Cell IFN-γ Responses Research Articles

A distinct subset of human NK cells expressing HLA‐DR expand in response to IL‐2 and can aid immune responses to BCG

Subsets of NK cells can have distinct functions. Here, we report that >25% of human peripheral blood NK cells express HLA-DR after culture with IL-2. This can be driven by an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLA-DR is upregulated on previously negative cells. HLA-DR-expressing NK cells showed enhanced degranulation to susceptible target cells and expressed chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggesting HLA-DR-expressing NK cells have an important role in an immune response, stimulation of PBMCs with Mycobacterium bovis BCG (BCG) triggered expansion of this subset. Importantly, the magnitude of an individual's NK cell IFN-γ response triggered by BCG was associated with the initial frequency of HLA-DR-expressing NK cells in PBMCs. More directly indicating the importance of HLA-DR-expressing NK cells, enriching the frequency of this subset in PBMCs substantially augmented the IFN-γ response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in circulating blood but readily expanded by IL-2, that can play an important role during immune responses to BCG.

NK cell IFNγ response to HLA-G: a role for myeloid DCs? (147.10)

Abstract Previous studies report that stimulation of freshly isolated Natural Killer (NK) cells with soluble (but not solid-phase) ligand to KIR2DL4 results in IFNγ production. We have found that stimulation of 13-day cultured, activated, KIR2DL4-expressing NK cells with solid-phase (but not soluble) ligand to KIR2DL4 also results in NK cell IFNγ production. Human Leukocyte Antigen-G (HLA-G) has been reported to be the ligand for KIR2DL4, but controversy persists due to the irreproducibility of interactions between HLA-G (in soluble or solid-phase form) and KIR2DL4. In this study, we took freshly isolated NK cells purified by two methods, stimulated with soluble recombinant HLA-G or control, and measured IFNγ response. We observed an IFNγ response only from RosetteSep purified NK cells, and not from Easysep purified cells. Antibody cocktail analysis predicted that dendritic cells would be removed by the Easysep method but not the RosetteSep method. We confirmed the presence of CD11c+ myeloid dendritic cells (mDCs) in the RosetteSep NK cell purification and showed that removal of mDCs by cell sorting negated the IFNγ response. We further found that addition of mDCs to 13-day cultured, activated, KIR2DL4-expressing NK cells enabled IFNγ production in response to soluble HLA-G. Lastly, we have observed HLA-G tetramers binding to mDCs but not NK cells. We therefore hypothesize that mDCs bind and subsequently present “soluble” HLA-G to NK cells in order to induce an IFNγ response.

Essential Role for Retinoic Acid in the Promotion of CD4+ T Cell Effector Responses via Retinoic Acid Receptor Alpha

Vitamin A and its metabolite, retinoic acid (RA) are implicated in the regulation of immune homeostasis via the peripheral induction of regulatory Tcells. Here we showed RA was also required to elicit proinflammatory CD4(+) helper Tcell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) was the critical mediator of these effects. Antagonism of RAR signaling and deficiency in RARα (Rara(-/-)) resulted in a cell-autonomous CD4(+) Tcell activation defect, which impaired intermediate signaling events, including calcium mobilization. Altogether, these findings reveal a fundamental role for the RA-RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive Tcell responses.

MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy

Sunitinib is a receptor tyrosine kinase inhibitor (TKI) that is front-line therapy for metastatic renal cell carcinoma (mRCC). Its antitumor activity is related to its ability to block tumor cell and tumor vasculature cell signaling via several TKI receptors (i.e. vascular endothelial growth factor receptors VEGFRs, platelet-derived growth factors (PDGFs), and stem cell factors). Sunitinib also targets myeloid derived suppressor cells (MDSCs) significantly reducing their accumulation in the peripheral blood and reversing T cell (IFNγ) suppression in both mRCC patients and in murine tumor models. This reduction in immune suppression provides a rationale for combining sunitinib with immunotherapy for the treatment of certain tumor types. Despite these encouraging findings, however, we have observed that sunitinib has variable impact at reducing MDSCs and restoring T cell function within the tumor microenvironment. Given the immunosuppressive and proangiogenic activities of MDSC, it seems plausible that their persistence may contribute to the resistance that develops in sunitinib-treated patients. While sunitinib reduced tumor infiltrating MDSCs in Renca and CT26-bearing mice, coinciding with strong to modest decreases in tumor size respectively, it was ineffective at reducing MDSCs (<35% reduction in Gr1+CD11b+) or tumor burden in 4T1-bearing mice. Persistence of intratumor MDSCs was paralleled by depressed intratumor T cell IFNγ response and increased GM-CSF expression. Additionally, in vitro and in vivo experiments showed that GM-CSF prolongs survival of MDSCs, thus protecting them from the effects of sunitinib via a pSTAT5-dependent pathway. Although preliminary, there is evidence of intratumor MDSC resistance in some mRCC patients following sunitinib treatment. Intratumor MDSC persistence and T cell IFNγ response post nephrectomy in patients receiving sunitinib in a neoadjuvant setting are being compared to RCC patients undergoing nephrectomy without prior sunitinib treatment. Tumors from untreated patients showed suppressed T cell IFNγ response along with substantial expression of MDSCs (5% of total digested cells). Thus far, tumors from 5/8 neoadjuvant patients showed persistence of intratumor MDSCs and low T cell IFNγ production post sunitinib treatment, findings that parallel results from untreated tumors. In the remaining 3 neoadjuvant patients, intratumor MDSCs were detected at low levels which coincided with a T cell IFNγ response similar to that observed with normal donor peripheral T cells. GM-CSF's role in promoting MDSC survival in patient tumors is supported by the observation that GM-CSF is produced in short-term RCC cultures at levels capable of protecting MDSCs from sunitinib-induced cell death. Additionally, persistence of MDSC also may be associated with increased expression of proangiogenic proteins, such as MMP9, MMP8, and IL-8 produced by tumor stromal cells or infiltrating MDSCs. Indeed our findings suggest that the most dominate MDSC subset in RCC patients is the neutrophilic population that produces proangiogenic proteins. We propose that the development of sunitinib resistance is partly mediated by the survival of MDSCs intratumorally, thereby providing sustained immune suppression and angiogenesis.

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4+ and CD8+ T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4+ and CD8+ T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.

CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

EAE is a demyelinating disease of the CNS and serves as a mouse model of MS. Expression of CCL22 in the draining LNs and spinal cord correlated with the onset of clinical EAE development and remained elevated. Administration of anti-CCL22 at the time of autoantigen immunization delayed the initiation of clinical disease and dampened the severity of peak initial disease and relapses. Reduced EAE severity correlated with the reduction of pathology and leukocytes in the CNS, particularly, activated CD11b+Ly6C(hi) macrophages. There were no differences in effector T cell-proliferative responses or effector T cell IFN-γ or IL-17 responses. However, treatment at the onset of disease did not reduce disease progression. Treatment of adoptive T cell transfer recipient mice with anti-CCL22 resulted in decreased clinical disease development accompanied by a decrease in CNS accumulation of CD11b+Ly6C(hi) macrophages. Neutralization of CCL22 resulted in a macrophage population whose effector cytokine expression consisted of decreased TNF and increased IL-10, a phenotype more consistent with M2 macrophages. This was corroborated by in vitro cultures of macrophages with CCL22. These results suggest that CCL22 functions to regulate development of EAE through macrophage chemoattraction and effector function.

Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein

BackgroundPlasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1.MethodsA recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes.ResultsFourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure.ConclusionsThis study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations.

Cross-Talk between T Cells and NK Cells Generates Rapid Effector Responses to Plasmodium falciparum -Infected Erythrocytes

Rapid cell-mediated immune responses, characterized by production of proinflammatory cytokines, such as IFN-gamma, can inhibit intraerythrocytic replication of malaria parasites and thereby prevent onset of clinical malaria. In this study, we have characterized the kinetics and cellular sources of the very early IFN-gamma response to Plasmodium falciparum-infected RBCs among human PBMCs. We find that NK cells dominate the early (12-18 h) IFN-gamma response, that NK cells and T cells contribute equally to the response at 24 h, and that T cells increasingly dominate the response from 48 h onward. We also find that although gammadelta T cells can produce IFN-gamma in response to P. falciparum-infected RBCs, they are greatly outnumbered by alphabeta T cells, and thus, the majority of the IFN-gamma(+) T cells are alphabeta T cells and not gammadelta T cells; gammadelta T cells are, however, an important source of TNF. We have previously shown that NK cell responses to P. falciparum-infected RBCs require cytokine and contact-dependent signals from myeloid accessory cells. In this study, we demonstrate that NK cell IFN-gamma responses to P. falciparum-infected RBCs are also crucially dependent on IL-2 secreted by CD4(+) T cells in an MHC class II-dependent manner, indicating that the innate response to infection actually relies upon complex interactions between NK cells, T cells, and accessory cells. We conclude that activation of NK cells may be a critical function of IL-2-secreting CD4(+) T cells and that standard protocols for evaluation of Ag-specific immune responses need to be adapted to include assessment of NK cell activation as well as T cell-derived IL-2.

Long-term immune characteristics of cow's milk allergy in a mouse model (141.12)

Abstract Specific changes in immune memory underlying the ability to outgrow cow’s milk allergy (CMA) are incompletely understood. We studied the long-term immune characteristics of CMA in an adjuvant-free mouse model we have recently published (Gonipeta et al 2009. J. Dairy Sci. 92:4738-4744). Thus, BALB/c mice were sensitized to cow’s milk whey protein using a transdermal sensitization protocol. Subsequently, exposure to whey protein was withdrawn for 3, 5 or 8 months. Fate of circulating IgE and IgG2a antibodies was monitored. Subsequently, mice were given booster exposures and examined for memory IgE, IgG2a, and spleen cell IL-4 and IFN-γ responses. Following allergen withdrawal, IgE antibody levels began to drop. Whereas, significant memory IgE response to booster exposures was evident after 3 months of allergen withdrawal, a weak or no memory IgE response was noted after 5 or 8 months of allergen withdrawal. Despite booster exposures, spleen cell memory IL-4 responses to whey protein was absent after 3, 5 or 8 months of allergen withdrawal. Milk sensitized mice, upon 8 months of allergen withdrawal, exhibited elevated IFN-γ and IgG2a responses suggesting a shift to a TH1 immune profile. In summary, these data suggest that: (i) upon withdrawal of allergen exposure, whey protein specific memory IgE, and memory IL-4 do not persist for long periods; and (ii) there occurs a shift in immune memory towards elevation of TH1 responses as mice age. Funding: US EPA STAR Grant#R833133

Vaccination with survivin as a self TAA and SA-4-1BBL is effective in eradicating established lung carcinomas in CD8+ T cell and NK cell dependent manner (131.29)

Abstract Costimulatory ligands of TNF family have great potential as adjuvant for therapeutic vaccines due to their critical role in the expansion of T cells, survival, and long-term memory. Particularly, 4-1BBL has the most potent T cell activity among the TNF costimulatory members. However, the use of 4-1BBL as the immunomodulatory component of conventional therapeutic vaccine has been limited due to its lack of activity in soluble form. We have recently generated a chimeric 4-1BBL with streptavidin (SA-4-1BBL) that exists as tetramers/oligomers with potent activity in soluble form. We herein formulated a subunit protein based vaccine containing SA-4-1BBL and survivin as an autologous tumor associated antigen. The therapeutic efficacy of this vaccine was tested in the transplantable survivin expressing 3LL metastatic lung tumor model. Vaccination with the survivin and SA-4-1BBL resulted in the eradication of established 3LL tumors in 75% of the animals. Importantly, animals with successful immunotherapy developed a robust NK cell cytotoxicity, antigen specific T cell proliferation, and IFN-γ and IL-2 responses. Depletion of various immune cell types demonstrated the indispensable role of CD8+ T cells and important role of NK cells for vaccine efficacy. Chimeric SA-4-1BBL hence prime CD8/NK cell axis for tumor eradication and has potential to be used as a specific immunological adjuvant to develop therapeutic vaccines against tumors with well characterized tumor associated antigens.

Temporal stability of naturally acquired immunity to Merozoite Surface Protein-1 in Kenyan Adults

BackgroundNaturally acquired immunity to blood-stage Plasmodium falciparum infection develops with age and after repeated infections. In order to identify immune surrogates that can inform vaccine trials conducted in malaria endemic populations and to better understand the basis of naturally acquired immunity it is important to appreciate the temporal stability of cellular and humoral immune responses to malaria antigens.MethodsBlood samples from 16 adults living in a malaria holoendemic region of western Kenya were obtained at six time points over the course of 9 months. T cell immunity to the 42 kDa C-terminal fragment of Merozoite Surface Protein-1 (MSP-142) was determined by IFN-γ ELISPOT. Antibodies to the 42 kDa and 19 kDa C-terminal fragments of MSP-1 were determined by serology and by functional assays that measure MSP-119 invasion inhibition antibodies (IIA) to the E-TSR (3D7) allele and growth inhibitory activity (GIA). The haplotype of MSP-119 alleles circulating in the population was determined by PCR. The kappa test of agreement was used to determine stability of immunity over the specified time intervals of 3 weeks, 6 weeks, 6 months, and 9 months.ResultsMSP-1 IgG antibodies determined by serology were most consistent over time, followed by MSP-1 specific T cell IFN-γ responses and GIA. MSP-119 IIA showed the least stability over time. However, the level of MSP-119 specific IIA correlated with relatively higher rainfall and higher prevalence of P. falciparum infection with the MSP-119 E-TSR haplotype.ConclusionVariation in the stability of cellular and humoral immune responses to P. falciparum blood stage antigens needs to be considered when interpreting the significance of these measurements as immune endpoints in residents of malaria endemic regions.

Killer Ig-Like Receptor (KIR) Genotype Predicts the Capacity of Human KIR-Positive CD56dim NK Cells to Respond to Pathogen-Associated Signals

IFN-gamma emanating from NK cells is an important component of innate defense against infection. In this study, we demonstrate that, following in vitro stimulation of human peripheral blood NK cells with a variety of microbial ligands, CD56(dim) as well as CD56(bright) NK cells contribute to the overall NK cell IFN-gamma response with, for most cell donors, IFN-gamma(+) CD56(dim) NK cells outnumbering IFN-gamma(+) CD56(bright) NK cells. We also observe that the magnitude of the human NK IFN-gamma response to microbial ligands varies between individuals; that the antimicrobial response of CD56(bright), but not CD56(dim), NK cells is highly correlated with that of myeloid accessory cells; and that the ratio of IFN-gamma(+) CD56(dim) to IFN-gamma(+) CD56(bright) NK cells following microbial stimulation differs between individuals but remains constant for a given donor over time. Furthermore, ratios of IFN-gamma(+) CD56(dim) to IFN-gamma(+) CD56(bright) NK cells for different microbial stimuli are highly correlated and the relative response of CD56(dim) and CD56(bright) NK cells is highly significantly associated with killer Ig-like receptor (KIR) genotype. These data reveal an influence of KIR genotype, possibly mediated via NK cell education, on the ability of NK cells to respond to nonviral infections and have implications for genetic regulation of susceptibility to infection in humans.

Could natural killer cells compensate for impaired CD4+ T-cell responses to CMV in HIV patients responding to antiretroviral therapy?

We evaluated NK cell subsets and functions in previously immunodeficient HIV patients responding to ART. Cytokine receptor mRNA was quantitated in purified CD56+ cells. Data were correlated with CD4+ T-cell counts and IFNgamma responses to CMV. NK cell IFNgamma responses to K562 cells and proportions of CD56lo NK cells were correlated in patients (p < 0.001) and both were lower than in controls (p < 0.001 and p = 0.008, respectively), so all patients had poor NK cell function. Proportions of CD56hi NK cells correlated inversely with current CD4+ T-cell counts (p = 0.006) and perforin expression in CD56hi NK cells was higher in patients than controls (p < 0.05). Hence increased proportions and cytolytic function of CD56hi NK cells may partially compensate for CD4+ T-cell deficiency. NK cell IFNgamma responses correlated inversely with expression of IL-10 and IL-12 receptor mRNA. Expression of these transcripts is reduced by exposure to the cytokines, which may reflect immune activation in immunodeficient patients.

Natural Killer Cell IFN-γ Levels Predict Long-term Survival with Imatinib Mesylate Therapy in Gastrointestinal Stromal Tumor–Bearing Patients

Clinical outcomes of gastrointestinal stromal tumor (GIST)-bearing patients treated with imatinib mesylate (IM) are variable. Other than the site of mutation within the c-kit gene, prognostic features of GIST remain undefined. IM can exhibit off-target effects such as triggering natural killer (NK) cell activity. We addressed whether NK cell functions could predict long term survival with IM. NK cell functions were followed up in 77 GIST patients enrolled onto two phase III trials. "Immunologic responders" were defined as patients whose NK cell IFN-gamma values after 2 months of IM were higher than or equal to the baseline value at entry into the trial. The prognostic effect of IFN-gamma on progression-free survival was assessed by a Wald test in a Cox regression analysis using the landmark method and stratified by trial and on the c-kit mutational status. Fifty-six patients were evaluable for the NK cell IFN-gamma responses at baseline and 2 months. Their median follow-up for progression-free survival was 3.7 years. Thirty-four of 56 patients were immunologic responders to IM. In the Cox regression analysis, immunologic responders possessed a hazard ratio of progression or death equal to 0.29 (95% confidence interval, 0.12-0.70; P = 0.006) compared with nonresponders. Kaplan-Meier 2-year survival estimates were 85% for immunologic responders and 50% for nonresponders. Moreover, the immunologic response added prognostic value to the c-kit mutation. The NK cell IFN-gamma production after 2 months of treatment could be considered an independent predictor of long term survival in advanced GISTs treated with IM.

Abstract LB-238: Immunotransplant filters out regulatory T cells and cures large lymphoma tumors.

Abstract Background: The potency of transferring ex vivo activated tumor-infiltrating lymphocytes into lymphodepleted recipients has been demonstrated in clinical trials, though the use of in vivo vaccine-primed anti-tumor lymphocytes could make this approach sufficiently feasible for broad clinical application. We previously described a vaccination maneuver against murine B-cell lymphoma combining chemotherapy (CTX) and intra-tumoral injection of an immunostimulant CpG, which induced T-cell immunity and eradicated established tumors. An ongoing clinical trial of this maneuver has demonstrated complete and partial responses in lymphoma patients, though immune-regulatory factors such as regulatory T cells (Tregs) and limiting amounts of “homeostatic” cytokines might block even more powerful antitumor immunity. Transfer of tumor-specific lymphocytes into lymphodepleted recipients may eliminate these immune-regulatory obstacles. Methods: Splenocytes and bone marrow from CpG/CTX vaccinated mice were transplanted into lethally irradiated recipients, i.e., “immunotransplant.” Recipients received high dose tumor challenge with A20 murine B-cell lymphoma and followed clinically for tumor growth and for tumor-specific CD8 T cell IFNγ responses by multi-color flow cytometry. Results: We observed selective filtering against Tregs, doubling of the transferred Teffector:Treg ratio, and preferential expansion of transferred tumor-specific CD8 T cells in immunotransplant recipients. Immunotransplant cures large subcutaneous (&amp;gt;100 mm2) and metastatic tumors in 100% of recipients and enhances tumor-specific T cell memory. The addition of a vaccine “boost” to the immunotransplant maneuver results in an additional 3-fold increase in tumor-specific immunity and the use of cured recipients as subsequent donors in serial immunotransplant experiments demonstrated even greater tumor protection. Conclusions: As hematopoietic stem cell transplantation is a standard therapy for lymphoma patients, pre-transplant vaccination followed by post-transplant transfer of tumor-specific T cells could be tested in clinical trials. Our model suggests that this immunotransplant approach would allow the benefit of cancer vaccines to be revealed and might lead to cure of otherwise resistant malignancies.

Evaluation of immune responses in HIV infected patients with pleural tuberculosis by the QuantiFERON®TB-Gold interferon-gamma assay

BackgroundDiagnosis of tuberculous (TB) pleuritis is difficult and better diagnostic tools are needed. New blood based interferon-gamma (IFN-γ) tests are promising, but sensitivity could be low in HIV positive patients. The IFN-γ tests have not yet been validated for use in pleural fluid, a compartment with higher level of immune activation than in blood.MethodsThe QuantiFERON TB®-Gold (QFT-TB) test was analysed in blood and pleural fluid from 34 patients presenting with clinically suspected pleural TB. Clinical data, HIV status and CD4 cell counts were recorded. Adenosine deaminase activity (ADA) analysis and TB culture were performed on pleural fluid.ResultsThe patients were categorised as 'confirmed TB' (n = 12), 'probable TB' (n = 16) and 'non-TB' pleuritis (n = 6) based on TB culture results and clinical and biochemical criteria. The majority of the TB patients were HIV infected (82%). The QFT-TB in pleural fluid was positive in 27% and 56% of the 'confirmed TB' and 'probable TB' cases, respectively, whereas the corresponding sensitivities in blood were 58% and 83%. Indeterminate results in blood (25%) were caused by low phytohemagglutinin (PHA = positive control) IFN-γ responses, significantly lower in the TB patients as compared to the 'non-TB' cases (p = 0.02). Blood PHA responses correlated with CD4 cell count (r = 0.600, p = 0.028). In contrast, in pleural fluid indeterminate results (52%) were caused by high Nil (negative control) IFN-γ responses in both TB groups. Still, the Nil IFN-γ responses were lower than the TB antigen responses (p < 0.01), offering a conclusive test for half of the patients. We did not find any correlation between blood CD4 cell count and IFN-γ responses in pleural fluid.ConclusionThe QFT-TB test in blood could contribute to the diagnosis of TB pleuritis in the HIV positive population. Still, the number of inconclusive results is too high to recommend the commercial QFT-TB test for routine use in pleural fluid in a TB/HIV endemic resource-limited setting.

Functional CD8+ T Cell Responses in Lethal Ebola Virus Infection

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4(+) and CD8(+) T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8(+) T cell IFN-gamma responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8(+) T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8(+) cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the 'impaired' immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.

T Regulatory Cells Contribute to the Attenuated Primary CD8+ and CD4+ T Cell Responses to Herpes Simplex Virus Type 2 in Neonatal Mice

The first weeks of life are characterized by immune tolerance and increased susceptibility to intracellular pathogens. The neonatal adaptive response to HSV is attenuated compared with adult control models in humans and mice. T Regulatory cells (Tregs) control autoimmunity and excessive immune responses to infection. We therefore compared Treg responses in the draining lymph nodes (LN) of HSV-infected neonatal and adult C57BL/6 mice with the effect of Treg depletion/inactivation by anti-CD25 (PC61) treatment before infection on Ag-specific T cell effector responses at this site. There was a small, but significant increase in the frequency of CD4(+)Foxp3(+) Tregs at day 3 postinfection (p.i.) in the LN of neonatal and adult mice, compared with age-matched mock-infected controls. Depletion of Tregs before HSV infection significantly enhanced HSV-specific CD8(+) T cell cytotoxicity in vivo, cell number, activation, and granzyme B expression 4 days p.i. only in neonatal mice, and significantly enhanced CD8(+) and CD4(+) T cell IFN-gamma responses in both infected adults and neonates. Treg depletion also reduced the titer of infectious virus in the draining LN and nervous system of infected neonates on days 2 and 3 p.i. Treg suppression of the neonatal CTL response p.i. with HSV was associated with increased expression of TGF-beta in the draining LN at day 4 p.i. compared with uninfected neonates, but IL-10 was increased in infected adults alone. These experiments support the notion that the newborn primary T cell effector responses to HSV are suppressed by Tregs.

TAP-1 indirectly regulates CD4+ T cell priming in Toxoplasma gondii infection by controlling NK cell IFN-γ production

To investigate if transporter associated with antigen processing (TAP)–1 is required for CD8+ T cell–mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP-1−/−, CD8−/−, and wild-type (WT) mice to infection with the parasite. Unexpectedly, TAP-1−/− mice displayed greater susceptibility than CD8−/−, β2-microglobulin−/− (β2m−/−), or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP-1−/− mice correlated with a reduction in the frequency of activated (CD62Llow CD44hi) and interferon (IFN)-γ–producing CD4+ T cells. Interestingly, infected TAP-1−/− mice also showed reduced numbers of IFN-γ–producing natural killer (NK) cells relative to WT, CD8−/−, or β2m−/− mice, and after NK cell depletion both CD8−/− and WT mice succumbed to infection with the same kinetics as TAP-1−/− animals and displayed impaired CD4+ T cell IFN-γ responses. Moreover, adoptive transfer of NK cells obtained from IFN-γ+/+, but not IFN-γ−/−, animals restored the CD4+ T cell response of infected TAP-1−/− mice to normal levels. These results reveal a role for TAP-1 in the induction of IFN-γ–producing NK cells and demonstrate that NK cell licensing can influence host resistance to infection through its effect on cytokine production in addition to its role in cytotoxicity.

The Induction and Persistence of T Cell IFN-γ Responses after Vaccination or Natural Exposure Is Suppressed byPlasmodium falciparum

Epidemiological observations suggest that T cell immunity may be suppressed in malaria-endemic areas. In vitro studies, animal models, and limited data in humans link immunosuppression with malaria, malnutrition, and other parasitic infections. However, there are no data to determine whether malaria-induced immunosuppression is significant in the long-term, or relative data comparing it with other factors in malaria-endemic areas, so as to measure the impact of malaria, other parasitic disease, nutritional status, age. and location on the acquisition and longevity of IFN-gamma responses in children in Kenya. We studied these factors in two cohorts of 1- to 6-year-old children in a malaria-endemic area. T cell responses were induced by vaccination in one cohort, and acquired as a result of natural exposure in a second cohort. Serial ELISPOT assays conducted over a 1-year period measured the induction and kinetics of IFN-gamma production in response to the malaria Ag thrombospondin-related adhesion protein. Induced responses in both cohorts and the longevity of response in the vaccinated cohort were fitted to potential explanatory variables. Parasitemia was prospectively associated with reduced IFN-gamma-producing T cells in both cohorts (by 15-25%), and both parasitemia and episodes of febrile malaria were associated with 19 and 31% greater attrition of T cell responses, respectively. Malaria may reduce the efficacy vaccinations such as bacillus Calmette-Guérin and investigational T cell-inducing vaccines, and may delay the acquisition of immunity following natural exposure to malaria and other pathogens.