton, A. L. Samson, and F. A. Ennis, J. Virol. 65:1823-1828, 1991). In the present experiments, we defined the epitope at the amino acid level, with 93 15-mer overlapping peptides which cover the entire NS3. A peptide 4 which contains amino acids 251 to 265 of NS3 sensitized the autologous B lymphoblastoid cell line (LCL) to the lysis by JK34. The smallest peptide recognized by JK34 was a 10-mer peptide which contains amino acids 255 to 264 (EIVDLMCHAT). A monoclonal antibody to HLA-DP inhibited the lysis of epitope peptide-pulsed autologous LCL by JK34. Genotypic typing revealed that the HLA-DP of this donor is DPA1*01, DPB1*0201, which is serologically defined as HLA-DPw2. JK34 lysed peptide 4-pulsed allogeneic LCL which carried HLA-DPw2. These results indicate that HLA-DPw2 is the restriction allele for recognition of this epitope by JK34.

Herpesvirus saimiri (HVS) was used to infect and transform human CD8+ cytotoxic T lymphocytes (CTL), and the phenotypic and functional consequences of HVS infection of CD8+ T lymphocytes were investigated. HVS-transformed CTL no longer require antigen restimulation yet maintain their phenotype and HLA-restricted cytolytic function and specificity. The ability of HVS to transform CTL may have an important role in the functional analysis of human antigen-specific CTL.

The AKXL-5 recombinant inbred mouse strain is positive for the endogenous ecotropic murine leukemia virus emv-14, the only emv present in its germ line. emv-14 is of particular interest because spleen cells expressing emv-14 virus escape recognition by anti-AKR/Gross virus-specific cytotoxic T lymphocytes. We report here the isolation and characterization of a replication-competent emv clone, pAK7, derived from an AKXL-5 mouse. This clone is novel in that it encodes a variant ecotropic murine leukemia virus that, when expressed in SC.Kb target cells, fails to be recognized efficiently by anti-AKR/Gross virus cytotoxic T lymphocytes. The pAK7 clone can therefore be used to further probe mechanisms of escape from cell-mediated immunity.

We have examined the in vitro induction and activity of feline immunodeficiency virus (FIV)-specific cytolytic T cells obtained from cats experimentally infected for 7 to 17 weeks or 20 to 22 months with the Petaluma isolate of FIV. Normal or FIV-infected autologous and allogeneic T lymphoblastoid cells were used as target cells in chromium-51 or indium-111 release assays. When effector cells consisted of either fresh peripheral blood mononuclear cells or concanavalin A- and interleukin-2-stimulated cells, only low levels of cytotoxicity were observed. However, the levels of FIV-specific cytotoxicity were consistently higher in both groups of cats following in vitro stimulation of the effector cells with irradiated, FIV-infected autologous T lymphoblastoid cells and interleukin-2. The effector cells lysed autologous but not allogeneic FIV-infected target cells and were composed predominantly of CD8+ T cells, indicating that the FIV-specific cytotoxicity measured in this system is mediated by CD8+, major histocompatibility complex class I-restricted T cells. These studies show that FIV-specific cytolytic T cells can be detected as early as 7 to 9 weeks postinfection, and they define a system to identify virus-encoded epitopes important in the induction of protective immunity against lentiviruses.

In a longitudinal study of HIV seropositive patients, there were fluctuations in the specificity of cytotoxic T cells for the virus. This was matched by variability in proviral gag DNA epitope sequences in the lymphocytes of these patients. Some of these viral variants are not recognized by autologous T cells. Accumulation of such mutations in T-cell antigenic targets would provide a mechanism for immune escape.

A series of recombinant vaccinia viruses expressing various parts of the entire Kunjin virus (KUN) coding region was used to analyse the cytotoxic T (Tc) cell responses to KUN. CBA/H mice inoculated with KUN or West Nile virus were shown to develop responses to KUN or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the Tc cells in vitro with KUN antigens. Tc cells from CBA mice showed the strongest response to target cells infected with recombinant vaccinia viruses expressing parts of the KUN NS3 and NS4A proteins, and only a weak response to the other structural or non-structural proteins. Further analysis of deleted versions of the NS3-NS4A region showed that the main epitope recognized was derived from a sequence of 99 amino acids spanning parts of NS3 and NS4A. No other major epitopes were detected by Tc cells from CBA mice in the remaining 3333 amino acids of the KUN polypeptide.

The identity of herpes simplex virus type 1 (HSV-1) antigens that serve as targets for cytotoxic T lymphocytes (CTL) and their ability to induce protective immunity remain uncertain. In this article, we report the identification of the immediate-early protein ICP27 as a CTL antigen in H-2d mice but not in H-2k or H-2b mice. Calculation of the frequencies of H-2d-restricted virus-specific CTL demonstrated that approximately one-fourth of the total HSV-1-specific response was directed against ICP27. To define the location of this CTL epitope, four truncated derivatives of the ICP27 gene which place the epitope in a 217-amino-acid region (amino acids 189 to 406) near the central portion of the protein were constructed. Mice immunized with ICP27 were able both to induce HSV-1-specific CTL and to survive a lethal intraperitoneal challenge with virulent HSV-1. However, neither appreciable antibody nor delayed-type hypersensitivity responses were induced in immunized mice, and they were also unable to clear a local epithelial virus challenge. It appears that ICP27, although capable of inducing several aspects of the immune response, is by itself unable to provide complete immunity.

Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype.

Bovine leukosis virus (BLV) is associated with the disease complex enzootic bovine leukosis. The infection may remain clinically silent in the form of an aleukaemic state or emerge as a persistent lymphocytosis and more rarely as lymphosarcroma. BLV has been considered classically to be a B lymphotropic virus, based upon the absolute increase in B lymphocytes in persistent lymphocytosis, the B lymphocyte phenotype of a majority of the cells making up lymphosarcomas and the identification of viral antigen expressed in B lymphocytes following in vitro culture of peripheral blood mononuclear leukocytes. This association of BLV with B lymphocytes is well established but the mechanism(s) of disease expression is not defined. To examine further the cellular tropism(s) of BLV, T lymphocyte subpopulations from 10 lymphocytotic cattle were established in vitro. Lymphocyte cultures were characterized by their subpopulation phenotype and DNA was extracted for identification of integrated provirus by Southern blot hybridization. Provirus was identified in T lymphocyte cultures derived from seven of 10 lymphocytotic cattle, with both T helper and T cytotoxic/suppressor subpopulations affected.

Virus-immune cytotoxic T cells can inhibit effectively growth of vaccinia virus in acutely infected target cells in vitro by destroying infected target cells before infectious virus progeny is assembled. Together with the fact that virus-specific T cells are demonstrable after 3 days, very early during infection, and with strong circumstantial evidence from adoptive transfer models in vivo, these data suggest that in some virus infections T cells may in fact act cytolytically in vivo to prevent virus growth and spread and be an important early antiviral effector mechanism.

The ability of herpes simplex virus type 1 to productively infect human T-cell subpopulations was examined. Unstimulated helper/inducer (T4+) and cytotoxic/suppressor (T8+) lymphocytes limited herpes simplex virus replication as effectively as unseparated peripheral blood T cells (T3+). Phytohemagglutinin stimulation before infection resulted in equivalently productive herpes simplex virus infections in the three cell fractions.

Human T-cell leukemia-lymphoma virus (HTLV) is a human C-type retrovirus that can transform T lymphocytes in vitro and is associated with certain T-cell neoplasms. Recent data suggest that, in the United States, patients with acquired immunodeficiency syndrome (AIDS), homosexual men with lymphadenopathy, and hemophiliacs have had significant exposure rates to HTLV, whereas matched and unmatched control American subjects have rarely been exposed to this agent. In the present experiments, T cells specifically reactive against HTLV were propagated from a patient whose HTLV-bearing lymphoma was in remission. The T cells were cloned in the presence of the virus and an HTLV-specific cytotoxic T-cell clone was isolated. This clone was infected and transformed by the virus, with one copy of an HTLV-I provirus being integrated into the genome. This T-cell clone did not exhibit the normal dependence on T-cell growth factor (interleukin-2) and proliferated spontaneously in vitro. Exposure of the clone to HTLV-bearing, autologous tumor cells specifically inhibited its proliferation and resulted in its death. These results may have implications for HTLV-associated inhibition of T-cell responses.

The small chain of influenza virus haemagglutinin, HA2 was isolated by a selective enzymic removal of HA1 or by preparative SDS-polyacrylamide gel electrophoresis. Anti-HA2 specific antisera and monoclonal antibodies were subtype-specific in immunodiffusion tests and radioimmunoassays. These antibodies did not inhibit haemagglutination or haemolysis, did not prevent virus release, did not neutralize infectivity, and HA2 did not induce a protective immunity. HA2-specific antigenic determinants could not be demonstrated on the surface of infected cells. Lymphocytes from pre-immunized mice could not be stimulated by HA2 to exert a cytotoxic effect.

The human T-cell leukemia virus type I (HTLV-I) is capable of chronically infecting various types of T cells and nonlymphoid cells. The effects of chronic infection on the specific functional activities and growth requirements of mature cytotoxic T lymphocytes (CTL) have remained poorly defined. We have, therefore, investigated the results of HTLV-I infection of both CD4+ and CD8+ human CTL clones. HTLV-I infection resulted in the establishment of functional CTL lines which propagated indefinitely in culture many months longer than the uninfected parental clone. The infected cells became independent of the need for antigen (target cell) stimulation as a requirement for proliferation and growth. Like their uninfected counterparts, however, these HTLV-I-infected clones remained strictly dependent on conditioned medium from mitogen-stimulated T lymphocytes for their growth. This growth factor requirement was not fulfilled by recombinant interleukin-2 alone. Furthermore, the infected lines remained functionally identical to their uninfected parental CTL clones in their ability to specifically recognize and lyse the appropriate target cells. Our findings indicate that the major effects of HTLV-I infection on mature CTL consist of (i) the capacity for proliferation in the absence of antigen stimulation and (ii) a prolonged or immortal survival in vitro, but they also indicate that the fine specificity and cytolytic capacity of these cells remain unaffected.

We have investigated virus-lymphocyte interactions by using cloned subpopulations of interleukin-2-dependent effector lymphocytes maintained in vitro. Cloned lines of H-2-restricted hapten- or virus-specific cytotoxic T lymphocytes (CTL) and alloantigen-specific CTL were resistant to productive infection by vesicular stomatitis virus (VSV). In contrast, cloned lines of natural killer (NK) cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral surface antigen, and produce infectious virus. Furthermore, persistently infected NK cells showed no marked alteration of normal cellular morphology and continued to lyse NK-sensitive target cells albeit at a slightly but significantly reduced level. The persistence of VSV in NK cells did not appear to be caused by the generation of temperature-sensitive viral mutants, defective interfering particles, or interferon. Consequently, studies comparing the intracellular synthesis and maturation of VSV proteins in infected NK and mouse L cells were conducted. In contrast to L cells, in which host cell protein synthesis was essentially totally inhibited by infection, the infection of NK cells caused no marked diminution in the synthesis of host cell proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of viral proteins from infected cells showed that the maturation rate and size of VSV surface G glycoprotein were comparable in L cells and NK cells. Nucleocapsid (N) protein synthesis also appeared to be unaffected in NK cells. In contrast, the viral proteins NS and M appeared to be selectively degraded in NK cell extracts. Mixing experiments suggested that a protease in NK cells was responsible for the selective breakdown of VSV NS protein. Finally, VSV-infected NK cells were resistant to lysis by virus-specific CTL, suggesting that persistently infected NK cells may harbor virus and avoid cell-mediated immune destruction in an immunocompetent host.

We studied the mechanism of lymphocytic choriomeningitis virus (LCMV) persistence and the suppression of cytotoxic T lymphocyte (CTL) responses in BALB/c WEHI mice infected at birth with LCMV Armstrong strain. Using adoptive transfer experiments we found that spleen cells from persistently infected (carrier) mice actively suppressed the expected LCMV-specific CTL response of spleen cells from normal adult mice. The suppression was specific for the CTL response and LCMV -specific antibody responses were not affected. Associated with the specific CTL suppression was the establishment of persistent LCMV infection. The transfer of spleen or lymph node cells containing LCMV -specific CTL resulted in virus clearance and prevented establishment of the carrier state. The suppression of LCMV -specific CTL responses by carrier spleen cells is not mediated by a suppressor cell, but is due to the presence of genetic variants of LCMV in spleens of carrier mice. Such virus variants selectively suppress LCMV-specific CTL responses and cause persistent infections in immunocompetent mice. In striking contrast, wild-type LCMV Armstrong, from which these variants were generated, induces a potent CTL response in immunocompetent mice and the LCMV infection is rapidly cleared. Our results show that LCMV variants that emerge during infection in vivo play a crucial role in the suppression of virus-specific CTL responses and in the maintenance of virus persistence.

The cytotoxic T-cell (Tc) response to varicella-zoster virus (VZV) is incompletely characterized. We investigated whether VZV-specific Tc restricted by class I products of the major histocompatibility complex can be generated from the peripheral blood of VZV-immune donors. Cell lines were established from peripheral blood lymphocytes (PBL) of seropositive donors by secondary in vitro restimulation. If cell-free VZV was used as the stimulating antigen, the resulting lines were predominantly CD4+ and did not show class I-restricted cytotoxicity; when autologous infected fibroblasts were used for in vitro stimulation, the resultant lines were usually cytotoxic, although in only 4 of 11 subjects tested was this cytotoxicity HLA restricted and virus specific. PBL were also tested for Tc activity without prior restimulation; VZV-specific Tc activity was only demonstrable in the PBL of a subject convalescent following zoster but not from subjects with recent varicella infection or from normal subjects. VZV-specific Tc precursor frequencies were then determined in six selected subjects by limiting-dilution analysis. A measurable frequency was detectable in four of the six seropositive subjects, ranging from 11/10(6) T cells in an asymptomatic carrier, to 63/10(6) T cells in a subject with recent zoster. We conclude that virus-specific major histocompatibility complex class I-restricted Tc precursors may be present in the peripheral blood of normal individuals seropositive for VZV but at a frequency lower than that for other herpesviruses with nonneuronal sites of latency.