Cell IFN-γ Responses Research Articles

CD8 T cells in innate immune responses: using STAT4-dependent but antigen-independent pathways to gamma interferon during viral infection.

ABSTRACTThe cytokine gamma interferon (IFN-γ), with antimicrobial and immunoregulatory functions, can be produced by T cells following stimulation through their T cell receptors (TCRs) for antigen. The innate cytokines type 1 IFNs and interleukin-12 (IL-12) can also stimulate IFN-γ production by natural killer (NK) but not naive T cells. High basal expression of signal transducer and activator of transcription 4 (STAT4), used by type 1 IFN and IL-12 to induce IFN-γ as well as CD25, contributes to the NK cell responses. During acute viral infections, antigen-specific CD8 T cells are stimulated to express elevated STAT4 and respond to the innate factors with IFN-γ production. Little is known about the requirements for cytokine compared to TCR stimulation. Primary infections of mice with lymphocytic choriomeningitis virus (LCMV) demonstrated that although the elicited antigen-specific CD8 T cells acquired STAT4-dependent innate cytokine responsiveness for IFN-γ and CD25 induction ex vivo, TCR stimulation induced these through STAT4-independent pathways. During secondary infections, LCMV-immune CD8 T cells had STAT4-dependent IFN-γ expression at times of innate cytokine induction but subsequently expanded through STAT4-independent pathways. At times of innate cytokine responses during infection with the antigen-distinct murine cytomegalovirus virus (MCMV), NK and LCMV-immune CD8 T cells both had activation of pSTAT4 and IFN-γ. The T cell IFN-γ response was STAT4 and IL-12 dependent, but antigen-dependent expansion was absent. By dissecting requirements for STAT4 and antigen, this work provides novel insights into the endogenous regulation of cytokine and proliferative responses and demonstrates conditioning of innate immunity by experience.

Proteome-wide screening of the European porcine reproductive and respiratory syndrome virus reveals a broad range of T cell antigen reactivity

The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-γ responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-γ ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-γ response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.

Monocytes sense hepatitis C virus-replicating cells and induce natural killer cell antiviral activity in an IL-18-mediated manner (INC8P.437)

Abstract Natural killer (NK) cells are important effectors in the innate immune defense against many viruses. However, NK cell IFN-γ production is attenuated in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is due to an NK cell-intrinsic or extrinsic mechanism. Hepatoma cells expressing luciferase-tagged subgenomic HCV-replicons (Huh7/HCV-replicons) or their HCV-negative counterparts were co-cultured with NK cells in the presence or absence of other PBMC subpopulations. RESULTS: The IFN-γ-mediated antiviral effect of NK cells was reduced when isolated NK cells rather than PBMC were co-cultured with Huh7/HCV-replicons suggesting that other subpopulations contributed to NK cell activation. Increased monokines levels implicated a role of activated monocytes. Indeed, depletion of CD14+ monocytes, siRNA knockdown of the monocyte NALP3 inflammasome and neutralization of the inflammasome product IL-18 decreased the IFN-γ-mediated antiviral activity of NK cells. Interestingly, monocytes from chronic HCV patients were less effective than monocytes from healthy controls in inducing NK cell IFN-γ production. Vice versa, monocytes from healthy controls improved antiviral function and IFN-γ production of NK cells from chronic HCV patients. CONCLUSION: Monocytes sense HCV-replicating cells and induce, via inflammasome-mediated IL-18 production, an NK-cell mediated decrease in HCV replication. Impaired monocyte function in HCV infection attenuates the NK cell IFN-γ response.

Frequency of Chlamydia trachomatis-specific T cell interferon-γ and interleukin-17 responses in CD4-enriched peripheral blood mononuclear cells of sexually active adolescent females

An evaluation of CD4 T cell responses to candidate Chlamydia trachomatis vaccine antigens was conducted in an adolescent female cohort exposed through natural infection to explore antigen immunogenicity and correlation with protection from reinfection. The frequency of peripheral blood CD4 T cell IFN-γ and IL-17 responses to three candidate vaccine antigens, polymorphic membrane protein G (PmpG), F (PmpF), and major outer membrane protein (MOMP), were determined by ELISPOT; responses to chlamydial heat shock protein 60 (HSP60) and to elementary bodies (EB) were included for comparison. Responses of Infected (n=8), Seropositive/Uninfected (n=13), and Seronegative/Uninfected (n=18) participants were compared. The median CD4 IFN-γ response to EB was significantly increased in Infected (P=0.003) and Seropositive/Uninfected (P=0.002) versus Seronegative/Uninfected female subjects. Higher rates of positive IFN-γ responders to EB, PmpF, and MOMP were detected in Seropositive/Uninfected versus Seronegative/Uninfected participants (P=0.021). IL-17 responses were generally low. A positive IFN-γ response to any of the antigens tested was associated with a trend toward a reduced risk of reinfection, although not statistically significant. Among this adolescent cohort, chlamydial-specific CD4 IFN-γ but not IL-17 responses were detected in acutely and previously infected participants and a positive CD4 IFN-γ response was associated with a non-significant reduced risk of reinfection.

Killer Ig-like Receptor 2DL4 Does Not Mediate NK Cell IFN-γ Responses to Soluble HLA-G Preparations

The MHC class Ib molecule HLA-G has previously been reported to be the ligand for the NK cell receptor killer Ig-like receptor (KIR)2DL4, but this remains controversial. In this study, we investigated IFN-γ production by freshly isolated NK cells in response to both soluble and solid-phase ligands, including anti-KIR2DL4 mAbs and rHLA-G. Although freshly isolated CD56(bright) NK cells produced IFN-γ in response to soluble HLA-G preparations, the response was found to be absolutely dependent on the presence of small numbers of contaminating CD56(-), CD14(-), CD11c(+) myeloid dendritic cells (mDCs). HLA-G tetramers bound only to the contaminating mDCs in the NK preparations, and Abs to KIR2DL4 and HLA-G did not block NK cell IFN-γ production. NK cells did not respond to plate-bound HLA-G. Freshly isolated NK cells also produced IFN-γ in response to unpurified soluble anti-KIR2DL4 mAb but not to low endotoxin affinity-purified Ab. The data suggest that previous reports of functional interactions between KIR2DL4 and HLA-G may have resulted from the use of purified NK cells that were contaminated with mDCs and HLA-G preparations that were contaminated with material capable of stimulating mDCs to produce cytokines that stimulate NK cells to produce IFN-γ.

Proteome-Wide Screening Reveals Immunodominance in the CD8 T Cell Response against Classical Swine Fever Virus with Antigen-Specificity Dependent on MHC Class I Haplotype Expression

Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core241–255 PESRKKLEKALLAWA and NS31902–1912 VEYSFIFLDEY, or minimal length antigenic peptides: E2996–1003 YEPRDSYF, NS21223–1230 STVTGIFL and NS5A3070–3078 RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS21223–1230 STVTGIFL and NS31902–1912 VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV.

Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection

Background: In a prior study, a DNA prime / adenovirus boost vaccine (DNA/Ad) expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) (NMRC-M3V-D/Ad-PfCA Vaccine) induced 27% protection against controlled human malaria infection (CHMI). To investigate the contribution of DNA priming, we tested the efficacy of adenovirus vaccine alone (NMRC-M3V-Ad-PfCA ) in a Phase 1 clinical trial.Methodology/Principal Findings: The regimen was a single intramuscular injection with two non-replicating human serotype 5 adenovectors encoding CSP and AMA1, respectively. One x 1010 particle units of each construct were combined prior to administration. The regimen was safe and well-tolerated. Four weeks later, 18 study subjects received P. falciparum CHMI administered by mosquito bite. None were fully protected although one showed delayed onset of parasitemia. Antibody responses were low, with geometric mean CSP ELISA titer of 381 (range < 50–1626) and AMA1 ELISA of 4.95 µg/mL (range 0.2–38). Summed ex vivo IFN-γ ELISpot responses to overlapping peptides were robust, with geometric mean spot forming cells/million peripheral blood mononuclear cells [sfc/m] for CSP of 273 (range 38–2550) and for AMA1 of 1303 (range 435–4594). CD4+ and CD8+ T cell IFN-γ responses to CSP were positive by flow cytometry in 25% and 56% of the research subjects, respectively, and to AMA1 in 94% and 100%, respectively.Significance: In contrast to DNA/Ad, Ad alone did not protect against CHMI despite inducing broad, cell-mediated immunity, indicating that DNA priming is required for protection by the adenovirus-vectored vaccine. ClinicalTrials.gov Identifier: NCT00392015.

Monocyte-derived IL-5 reduces TNF production by Mycobacterium tuberculosis-specific CD4 T cells during SIV/M. tuberculosis coinfection.

HIV-infected individuals are significantly more susceptible to tuberculosis (TB) than uninfected individuals. Although it is established that HIV reduces Mycobacterium tuberculosis-specific T cell responses, the causes of this dysfunction are not known. We used the cynomolgus macaque model of TB to demonstrate that ex vivo SIV reduces the frequency of M. tuberculosis-specific TNF and IFN-γ-producing T cells within 24 h after infection. In vivo, T cell IFN-γ responses in granulomas from animals with SIV/M. tuberculosis coinfection were lower than SIV-negative animals with active TB. The SIV effects on the inhibition of T cell responses were primarily on APCs and not the T cells directly. Specifically, reductions in the frequency of TNF-producing M. tuberculosis-specific CD4 T cells were caused, at least in part, by SIV-induced production of monocyte derived IL-5.

Location of CD4+ T Cell Priming Regulates the Differentiation of Th1 and Th17 Cells and Their Contribution to Arthritis

Th cytokines IFN-γ and IL-17 are linked to the development of autoimmune disease. In models of rheumatoid arthritis, that is, proteoglycan (PG)-induced arthritis, IFN-γ is required, whereas in collagen-induced arthritis, IL-17 is necessary for development of arthritis. In this study we show that the route of immunization determines the requirement for either IFN-γ or IL-17 in arthritis. Intraperitoneal immunization with PG induces a CD4(+) T cell IFN-γ response with little IL-17 in the spleen and peripheral lymph nodes. However, s.c. immunization induces both an IFN-γ and an IL-17 CD4(+) T cell response in spleen and lymph nodes. The failure to induce a CD4(+) T cell IL-17 response after i.p. immunization is associated with T cell priming, as naive T cells activated in vitro were fully capable of producing IL-17. Moreover, PG-induced arthritis is converted from an IFN-γ to an IL-17-mediated disease by altering the route of immunization from i.p. to s.c. The histological appearance of joint inflammation (cellular inflammation and bone erosion) is similar in the i.p. versus s.c. immunized mice despite the presence of CD4(+) T cells producing IL-17 in joint tissues only after s.c. immunization. These data indicate a critical role for the site of initial T cell priming and the Th cytokines required for susceptibility to arthritis. Our findings suggest that T cell activation at different anatomical sites in rheumatoid arthritis patients may skew the T cells toward production of either IFN-γ or IL-17.

Virus-specific CD4 and CD8 T cell responses in the absence of Th1-associated transcription factors (P6052)

Abstract The eradication of various types of pathogens is mediated by the coordinated efforts of both effector CD4 and CD8 T cells. Following pathogen control, the majority of CD4 and CD8 T cell effector populations undergo apoptosis, but a subset of these cells persists, giving rise to the memory T cell pool. The requirements for the generation of T cell memory have not been completely elucidated, hence we were interested in understanding importance of the Th1 transcription factors Tbet and STAT4 in this process. To interrogate this, we utilized lymphocytic choriomeningitis virus infection of BALB/c mice. Not surprisingly, we observed that in the absence of Tbet, the virus-specific CD4 T cell IFNγ response was ablated but the IL-2 response remained unperturbed. In contrast, we found that STAT4 was not required for IFNγ production by virus-specific CD4 T cells. Conversely, the functional virus-specific CD8 T cell response was intact in the absence of Tbet, STAT4, or both, although increases in the proportion of effector cells competent for IL-2 production were noted in mice that lacked Tbet expression. We did detect marked differences in the expression of CD127 and KLRG1 on virus-specific effector CD8 T cells, however this did not translate into alterations in the numbers of memory CD8 T cells after the resolution of the infection. Collectively, we discovered distinct roles for Tbet and STAT4 in shaping the phenotype and function of virus-specific T cell responses.

Recombinant salmonella-based 4-1BBL vaccine enhances T cell immunity and inhibits the development of colorectal cancer in rats: in vivo effects of vaccine containing 4-1BBL

BackgroundImmunotherapy with vaccines is attractive for the treatment of cancer. This study is aimed at determining the effect of recombinant Salmonella (SL3261)-based 4-1BB ligand (4-1BBL) vaccine on the development of colorectal cancers and the potential immune mechanisms in rats.ResultsIn comparison with that in the PBS group, similar levels of 4-1BBL expression, the frequency of T cells, IFN-γ responses, and comparable numbers of tumors were detected in the SL3261 and SL3261C groups of rats. In contrast, significantly fewer numbers of tumors, increased levels of 4-1BBL expression in the spleens and colorectal tissues, higher frequency of peripheral blood and splenic CD3+CD25+ T cells, and stronger splenic T cell IFN-γ responses were detected in the SL3261R group of rats.ConclusionOur results indicated that vaccination with recombinant attenuated Salmonella harboring the 4-1BBL gene efficiently enhanced T cell immunity and inhibited the development of carcinogen-induced colorectal cancers in rats.

Natural killer cell function in women at high risk for HIV acquisition

To assess the role of natural killer (NK) cells in HIV acquisition. We conducted a nested case-control substudy to the Center for the AIDS Programme of Research in South Africa (CAPRISA004) tenofovir gel trial. Thirty women who acquired HIV infection (cases) and 30 women with high-risk sexual activity who remained HIV-negative (controls) were selected. Proliferation, degranulation and interferon-γ (IFNγ) secretion were measured by multiparametric flow cytometry after culture of recombinant human interleukin-2 (rhIL-2)-activated peripheral blood mononuclear cells with 721.221 cells or in-vitro HIV-infected, autologous CD4 T-cell blasts. Relationships between pre-acquisition NK cell responses and HIV acquisition were modeled with logistic regression models. NK cells from cases had lower IFNγ responses to human leukocyte antigen-deficient 721.221 cells than controls (median %IFNγposNK cells: 13.7 vs. 21.6%, P = 0.03). rhIL-2-activated NK cells from cases had responses to autologous HIV-infected target cells distinct from controls: cases had fewer proliferating and more frequent degranulating NK cells. NK cells from cases had significantly lower IFNγ responses to in-vitro HIV-infected autologous T cells than controls even after adjusting for responses to uninfected blasts (median %IFNγposNK-cells: 0.53 vs. 2.09%, P = 0.007). Responses to in-vitro HIV-infected autologous T cells were significantly lower in herpes simplex virus 2 (HSV-2)-infected women (P = 0.003). IFNγ NK cell responses to autologous HIV-infected cells were associated with lower risk of HIV acquisition (odds ratio adjusted for age, gel arm, HSV-2 and immune activation: 0.582, 95% confidence interval 0.347-0.977, P = 0.04). At the time of exposure to HIV, women with impaired NK cell IFNγ responses were more likely to acquire HIV infection. NK cells, as early responders to viral exposure, were associated with lower risk of HIV acquisition, independent of the intercalated effect of HSV-2 infection suppressing NK cell responses.

Natural killer cells display impaired responses to toll like receptor 9 that support viral persistence in chronic hepatitis B

Toll like receptors (TLR) are crucial mediators of innate immune responses, but their influence on natural killer (NK) cell function in chronic hepatitis B infection (CHB) is not well understood. Here we evaluated the responses of peripheral NK cells from CHB patients to multiple TLR agonists. CHB was associated with an impaired NK cell IFN-γ response to TLR9 stimulation compared to controls. This deficiency corrected with recombinant IFN-alpha, while anti-IFN-alpha neutralizing antibody diminished NK IFN-γ production in controls. NK cell CD69 upregulation in response to TLR9 was maintained in CHB. No differences were noted in responses to the other TLR ligands. Our results demonstrate a dichotomous NK cell response to TLR9 that is mediated by IFN-alpha and reflect the multiple mechanisms involved with NK activation. Consequently, it is possible that when activated these cells are unable to contribute to viral clearance while still having the potential to mediate tissue injury.

Cutting Edge: In the Absence of TGF-β Signaling in T Cells, Fewer CD103+ Regulatory T Cells Develop, but Exuberant IFN-γ Production Renders Mice More Susceptible to Helminth Infection

Multiple factors control susceptibility of C57BL/6 mice to infection with the helminth Heligmosomoides polygyrus, including TGF-β signaling, which inhibits immunity in vivo. However, mice expressing a T cell-specific dominant-negative TGF-β receptor II (TGF-βRII DN) show dampened Th2 immunity and diminished resistance to infection. Interestingly, H. polygyrus-infected TGF-βRII DN mice show greater frequencies of CD4(+)Foxp3(+)Helios(+) Tregs than infected wild-type mice, but levels of CD103 are greatly reduced on both these cells and on the CD4(+)Foxp3(+)Helios(-) population. Although Th9 and Th17 levels are comparable between infected TGF-βRII DN and wild-type mice, the former develop exaggerated CD4(+) and CD8(+) T cell IFN-γ responses. Increased susceptibility conferred by TGF-βRII DN expression was lost in IFN-γ-deficient mice, although they remained unable to completely clear infection. Hence, overexpression of IFN-γ negatively modulates immunity, and the presence of Helios(+) Tregs may maintain susceptibility on the C57BL/6 background.

Antigen-Specific IL-2 Secretion Correlates with NK Cell Responses after Immunization of Tanzanian Children with the RTS,S/AS01 Malaria Vaccine

RTS,S/AS01, a vaccine targeting pre-erythrocytic stages of Plasmodium falciparum, is undergoing clinical trials. We report an analysis of cellular immune response to component Ags of RTS,S-hepatitis B surface Ag (HBs) and P. falciparum circumsporozoite (CS) protein-among Tanzanian children in a phase IIb RTS,S/AS01(E) trial. RTS,S/AS01 (E) vaccinees make stronger T cell IFN-γ, CD69, and CD25 responses to HBs peptides than do controls, indicating that RTS,S boosts pre-existing HBs responses. T cell CD69 and CD25 responses to CS and CS-specific secreted IL-2 were augmented by RTS,S vaccination. Importantly, more than 50% of peptide-induced IFN-γ(+) lymphocytes were NK cells, and the magnitude of the NK cell CD69 response to HBs peptides correlated with secreted IL-2 concentration. CD69 and CD25 expression and IL-2 secretion may represent sensitive markers of RTS,S-induced, CS-specific T cells. The potential for T cell-derived IL-2 to augment NK cell activation in RTS,S-vaccinated individuals, and the relevance of this for protection, needs to be explored further.

Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus

Live attenuated C-strain classical swine fever viruses (CSFV) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in CSF outbreaks. Since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood T cell responses from pigs vaccinated with a C-strain vaccine and challenged after five days with a genotypically divergent CSFV isolate. Vaccinated animals displayed virus-specific IFN-γ responses from day 3 post-challenge, whereas, unvaccinated challenge control animals failed to mount a detectable response. Both CD4+ and cytotoxic CD8+ T cells were identified as the cellular source of IFN-γ. IFN-γ responses showed extensive cross-reactivity when T cells were stimulated with CSFV isolates spanning the major genotypes. To determine the specificity of these responses, T cells were stimulated with recombinant CSFV proteins and a proteome-wide peptide library from a related virus, BVDV. Major cross-reactive peptides were mapped on the E2 and NS3 proteins. Finally, IFN-γ was shown to exert potent antiviral effects on CSFV in vitro. These data support the involvement of broadly cross-reactive T cell IFN-γ responses in the rapid protection conferred by the C-strain vaccine and this information should aid the development of the next generation of CSFV vaccines.

Challenge of Pigs with Classical Swine Fever Viruses after C-Strain Vaccination Reveals Remarkably Rapid Protection and Insights into Early Immunity

Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser® C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-γ responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-γ response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination.

Autoreactive T Cells in Human Smokers is Predictive of Clinical Outcome.

Cross-sectional studies have suggested a role for activation of adaptive immunity in smokers with emphysema, but the clinical application of these findings has not been explored. Here we examined the utility of detecting autoreactive T cells as a screening tool for emphysema in an at-risk population of smokers. We followed 156 former and current (ever)-smokers for 2 years to assess whether peripheral blood CD4 T cell cytokine responses to lung elastin fragments (EFs) could discriminate between those with and without emphysema, and to evaluate the relevance of autoreactive T cells to predict changes during follow-up in lung physiological parameters. Volunteers underwent baseline complete phenotypic assessment with pulmonary function tests, quantitative chest CT, yearly 6-min walk distance (6MWD) testing, and annual measurement of CD4 T cell cytokine responses to EFs. The areas under the receiver operating characteristic curve to predict emphysema for interferon gamma (IFN-γ), and interleukin 6 (IL-6) responses to EFs were 0.81 (95% CI of 0.74–0.88) and 0.79 (95% CI of 0.72–0.86) respectively. We developed a dual cytokine enzyme-linked immunocell spot assay, the γ-6 Spot, using CD4 T cell IFN-γ and IL-6 responses and found that it discriminated emphysema with 90% sensitivity. After adjusting for potential confounders, the presence of autoreactive T cells was predictive of a decrease in 6MWD over 2 years (decline in 6MWD, −19 m per fold change in IFN-γ; P = 0.026, and −26 m per fold change in IL-6; P = 0.003). In support of the human association studies, we cloned CD4 T cells with characteristic T helper (Th)1 and Th17 responses to EFs in the peripheral blood of ever-smokers with emphysema, confirming antigenicity of lung elastin in this population. These findings collectively suggest that the EF-specific autoreactive CD4 T cell assay, γ-6 Spot, could provide a non-invasive diagnostic tool with potential application to large-scale screening to discriminate emphysema in ever-smokers, and predict early relevant physiological outcomes in those at risk.

Natural Killer (NK) Cells Respond to CMV Reactivation After Allogeneic Transplantation with An Increase in NKG2C+CD57+ Self-KIR+ NK Cells with Potent IFNγ Production

Abstract 356In healthy individuals human cytomegalovirus (hCMV) infection is asymptomatic or a mild illness. In immunosuppressed patients after hematopoietic cell transplantation, however, CMV is potentially life-threatening complication. We have previously shown that upon mouse CMV infection, a population of Ly49H+ NK cells expand and are responsible for disease clearance through the induction of a “memory NK cell response”. Whether similar events occur in hCMV infection is unknown. Some studies suggest that the C-type lectin-like receptor NKG2C and the killer cell immunoglobulin-like receptor (KIR) family may be involved. We studied NK cell function after hCMV reactivation. Cohort 1 consisted of patients who received umbilical cord blood grafts (n=23). Recipients were CMV-seronegative (n=10) and -seropositive (n=13), with 8 patients developing detectable hCMV in the blood 22 to 82 days after transplant. For this cohort, peripheral blood mononuclear cells (PBMC) were collected at day 28, day 60 and day 100 after transplant. Cryopreserved PBMC were thawed and rested overnight in cytokine-free media. After incubation with K562 cells a functional analysis was performed using multi-color flow cytometry for CD107a (a surrogate for cytotoxicity), IFNγ, CD56, CD3, CD158a, CD158b, CD158e, CD57, NKG2A, and NKG2C. In cohort 1 the frequency of NKG2C+ NK cells significantly increased between day 28 and day 60 (10±1.3% v 26±5%, p=0.01), and this increase persisted at day 100 in seropositive patients who reactivated CMV (32±4.8%, p=0.001). No change in NKG2C expression was detected in seropositive patients who did not reactivate CMV or in seronegative recipients, showing that this response was specific. There was no difference in the expression of CD107a by NKG2C+ NK cells in the three groups. In marked contrast, IFNγ production by NKG2C+ NK cells was low prior to CMV reactivation (0.8±1.4%) and increased at day 60 and day 100 (7.9±2.1%, p=0.012). The IFNγ response was specific to NKG2C+ NK cells as the NKG2C− population produced minimal IFNγ at day 60 and day 100 (NKG2C+: 7.9±2.1% vs. NKG2C−: 0.4±0.25%, p=0.003). The frequency of NKG2C+ NK cells with increased cytokine production correlated with CMV reactivation in this cohort. The second cohort included PBMCs collected at the time of viral reactivation (monitored weekly) and 2, 4, and 8 weeks later (n=10). The frequency of NKG2C+ NK cells increased following the detection hCMV viremia. The peak response (i.e., rise in NKG2C+ NK cells compared to baseline) was at 4 weeks post infection (23±3.7% vs. 13±1.8%, p=0.046), followed by contraction at 8 weeks. NKG2C+NK cells produced significantly more IFNγ than did NKG2C− cells at 2, 4 (NKG2C+: 9.4±1.2% vs. NKG2C−: 2.4±0.3%, p=0.0002) and 8 weeks post infection. The maximum IFNγ production occurred at 4 weeks, with the peak NKG2C+ frequency. We have previously demonstrated that KIR expression is essential to educate NK cells to produce IFNγ. Therefore, we measured coexpression of NKG2C and KIR in these cohorts. Four weeks after infection, NK cells that coexpressed NKG2C and KIR produced significantly more IFNγ than did the KIR+ NKG2C−NK cells (11.4±1.66% vs. 5.6±0.83%, p=0.0078). Importantly, only the NKG2C+NK cells expressing KIR that recognized recipient MHC ligands made IFNγ. In contrast, NKG2C+ NK cells coexpressing KIR that did not recognize recipient ligands were hyporesponsive. Lastly, we tested whether coexpression of CD57, a phenotypic marker of NK cells maturity, might be present on NKG2C+ cells after CMV reactivation. Over the 8 weeks following CMV reactivation the NKG2C+ NK cells but not the NKG2C−NK cells progressively acquired CD57, suggesting that CD57 may mark the human memory NK cell in response to CMV reactivation. In summary, our data demonstrate that CMV reactivation induces an NK cell IFNγ response specific to NKG2C+cells educated by recipient KIR ligands that then acquire CD57+. These findings support the emerging concept that hCMV specific, innate memory cell populations could be exploited for therapeutic purposes. Disclosures:No relevant conflicts of interest to declare.

Adenovirus 5-Vectored P. falciparum Vaccine Expressing CSP and AMA1. Part A: Safety and Immunogenicity in Seronegative Adults

BackgroundModels of immunity to malaria indicate the importance of CD8+ T cell responses for targeting intrahepatic stages and antibodies for targeting sporozoite and blood stages. We designed a multistage adenovirus 5 (Ad5)-vectored Plasmodium falciparum malaria vaccine, aiming to induce both types of responses in humans, that was tested for safety and immunogenicity in a Phase 1 dose escalation trial in Ad5-seronegative volunteers.Methodology/Principal FindingsThe NMRC-M3V-Ad-PfCA vaccine combines two adenovectors encoding circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). Group 1 (n = 6) healthy volunteers received one intramuscular injection of 2×10∧10 particle units (1×10∧10 each construct) and Group 2 (n = 6) a five-fold higher dose. Transient, mild to moderate adverse events were more pronounced with the higher dose. ELISpot responses to CSP and AMA1 peaked at 1 month, were higher in the low dose (geomean CSP = 422, AMA1 = 862 spot forming cells/million) than in the high dose (CSP = 154, p = 0.049, AMA1 = 423, p = 0.045) group and were still positive at 12 months in a number of volunteers. ELISpot depletion assays identified dependence on CD4+ or on both CD4+ and CD8+ T cells, with few responses dependent only on CD8+ T cells. Intracellular cytokine staining detected stronger CD8+ than CD4+ T cell IFN-γ responses (CSP p = 0.0001, AMA1 p = 0.003), but similar frequencies of multifunctional CD4+ and CD8+ T cells secreting two or more of IFN-γ, TNF-α or IL-2. Median fluorescence intensities were 7–10 fold higher in triple than single secreting cells. Antibody responses were low but trended higher in the high dose group and did not inhibit growth of cultured P. falciparum blood stage parasites.SignificanceAs found in other trials, adenovectored vaccines appeared safe and well-tolerated at doses up to 1×10∧11 particle units. This is the first demonstration in humans of a malaria vaccine eliciting strong CD8+ T cell IFN-γ responses.Trial Registration ClinicalTrials.gov NCT00392015