Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).

Galectins are a family of animal lectins defined by two properties: shared amino acid sequences in their carbohydrate-recognizing domain, and beta-galactoside affinity. A wide variety of biological phenomena are related to galectins, i.e., development, differentiation, morphogenesis, tumor metastasis, apoptosis, RNA splicing, and immunoregulatory function. In this review, we will focus on galectin-1 receptors, and some of the mechanisms by which this lectin affects different cell types. Several galectin-1 receptors are discussed such as CD45, CD7, CD43, CD2, CD3, CD4, CD107, CEA, actin, extracellular matrix proteins such as laminin and fibronectin, glycosaminoglycans, integrins, a beta-lactosamine glycolipid, GM1 ganglioside, polypeptide HBGp82, glycoprotein 90 K/MAC-2BP, CA125 cancer antigen, and pre-B cell receptor.

Mesenchymal stromal cells (MSCs) are multipotent cells that possess broad immunomodulatory properties; the mechanisms underlying these properties have not been completely clarified. Aim of this study was to compare in vitro immunomodulatory effects of MSCs with those of microvesicles (MVs) released in supernatants from the same MSCs. MSCs were generated from bone marrow of 12 healthy donors (HDs) and MVs were isolated from their supernatant by serial ultracentrifugation according to two different procedures. Both MSCs and MVs were characterized by flow cytometry and incubated in vitro with peripheral blood mononuclear cells (PBMCs) of 12 HDs after stimulation with PHA and CpG. Growth factors and cytokines were quantified by ELISA. MVs were identified as 0.1-1 μm particles positive for CMFDA, CD107, and CD13. MSCs were significantly more capable to inhibit in vitro PHA-induced T-cell proliferation as compared with the corresponding MVs (P<0.01 and P<0.05 for MSC:PBMC ratio 1:2 and 1:10, respectively). While MVs displayed similar inhibitory activity on B-cell proliferation (P=0.43 as compared with PBMCs/CpG/MSCs; MSC:PBMC ratio 1:10) they induced lower inhibitory effect on plasmacell differentiation and antibody secretion (P<0.05 as compared with PBMCs/CpG/MSCs). For both T and B cells, MSC co-colture induced a statistically significant increase in IL-10 and TGFβ and decrease of GM-CSF and IFNγ, as compared with MV incubation. Our data indicate a lower in vitro immunomodulatory effect of MVs on T-cell proliferation and antibody formation, as compared with their cellular counterpart. The relative clinical benefit of either MSCs or MVs needs to be compared in proper prospective studies.

T cells are likely to play an important role in the host defense against Salmonella enterica serovar Typhi, the causative agent of typhoid fever. We have shown that HLA-E can function as a restriction element for S. Typhi-specific CD8(+) T cells. Because of the potential importance of HLA-E-restricted CD8(+) responses in resistance to Salmonella infection, we characterized these responses and investigated their kinetics of appearance and persistence in volunteers immunized orally with the licensed attenuated Ty21a strain typhoid vaccine. Cells were obtained from volunteers before and at days 2, 4, 7, 10, 14, 28, 42, 56, 120, 180, 360, and 720 after immunization. An ex vivo multicolor staining panel including antibodies to CD107a and -b, interleukin-2, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) was used to functionally assess memory T-cell subsets by flow cytometry. Increases in cytokine-secreting CD8(+) cells were observed in the T effector/memory (T(EM)) and CD45RA(+) T(EM) (T(EMRA)) subsets as early as 4 days after immunization and persisted, particularly in the T(EMRA) subset, up to 2 years after immunization. The majority of HLA-E-restricted CD8(+) cells 28 to 56 days after immunization coexpressed CD107, IFN-gamma, and TNF-alpha, showing characteristic features of multifunctional T cells. In summary, the multifunctionality and longevity of the HLA-E-restricted CD8 responses observed in this study highlight their significance in adaptive immunity to S. Typhi. Finally, this is the first demonstration, in either animals or humans, of the presence of long-term multifunctional HLA-E-restricted CD8(+) cells after immunization.

BK polyomavirus (BKV) reactivation is associated with a failure of T cell immunity in kidney transplant patients, and may lead to BKV-associated nephropathy (BKVN) and loss of the allograft. BKV reactivation in hematopoietic stem cell transplant recipients is associated with hemorrhagic cystitis. We have investigated T cell responses to overlapping peptide mixtures corresponding to the whole BKV major T antigen (TAg) and major capsid protein (VP1) in peripheral blood mononuclear cell samples from a cohort of healthy BKV-seropositive subjects. The majority of these individuals possessed populations of both CD8(+) and CD4(+) T cells specific for these BKV antigens. After expansion in culture, the majority of the BKV-specific CD4(+) T cells, in addition to expressing CD40L (CD154), secreted both interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, contained both granzyme A and granzyme B, and degranulated/mobilized CD107 in response to antigen-specific stimulation. These T cells thus represent potentially functional BKV-specific cytotoxic CD4(+) T lymphocytes. Secretion of both TNF-alpha and IFN-gamma by CD154(+)CD4(+) T cells on BKV-specific stimulation was associated with higher levels of granzyme B and a higher proportion of degranulating cells compared with CD154(+)CD4(+) T cells producing only IFN-gamma or neither cytokine. These healthy subjects also harbored populations of functional CD8(+) T cells specific for one or more of three newly defined HLA-A 02-restricted cytotoxic T lymphocyte epitopes within the BKV TAg as well as two HLA-A 02-restricted epitopes within the BKV VP1 we have previously described. The BKV-specific CD4(+) T cells characterized in this study may play a part in maintaining persistent memory T cell responses to the virus and thus contribute to the immune control of BKV in healthy individuals.

OBJECTIVE

There is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets.

METHODS

GDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr(51)-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry.

RESULTS

Longer initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1).

CONCLUSIONS

Our ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.

The recently reported FACS-based CD107 assay has been used in human HIV and CMV antigen models as well as in the ex vivo analysis of tumor cytolytic T cells in a melanoma model by a single group. The purpose of our study was to validate this assay and to use it in previously untested viral and tumor antigen models. Specifically, we investigated the use of the novel CD107 cytotoxicity assay in the detection of influenza and HER2/neu tumor-specific cytolytic CD8+ T cells. CD8+ T cells from HLA-A2+ healthy donors were stimulated with autologous dendritic cells pulsed with FluM or the HER2/neu peptides, E75 or GP2. These CD8+ T cells were then tested in cytotoxicity assays at varying effector:target (E:T) ratios against T2 targets. Cytotoxicity was measured by detection of CD107a and b on the surface of CD8+ T cells. An E:T of 1:5 was found to optimize the resulting percentage of CD8+CD107+ T cells. E75- and GP2-stimulated CD8+ T cells were then tested in cytotoxicity assays with MCF-7 (HER2/neu+HLA-A2+) and AU565 (HER2/neu+HLA-A2-) tumor cells. Cytotoxicity was measured by both the CD107 assay and the (51)Cr release assay. Results of cytotoxicity were then correlated between these two assays. In representative experiments, the CD107 assay identified average specific increases for E75- and GP2-stimulated cells of 4.26 and 3.57%, respectively. These results correlated favorably with cytotoxicity as measured by the traditional (51)Cr assay. These findings confirm preliminary reports of the CD107 assay and suggest its usefulness for monitoring cancer trials.

The HSV type 1 tegument virion phosphoprotein (VP) 11/12 (VP11/12) is a major Ag targeted by CD8(+) T cells from HSV-seropositive individuals. However, whether and which VP11/12 epitope-specific CD8(+) T cells play a role in the "natural" protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8(+) T cell epitopes from the 718-aa sequence of VP11/12. Three of 10 epitopes exhibited high-to-moderate binding affinity to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive and HSV-1-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional effector CD8(+) T cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107(a/b) cytotoxic degranulation, IFN-γ, and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266-74, VP11/12220-228, and VP11/12702-710. Interestingly, ASYMP individuals had a significantly higher proportion of CD45RA(low)CCR7(low)CD44(high)CD62L(low)CD27(low)CD28(low)CD8(+) effector memory CD8(+) T cells (TEMs) specific to the three epitopes, compared with symptomatic individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8(+) TEM cell epitopes induced robust and polyfunctional epitope-specific CD8(+) TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8(+) T cells that should guide the development of an effective T cell-based herpes vaccine.

Immunologic memory induced by pathogenic agents or vaccinations is inextricably linked to long-lasting protection. Adequately maintained memory T and B cell pools assure a fast, effective, and specific response against re-infections. Studies of immune responses amongst residents of malaria endemic areas suggest that memory responses to Plasmodia antigens appear to be neither adequately developed nor maintained, because persons who survive episodes of childhood malaria remain vulnerable to persistent or intermittent malaria infections. By contrast, multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodia sporozoites (γ-spz) induces sterile and long-lasting protection against experimental sporozoite challenge. Protection is associated with MHC-class I-dependent CD8 T cells, the key effectors against pre-erythrocytic stage infection. We have adopted the P. berghei γ-spz mouse model to study memory CD8 T cells that are specific for antigens expressed by Pb liver-stage (LS) parasites and are found predominantly in the liver. On the basis of phenotypic and functional characteristics, we have demonstrated that liver CD8 T cells form two subsets: CD44(hi)CD62L(lo)KLRG-1(+)CD107(+)CD127(-)CD122(lo)CD8 T effector/effector memory (T(E/EM)) cells that are the dominant IFN-γ producers and CD44(hi)CD62L(hi)KLRG-1(-)CD107(-)CD127(+)CD122(hi)CD8 T central memory (T(CM)) cells. In this review, we discuss our observations concerning the role of CD8 T(E/EM) and CD8 T(CM) cells in the maintenance of protracted protective immunity against experimental malaria infection. Finally, we present a hypothesis consistent with a model whereby intrahepatic CD8 T(CM) cells, that are maintained in part by LS-Ag depot and by IL-15-mediated survival and homeostatic proliferation, form a reservoir of cells ready for conscription to CD8 T(E/EM) cells needed to prevent re-infections.

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8(+) T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8(+) T cells play a key role in the "natural" protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8(+) T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)). In contrast, SYMP patients had frequent less-differentiated central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8(+) T cells which responded mainly to gBBCD107(a/b), granzyme B, and perforin. In contrast, effector CD8(+) T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gBBbut not with "SYMP" CD8(+) TCM cell epitopes, induced a strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8(+) TEM cells in protection against herpes and should be considered in the development of an effective vaccine.

OBJECTIVE

A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)) were found in healthy ASYMP individuals who are seropositive for HSV-1 but never had any recurrent herpetic disease, while there were frequent less-differentiated and monofunctional central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)) in SYMP patients. Immunization with "ASYMP" CD8(+) TEM cell epitopes, but not with "SYMP" CD8(+) TCM cell epitopes, induced a strong protective HSV-specific CD8(+) T cell response in HLA-A*02:01 transgenic mice. These findings are important for the development of a safe and effective T cell-based herpes vaccine.

We previously demonstrated that autologous dendritic cells that have endocytosed apoptotic bodies of chronic lymphocytic leukemia (CLL) cells (Apo-DC) can stimulate antileukemic T cell responses in vitro. In this phase I study, we vaccinated 15 asymptomatic CLL patients at five time points with Apo-DC administered intradermally either alone (cohort I), or in combination with subcutaneous granulocyte-macrophage-colony-stimulating-factor (GM-CSF) (cohort II) or with GM-CSF and intravenous low-dose cyclophosphamide (cohort III). Aim of the study was to evaluate the safety and immunogenicity of Apo-DC alone or in combination with GM-CSF and low-dose cyclophosphamide in CLL patients. All patients completed the vaccination schedule without dose-limiting toxicity. No objective clinical responses were seen. Vaccine-induced leukemia-specific immune responses were evaluated by IFN-γ ELISpot and proliferation assays over a 52 weeks observation period and immune response criteria were defined. According to these criteria, 10/15 patients were defined as immune responders. The frequency of immune-responding patients was higher in cohorts II (3/5) and III (5/5) than in cohort I (2/5). In order to further characterize the induced immune response, estimation of secreted cytokines and CD107-degranulation assay were performed. Clustering of T and CLL cells was observed in CD107-degranulation assay and visualized by confocal microscopy. Additionally, assessment of regulatory T cells (T(regs)) revealed their significantly lower frequencies in immune responders versus non-responders (P < 0.0001). Cyclophosphamide did not reduce T(regs) frequency. In conclusion, vaccination with Apo-DC + GM-CSF and cyclophosphamide was safe and elicited anti-CLL immune responses that correlated inversely with T(regs) levels. Lack of clinical responses highlights the necessity to develop more potent vaccine strategies in B cell malignancies.

The immune control of hepatitis B virus (HBV) infection is essential for viral clearance. Therefore, restoring functional anti-HBV immunity is a promising immunotherapeutic approach to treatment of chronic infection. Plasmacytoid dendritic cells (pDCs) play a crucial role in triggering antiviral immunity through their ability to capture and process viral antigens and subsequently induce adaptive immune responses. We investigated the potential of pDCs to trigger antiviral cellular immunity against HBV. We used a human leukocyte antigen A (HLA-A)*0201(+) pDC line loaded with HLA-A*0201-restricted peptides derived from hepatitis B core/hepatitis B surface (HBc/HBs) antigens to amplify specific CD8 T cells ex vivo from chronic HBV patients and established a Hepato-HuPBL mouse model to address the therapeutic potential of the strategy in vivo. Stimulation of PBMCs or liver-infiltrating lymphocytes from HLA-A*0201(+) chronic HBV patients by HBc peptide-loaded pDCs elicited up to 23.1% and 76.1% HBV-specific CD8 T cells in 45.8% of cases. The specific T cells from the "responder" group secreted interferon-γ, expressed CD107 upon restimulation, and efficiently lysed HBV antigen-expressing hepatocytes. Circulating hepatitis B e antigen (HBeAg) was found to distinguish the group of patients not responding to the pDC stimulation. The therapeutic efficacy of the pDC vaccine was evaluated in immunodeficient NOD-SCID β(2) m(-/-) mice reconstituted with HBV patients' PBMCs and xenotransplanted with human HBV-transfected hepatocytes. Vaccination of Hepato-HuPBL mice with the HBc/HBs peptide-loaded pDCs elicited HBV-specific T cells able to specifically lyse the transfected hepatocytes and reduce the systemic viral load.

CONCLUSIONS

pDCs loaded with HBV-derived peptides can elicit functional virus-specific T cells. HBeAg appears to be critical in determining the outcome of immunotherapies in chronic HBV patients. A pDC-based immunotherapeutic approach could be of interest in attempts to restore functional antiviral immunity, which is critical for the control of the virus in chronic HBV patients.

BACKGROUND

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers.

METHODS

HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA.

RESULTS

The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients.

CONCLUSIONS

Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use.

BACKGROUND

International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1 dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-β mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4 dpi. In contrast, IFN-γ production in blood was highest at 1 dpi and decreased over time down to levels comparable to uninfected birds at 4 dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-β compared to uninfected birds.

OBJECTIVE

A clinical vaccine that protects from ocular herpes simplex virus type 1 (HSV-1) infection and disease still is lacking. In the present study, preclinical vaccine trials of nine asymptomatic (ASYMP) peptides, selected from HSV-1 glycoproteins B (gB), and tegument proteins VP11/12 and VP13/14, were performed in the "humanized" HLA-transgenic rabbit (HLA-Tg rabbit) model of ocular herpes. We recently reported that these peptides are highly recognized by CD8+ T cells from "naturally" protected HSV-1-seropositive healthy ASYMP individuals (who have never had clinical herpes disease).

METHODS

Mixtures of three ASYMP CD8+ T-cell peptides derived from either HSV-1 gB, VP11/12, or VP13/14 were delivered subcutaneously to different groups of HLA-Tg rabbits (n = 10) in incomplete Freund's adjuvant, twice at 15-day intervals. The frequency and function of HSV-1 epitope-specific CD8+ T cells induced by these peptides and their protective efficacy, in terms of survival, virus replication in the eye, and ocular herpetic disease were assessed after an ocular challenge with HSV-1 (strain McKrae).

RESULTS

All mixtures elicited strong and polyfunctional IFN-γ- and TNF-α-producing CD107+CD8+ cytotoxic T cells, associated with a significant reduction in death, ocular herpes infection, and disease (P < 0.015).

CONCLUSIONS

The results of this preclinical trial support the screening strategy used to select the HSV-1 ASYMP CD8+ T-cell epitopes, emphasize their valuable immunogenic and protective efficacy against ocular herpes, and provide a prototype vaccine formulation that may be highly efficacious for preventing ocular herpes in humans.

Alzheimerś disease (AD) is a progressive irreversible neurological brain disorder characterized by accumulation of amyloid-β, amyloid plaques, and neurofibrillary tangles. Inflammation and immune alterations have been linked to AD, suggesting that the peripheral immune system plays a role during the asymptomatic period of AD. NK cells participate in innate immune surveillance against intracellular pathogens and malignancy but their role in AD remains controversial. We have investigated changes in peripheral NK cell phenotypes and functions in amnestic mild cognitive impairment (aMCI, n = 10), patients with mild AD (mAD, n = 11), and healthy elderly controls (n = 10). Patients selected according to NINCDS-ADRDA criteria were classified using neuropsychological assessment tests. Phenotype analysis revealed differences in expression of CD16 (increased in mAD), NKG2A (decreased in aMCI), and TLR2 and TLR9 (both decreased in mAD). Functional assays revealed that NK cell killing activity and degranulation (CD107 expression) were unchanged in the three groups. In contrast, expression of the CD95 receptor was increased in aMCI and mAD. Granzyme B expression and cytokine production (TNFα, IFNγ) were increased in aMCI but not in mAD. CCL19- but not CCL21-dependent chemotaxis was decreased in aMCI and mAD, despite the fact that CCR7 expression was increased in aMCI. Our data suggest that the number of alterations observed in peripheral NK cells in aMCI represent an activation state compared to mAD patients and that may reflect an active immune response against a still to be defined aggression.

(<em>b</em>)Background:</<em>b</em>) Natural Killer (NK) cell-<em>b</em>ased immunotherapy used to treat cancer requires the adoptive transfer of a large num<em>b</em>er of activated NK cells. Here, we report a new effective method to expand human NK cells <i>ex vivo</i> using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in com<em>b</em>ination with a short exposure to solu<em>b</em>le IL-21. In addition, we descri<em>b</em>e a possi<em>b</em>le mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction. (<em>b</em>)Methods:</<em>b</em>) K562-OX40L cells were generated <em>b</em>y lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Solu<em>b</em>le IL-21 was also added in various concentrations only once at the <em>b</em>eginning of the culture. NK cells were expanded for 4-5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, anti<em>b</em>ody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, <em>CD107</em>a degranulation) of these expanded NK cells were compared to those generated <em>b</em>y using K562 feeder cells. (<em>b</em>)Results:</<em>b</em>) The culture of NK cells with K562-OX40L cells in com<em>b</em>ination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2,000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis <em>b</em>etween the feeder cells and NK cells as well as the homotypic interaction <em>b</em>etween NK cells through the OX40-OX40L axis were <em>b</em>oth necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and <em>CD107</em> degranulation. (<em>b</em>)Conclusion:</<em>b</em>) Our data suggest that OX40 ligand is a potent co-stimulant for the ro<em>b</em>ust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.

Chronic viruses such as herpes simplex virus 1 (HSV-1) evade the hosts' immune system <em>b</em>y inducing the exhaustion of antiviral T cells. In the present study, we found that exhausted HSV-specific CD8⁺ T cells, with elevated expression of programmed death ligand-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) receptors were frequent in symptomatic patients, with a history of numerous episodes of recurrent corneal herpetic disease, compared to asymptomatic patients who never had corneal herpetic disease. Su<em>b</em>sequently, using a ra<em>b</em><em>b</em>it model of recurrent ocular herpes, we found that the com<em>b</em>ined <em>b</em>lockade of PD-1 and LAG-3 pathways with antagonist anti<em>b</em>odies significantly restored the function of tissue-resident antiviral CD8⁺ T<su<em>b</em>)RM</su<em>b</em>) cells in <em>b</em>oth the cornea and the trigeminal ganglia (TG). An increased num<em>b</em>er of functional tissue-resident HSV-specific CD8⁺ T<su<em>b</em>)RM</su<em>b</em>) cells in latently infected ra<em>b</em><em>b</em>its was associated with protection against recurrent herpes infection and disease. Compared to the PD-1 or LAG-3 <em>b</em>lockade alone, the com<em>b</em>ined <em>b</em>lockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional Ki-67⁺, IFN-γ⁺, <em>CD107</em>⁺, and CD8⁺ T cells. Moreover, using the human leukocyte antigen (HLA) transgenic ra<em>b</em><em>b</em>it model, we found that dual <em>b</em>lockade of PD-1 and LAG-3 reinforced the effect of a multiepitope vaccine in <em>b</em>oosting the frequency of HSV-1-specific CD8⁺ T<su<em>b</em>)RM</su<em>b</em>) cells and reducing disease severity. Thus, <em>b</em>oth the PD-1 and the LAG-3 exhaustion pathways play a fundamental role in ocular herpes T cell immunopathology and provide important immune checkpoint targets to com<em>b</em>at ocular herpes.(<em>b</em>)IMPORTANCE</<em>b</em>) HSV-specific tissue-resident memory CD8⁺ T<su<em>b</em>)RM</su<em>b</em>) cells play a critical role in preventing virus reactivation from latently infected TG and su<em>b</em>sequent virus shedding in tears that trigger the recurrent corneal herpetic disease. In this report, we determined how the dual <em>b</em>lockade of PD-1 and LAG-3 immune checkpoints, com<em>b</em>ined with vaccination, improved the function of CD8⁺ T<su<em>b</em>)RM</su<em>b</em>) cells associated with a significant reduction in recurrent ocular herpes in HLA transgenic (Tg) ra<em>b</em><em>b</em>it model. The com<em>b</em>ined <em>b</em>lockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional CD8⁺ T<su<em>b</em>)RM</su<em>b</em>) cells that infiltrated <em>b</em>oth the cornea and the TG. The preclinical findings using the esta<em>b</em>lished HLA Tg ra<em>b</em><em>b</em>it model of recurrent herpes highlight that <em>b</em>locking immune checkpoints com<em>b</em>ined with a T cell-<em>b</em>ased vaccine would provide an important strategy to com<em>b</em>at recurrent ocular herpes in the clinic.

Human glioma facilitates an impaired anti-tumor immunity response, including defects in circulation of T lymphocytes. The level of CD8⁺ T-cell activation acts as an immune regulator associated with disease progression. However, little is known about the characteristics of peripheral and tumor-infiltrating CD8⁺ T cells in patients with glioma. In this study, we examined the level of CD8⁺ T-cell activation in a group of 143 patients with glioma and determined that peripheral CD3⁺ T cells decreased in accordance with disease severity. The patients' peripheral CD8⁺ T-cell populations were similar to that of healthy donors, and a small amount of CD8⁺ tumor-infiltrating lymphocytes was identified in glioma tissues. An increase in activated CD8⁺ T cells, characterized as CD38⁺HLA-DR⁺, and their association with disease progression were identified in the patients' peripheral blood and glioma, and shown to display enriched CCR5⁺ and TNFR2⁺ expression levels. Ex vivo examination of CD38⁺HLA-DR⁺CD8⁺ T cells indicated that this subset of cells displayed stronger secretion of IFN-γ and IL-2 before and after a 6-h stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) relative to healthy CD38⁺HLA-DR⁺CD8⁺ T cells, indicating the functional feasibility of CD38⁺HLA-DR⁺CD8⁺ T cells. Higher CCL5 protein and mRNA levels were identified in glioma tissues, which was consistent with the immunohistochemistry results revealing both CCL5 and CD38⁺HLA-DR⁺CD8⁺ T cell expression. Patients' CCR5⁺CD38⁺HLA-DR⁺CD8⁺ T cells were further validated and shown to display increases in CD45RA⁺CCR7^- and T-bet⁺ accompanied by substantial CD107-a, IFN-γ, and Granzyme B levels in response to glioma cells.

Chronic hepatitis C virus (HCV) infection disrupts immune functions, including that of cytotoxic CD8⁺ T-cells which are important mediators of immune response. While HCV cure aims to eliminate long term sequelae of infection, whether direct-acting antiviral (DAA) treatment results in immune reconstitution remains unclear. We and others have reported generalized CD8⁺ T-cell dysfunction in chronic HCV infection and our research suggests that the degree of liver damage is a factor in this process. Our recent research indicates that liver fibrosis is not readily reversed after DAA-mediated clearance of chronic HCV infection. We therefore examined the function of circulating CD8⁺ T-cell subsets in chronic HCV infection in the context of liver fibrosis severity, determined by ultrasound elastography and Metavir F-score system. We observed progressive shifts in CD8⁺ T-cell subset distribution in HCV-infected individuals with advanced liver fibrosis (F4) compared to minimal fibrosis (F0-1) or uninfected controls, and this remained unchanged after viral cure. Impaired CD8⁺ T-cell function was observed as a reduced proportion of CD107⁺ and perforin⁺ late effector memory cells in HCV⁺(F4) and HCV⁺(F0-1) individuals, respectively. In HCV⁺(F4) individuals, nearly all CD8⁺ T-cell subsets had an elevated proportion of perforin⁺ cells while naïve cells had increased proportions of IFN-γ⁺ and CD107⁺ cells. These exaggerated CD8⁺ T-cell activities were not resolved when evaluated 24 weeks after completion of DAA therapy and HCV clearance. This was further supported by sustained, high levels of cell proliferation and cytolytic activity. Furthermore, DAA therapy had no effect on elevated concentrations of systemic inflammatory cytokines and decreased levels of inhibitory TGF-β in the plasma of HCV⁺(F4) individuals, suggesting HCV infection and advanced liver disease result in a long-lasting immune activating microenvironment. These data demonstrate that in chronic HCV infection, liver fibrosis severity is associated with generalized hyperfunctional CD8⁺ T-cells, particularly with perforin production and cytotoxicity, and this persists after viral clearance. Whether DAA therapy will eliminate other related long-term sequelae in HCV⁺(F4) individuals remains an important research question.

<A<em>b</em>stractText>This study was aimed at evaluating the feasi<em>b</em>ility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNα and GM-CSF (IFN-DC) in com<em>b</em>ination with low doses of rituxima<em>b</em>.</A<em>b</em>stractText><p><div>(<em>b</em>)PATIENTS AND METHODS</<em>b</em>)</div>Firstly, we analyzed <i>in vitro</i> and <i>in vivo</i> the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma <em>b</em>ased on sequential intranodal injections of low-dose of rituxima<em>b</em> and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients.</p><A<em>b</em>stractText>Preclinical studies indicated that IFN-DC can synergize with rituxima<em>b</em> leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the com<em>b</em>ined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11-47). Nota<em>b</em>ly, following the com<em>b</em>ined therapy all patients showed induction/enhancement of T-cell responses <em>b</em>y <em>CD107</em> degranulation or IFNγ ELISPOT assay against patient-specific tumor IGHV sequences.</A<em>b</em>stractText><A<em>b</em>stractText>These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing dura<em>b</em>le clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can <em>b</em>e successfully exploited to induce/enhance antitumor responses, thus representing a valua<em>b</em>le antitumor strategy for novel and more effective com<em>b</em>ination therapies in patients with cancer.</A<em>b</em>stractText>

Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) are critical for the immune response against virus infections or transformed cells. They kill target cells via polarized exocytosis of lytic proteins from secretory lysosomes (SL). Rab27a and munc13-4 interact directly and are required for target cell killing. How they cooperate in the intricate degranulation process is not known. We identified critical residues in munc13-4 for rab27 interaction and tested binding mutants in several complementation assays. In a rat mast cell line we replaced endogenous munc13-4 with ectopically expressed munc13-4 constructs. Unlike wild type munc13-4, binding mutants fail to rescue β-hexosaminidase secretion. In accord, expression of binding mutants in CTL of Familial Hemophagocytic Lymphohistiocytosis type 3 patients, does not rescue CD107 appearance on the plasma membrane. Total Internal Reflection Fluorescence (TIRF) imaging shows that munc13-4*rab27a restricts motility of SL in the subapical cytoplasm. We propose that rab27*munc13-4 tethers SL to the plasma membrane, a requirement for formation of a cognate SNARE complex for fusion.

γδ T cells act as a first line of defense against invading pathogens. However, despite their abundance in mucosal tissue, little information is available about their functionality in this compartment in the context of HIV/SIV infection. In this study, we evaluated the frequency, phenotype, and functionality of Vδ1 and Vδ2 T cells from blood, rectum, and the female reproductive tract (FRT) of rhesus macaques to determine whether these cells contribute to control of SIV infection. No alteration in the peripheral Vδ1/Vδ2 ratio in SIV-infected macaques was observed. However, CD8+ and CD4+CD8+ Vδ1 T cells were expanded along with upregulation of NKG2D, CD107, and granzyme B, suggesting cytotoxic function. In contrast, Vδ2 T cells showed a reduced ability to produce the inflammatory cytokine IFN-γ. In the FRT of SIV+ macaques, Vδ1 and Vδ2 showed comparable levels across vaginal, ectocervical, and endocervical tissues; however, endocervical Vδ2 T cells showed higher inflammatory profiles than the two other regions. No sex difference was seen in the rectal Vδ1/Vδ2 ratio. Several peripheral Vδ1 and/or Vδ2 T cell subpopulations expressing IFN-γ and/or NKG2D were positively correlated with decreased plasma viremia. Notably, Vδ2 CD8+ T cells of the endocervix were negatively correlated with chronic viremia. Overall, our results suggest that a robust Vδ1 and Vδ2 T cell response in blood and the FRT of SIV-infected macaques contribute to control of viremia.

The safe and effective activation of the innate and adaptive immune systems are crucial in the implementation of immunotherapeutic modalities for the prevention and treatment of human diseases. Eimeria antigen (EA) and its recombinantly expressed analog (rEA) are extremely effective activators of innate immunity in mice. The effects of rEA in the mouse are primarily mediated through the TLR11/12 MyD88 signaling system. Human cells lack functional TLR11 and TLR12, suggesting that rEA would not be effective in providing beneficial immune activation in humans. In the current report we provide definitive evidence that the treatment of human peripheral blood mononuclear cell (PBMC) cultures with rEA significantly up regulates CD69, CD107, NKG2D levels on NK cells. Furthermore, rEA stimulates human NK cell effector functions including increasing intracellular levels of IFNγ and Granzyme B. These responses are positively correlated with an improved capacity of rEA stimulated human PBMCs to kill NK cell-sensitive human K562 tumor cells. Importantly, rEA-triggered innate immune responses was not associated with increased pro-inflammatory cytokines and chemokines production. These data confirm a previously unidentified role for rEA in human immune cell activation, and suggests the utilization of rEA in immunotherapies against a variety of infectious diseases and cancers.