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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.

Background: Identification of Staphylococcus aureus intracellularly in chronic rhinosinusitis (CRS) suggests an underlying cellular immunodeficiency. Supporting this, we have previously reported low CD8+ (cytotoxic) T-lymphocyte levels in a subpopulation of CRS patients and identified polymorphisms in the CD8A gene associated with CRS. In order to better understand the role of low CD8+ in CRS, we wished to determine the phenotype for CRS/Low CD8+ in comparison to that of conventional CRS., Methods: Sixty-seven low CD8+ CRS patients identified during investigation of CRS were compared for demographics, disease evolution, and bacteriology on endoscopic culture were compared with an existing population of 480 patients with CRS with nasal polyposis previously recruited for genetic association studies., Results: Mean level of CD8+ in the CRS/Low CD8+ population was 0.15 × 10(9)/L (range, 0.20-1.5 × 10(9)/L). There was no difference between both groups in terms of history of allergy, asthma, eczema, acetylsalicylic acid (ASA) intolerance or smoking. The bacteriology was similar between both groups (S. aureus: CRS/Low CD8+: 35%; CRS 32%, p = 0.643). Evolution of disease was somewhat milder in CRS/Low CD8+, with fewer patients requiring surgery, and first surgery performed at a more advanced age. However, antibiotic use was higher in CRS/Low CD8+. Subgroup analysis restricted to CRS with nasal polyposis (CRSwNP)/Low CD8 or CRS without nasal polyposis (CRSsNP)/Low CD8 phenotypes did not substantially alter these results., Conclusion: Low CD8+ levels are often identified in CRS patients; however, these patients have disease remarkably similar to those with conventional CRS. This suggests that immune deficiency, whether systemic or locally mediated, is well tolerated and may be present in other forms in CRS. CRS patients with low CD8+ levels may possibly require antibacterial therapies as part of ongoing management., (© 2014 ARS-AAOA, LLC.)

Following stimulation, memory T (T(M)) cells rapidly express many effector functions, a hallmark feature that allows them to provide protective immunity. Recent studies suggest that genes involved in this rapid recall response may maintain an open chromatin structure in resting T(M) cells via epigenetic modifications. However, these studies have mostly focused on a few loci, and the techniques used required a large number of cells. We have developed a flow cytometric assay measuring histone modifications in individual murine T cells in combination with lineage-specific markers. In this study, we show that the per-cell level of a marker of open chromatin, diacetylated histone H3 (diAcH3), increases as naive CD8(+) T cells develop into T(M) cells, demonstrating a novel correlation between the differentiation state of a CD8(+) T cell and its abundance of a specific histone modification. Furthermore, our results show that T(M) cells defective in rapid recall ability have less diAcH3 than their fully functional counterparts, indicating that the diAcH3 level of individual T(M) cells is a useful marker for assessing their functionality.

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.

Our previous studies in volunteers immunized with Salmonella enterica serovar Typhi (S. Typhi) have suggested an important role for CD8+ T cells in host defense. In this study we describe a novel subset of nonclassical human HLA-E-restricted S. Typhi-specific CD8+ T cells derived from PBMC of Ty21a typhoid vaccinees. CD3+CD8+CD4-CD56- T cells effectively killed S. Typhi-infected targets regardless of whether they share classical HLA class I molecules with them, by a FAS-independent, granule-dependent mechanism, as evidenced by induction of granzyme B release and the blocking effects of concanamycin and strontium ions. The expression of HLA-E Ags, but not CD1-a, -b, or -c, on the membrane of S. Typhi-infected targets rendered them susceptible to lysis. Moreover, anti-HLA-E Abs partially blocked these responses. We also demonstrated that presentation of S. Typhi Ags via HLA-E could stimulate IFN-gamma production. Increases in the net frequency of IFN-gamma spot-forming cells were observed in the presence of targets coated with peptides that contain S. Typhi GroEL HLA-E binding motifs. These results demonstrate that HLA-E binds nonamer peptides derived from bacterial proteins and trigger CD8+-mediated lysis and IFN-gamma production when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections.

Type 1 cell-mediated immunity might play an important role in protection from typhoid fever. We evaluated whether immunization with Salmonella enterica serovar Typhi (S. Typhi) strain CVD 908-htrA (a Delta aroC Delta aroD Delta htrA mutant), a leading live oral typhoid vaccine candidate, elicits specific CD4(+) and CD8(+) S. Typhi immune responses. Potent CTL responses and IFN-gamma secretion by CD8(+) T cells were detected following immunization with CVD 908-htrA in high (4.5 x 10(8) CFU) and low (5 x 10(7) CFU) dosages. S. Typhi-specific CTL were observed in six of eight vaccinees (four high and two low dose) after immunization. Mean increases in the frequency of IFN-gamma spot-forming cells (SFC) in the presence of S. Typhi-infected targets were 221 +/- 41 SFC/10(6) PBMC and 233 +/- 87 SFC/10(6) PBMC, in the high and low dose groups, respectively. Strong CD4(+) T cell responses were also observed. Increases in the IFN-gamma production to soluble S. Typhi flagella (STF) occurred in 82 and 38% of the volunteers who received the high and low doses, respectively. Robust correlations were observed between volunteers that responded with IFN-gamma SFC to stimulation with S. Typhi-infected cells and IFN-gamma released in response to stimulation with STF Ags (r = 0.822, p < 0.001) and between CTL and IFN-gamma production to STF (r = 0.818, p = 0.013). These data demonstrating the concomitant induction of both CD4- and CD8-mediated CMI are consistent with a significant role for type 1 immunity in controlling typhoid infection and support the continuing evaluation of CVD 908-htrA as a typhoid vaccine candidate.

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.

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.