Response Of Peripheral Blood Leukocytes Research Articles

First characterization of a teleost Epstein-Barr virus-induced gene 3 (EBI3) reveals a regulatory effect of EBI3 on the innate immune response of peripheral blood leukocytes

Epstein-Barr virus-induced gene 3 (EBI3) encodes a protein that in mammals is known to be a subunit of interleukin (IL)-27 and IL-35, both which regulate cytokine production and inflammatory response. To date, no studies on fish EBI3 have been documented. In this work, we report the identification of an EBI3 homologue, CsEBI3, from tongue sole (Cynoglossus semilaevis) and analysis of its expression and biological effect. CsEBI3 is composed of 245 amino acid residues and possesses a Fibronectin type 3 (FN3) domain that is preserved in lower and higher vertebrates. Expression of CsEBI3 was detected in a wide range of tissues, in particular those of immune relevant organs, and upregulated in a time-dependent manner by experimental challenge with bacterial and viral pathogens. Bacterial infection of peripheral blood leukocytes (PBL) enhanced CsEBI3 expression and caused extracellular secretion of CsEBI3. Purified recombinant CsEBI3 (rCsEBI3) stimulated the respiratory burst activity of PBL and upregulated the expression of IL-1β, IL-8, Myd88, interferon-induced gene 15, CD28, and chemokines. In contrast, rCsEBI3M, a mutant CsEBI3 that lacks the FN3 domain failed to activate PBL and induced much weaker expression of the immune genes. Treatment of PBL with rCsEBI3, but not with the mutant rCsEBI3M, enhanced cellular resistance against bacterial invasion, whereas antibody blocking of CsEBI3 on PBL significantly reduced cellular resistance against bacterial infection. Taken together, these results indicate for the first time that a teleost EBI3 possesses immunoregulatory property in a manner that is dependent on the conserved FN3 domain, and that CsEBI3 is involved in the innate immune defense of PBL against microbial pathogens.

SmLMWPTP, a teleost low molecular weight protein tyrosine phosphatase, inhibits the immune response of peripheral blood leukocytes in a manner that depends on the conserved P-loop

Protein tyrosine phosphatases (PTPs) are a family of enzymes that play a key role in cellular signal transduction. Low molecular weight PTPs (LMWPTPs) are a subfamily of PTPs that are characterized by the presence of a conserved phosphate-binding loop (P-loop) with the signature sequence of (V/I)CXGNXCRS. To date, very little study on teleost LMWPTPs has been documented, and, as a result, the function of LMWPTPs in fish is essentially unknown. In this study, we identified a LMWPTP from turbot (Scophthalmus maximus) and examined its biological activity and functionality. The turbot LMWPTP (SmLMWPTP) is composed of 158 residues and possesses a typical P-loop sequence in the form of 12VCLGNICRS20. Purified recombinant SmLMWPTP (rSmLMWPTP) exhibited apparent phosphatase activity, which was optimal at pH 5 and 50°C. The activity of SmLMWPTP was abolished when C13 and, in particular, R19 of the P-loop were mutated. SmLMWPTP expression was detected in a wide range of tissues and upregulated by bacterial and viral infection. Subcellular localization analysis showed that SmLMWPTP was secreted by peripheral blood leukocytes (PBL) into the extracellular milieu. When PBL were treated with rSmLMWPTP, the cells exhibited significant reductions in (i) proliferative and respiratory burst activity, (ii) expression levels of multiple immune relevant genes, and (iii) phagocytic activity. In contrast, the mutant SmLMWPTP bearing R19 mutation had no effect on PBL activity. Taken together, these results indicate that SmLMWPTP is a secreted PTP that exerts a negative regulatory effect on the innate immune response of PBL in a manner that depends on the structural integrity of the P-loop.

Microarray analysis of gene expression in peripheral blood leucocytes from rock bream (Oplegnathus fasciatus) after stimulation by LPS, ConA/PMA, and poly I:C

The responses of peripheral blood leucocytes (PBLs) from the rock bream Oplegnathus fasciatus stimulated with lipopolysaccharides (LPS), concanavalin A/phorbol myristate acetate (Con A/PMA), and/or polyriboinosinic polyribocytidylic acid (poly I:C) were investigated using a cDNA microarray consisting of 2,400 clones. A total of 336 unique genes were more than twofold up-regulated: 169 in LPS-stimulated PBLs, 209 in Con A/PMA-stimulated PBLs, and 146 in poly I:C-stimulated PBLs. Moreover, other genes were similarly up-regulated with combinations of stimulants: 85 genes in PBLs stimulated with both LPS and Con A/PMA, 79 in PBLs stimulated with both LPS and poly I:C, and 66 in PBLs stimulated with both Con A/PMA and poly I:C. Furthermore, 42 other genes were more than twofold up-regulated in PBLs stimulated with all tested mitogens. Other genes were not significantly induced. Overall, these results suggest that certain genes in rock bream play important roles in physiological and pathological processes. Furthermore, the microarray analysis showed promise as a tool for investigating immune mechanisms in teleost fish.

Coassociations between IL10 polymorphisms, IL-10 production, helminth infection, and asthma/wheeze in an urban tropical population in Brazil

Helminth infections are associated with protection against allergies. It is postulated that IL-10 production after helminth infection suppresses skin hypersensitivity and increases IgG₄ production, protecting against allergies. We aimed to determine whether IL10 polymorphisms are associated with helminth infection and the risk of wheeze and allergy. Twelve IL10 single nucleotide polymorphisms were genotyped in 1353 children aged 4 to 11 years living in a poor urban area in Salvador, Brazil. Wheezing status, Ascaris lumbricoides and Trichuris trichiura infection, IL-10 production by peripheral blood leukocytes stimulated with A lumbricoides extract, serum total IgE levels, specific IgE levels, skin prick test responses to common aeroallergens, and IgG4 and IgE anti-A lumbricoides antibody levels were measured in all children. Association tests were performed by using logistic or linear regression when appropriate, including sex, age, helminth infection, and principal components for ancestry informative markers as covariates by using PLINK. Allele G of marker rs3024496 was associated with the decreased production of IL-10 by peripheral blood leukocytes in response to A lumbricoides stimulation. Allele C of marker rs3024498 was negatively associated with helminth infection or its markers. Marker rs3024492 was positively associated with the risk of atopic wheeze, total IgE levels, and skin prick test responses to cockroach. Our findings suggest that IL10 polymorphisms might play a role in the production of IL-10, helminth infection, and allergy. We hypothesize that polymorphisms related to protection against helminths, which would offer an evolutionary advantage to subjects in the past, might be associated with increased risk of allergic diseases.

Strong Concordance Between Transcriptomic Patterns of Spleen and Peripheral Blood Leukocytes in Response to Avian Pathogenic Escherichia coli Infection

Avian pathogenic Escherichia coli (APEC) causes morbidity in chickens and exhibits zoonotic potential. Understanding host transcriptional responses to infection aids the understanding of protective mechanisms and serves to inform future colibacillosis control strategies. Transcriptomes of spleen and peripheral blood leukocytes (PBLs) of the same individual birds in response to APEC infection were compared to identify common response patterns and connecting pathways. More than 100 genes in three contrasts examining pathology and infection status were significantly differentially expressed in both tissues and similarly regulated. Tissue-specific differences in catalytic activity, however, appear between birds with mild and severe pathology responses. Early expression differences, between birds with severe pathology and uninfected controls, in the mitogen-activated protein kinase pathway in PBLs precede spleen responses in the p53 and cytokine-cytokine receptor pathways. Tissue bianalysis is useful in identifying genes and pathways important to the response to APEC, whose role might otherwise be underestimated in importance.

Strong Concordance Between Transcriptomic Patterns of Spleen and Peripheral Blood Leukocytes in Response to Avian Pathogenic Escherichia coli Infection

Strong Concordance Between Transcriptomic Patterns of Spleen and Peripheral Blood Leukocytes in Response to Avian Pathogenic Escherichia coli Infection

Identification of Haplotype Tag SNPs Within the Whole Myeloid Differentiation 2 Gene and Their Clinical Relevance in Patients With Major Trauma

Myeloid differentiation 2 (MD-2) plays a critical role in orchestrating the innate immune response and the development of sepsis and subsequent organ dysfunction after trauma. The objectives of this prospective study were to identify haplotype tag single-nucleotide polymorphisms (htSNPs) within the entire MD-2 gene and to investigate their clinical relevance in patients with major trauma. A total of 726 patients with major trauma were prospectively recruited and composed of two different geographic populations (Chongqing in southwestern China and Zhejiang in eastern China). The htSNPs of the MD-2 gene were determined using HapMap database and linkage disequilibrium analysis. The htSNPs were genotyped using pyrosequencing method. The whole peripheral blood samples obtained immediately after admission were stimulated with bacterial lipopolysaccharide and then determined for production of tumor necrosis factor α. Sepsis morbidity rate and multiple organ dysfunction (MOD) scores were accessed. Three SNPs (rs7843858, rs11465996, and rs2114169) were identified as htSNPs for the MD-2 gene. All of them were shown to be high-frequent SNPs in this study cohort. However, only the rs11465996 polymorphism was shown to be significantly associated with higher sepsis morbidity rate and MOD scores in patients with major trauma in both Chongqing and Zhejiang districts. In addition, the rs11465996 polymorphism was significantly associated with tumor necrosis factor α production by peripheral blood leukocytes in response to bacterial lipoprotein stimulation. Among the three htSNPs of the entire MD-2 gene, only the rs11465996 might be used as relevant risk estimate for the development of sepsis and MOD syndrome in patients with major trauma.

Expression analysis of lipopolysaccharide-and/or concanavalin A/phorbol myristate acetate-stimulated black rockfish peripheral blood leukocytes using a cDNA microarray to identify up-regulated genes

The responses of peripheral blood leucocytes (PBLs) from the black rockfish Sebastes schlegelii stimulated with lipopolysaccharides (LPS) and/or concanavalin A/phorbol myristate acetate (Con A/PMA) were investigated using a cDNA microarray consisting of 5,088 clones. This analysis showed that 254 unique genes were more than twofold upregulated, and they were selected for sequencing. Among the mitogens, 84 genes were more than twofold upregulated in LPS-stimulated PBLs, and 112 genes were induced in Con A/PMA-stimulated PBLs. Moreover, 58 other genes were more than twofold upregulated in PBLs stimulated with both LPS and Con A/PMA. Other genes were not significantly induced. Overall, these results suggest that certain genes in black rockfish have important roles in physiological and pathological processes. Furthermore, the microarray analysis suggested a promising tool for immune mechanisms in teleost fish.

Systemic inflammation and priming of peripheral blood leukocytes persist during clinical remission in horses with heaves

ObjectiveTo compare innate immune responses of peripheral blood leukocytes from healthy and asymptomatic heaves-affected horses. AnimalsHeaves-affected horses (n=5-6) and healthy controls (n=4–5) kept under low dust environments (pasture or shavings and pellets). MethodsBlood neutrophil and neutrophil-depleted cell populations were isolated using MACS system. Cells were incubated with or without bacterial products (lipopolysaccharide (LPS), 100ng/mL and fMLP, 5ng/mL, 5h). Cytokine (IL-1β, IL-8, TNF, IL-4, INFγ and IL-10) and receptor (TLR4) mRNA expression was assessed by qPCR. TNF concentration in culture supernatants and serum samples was assessed using equine specific ELISA. Apoptotic rate of resting and stimulated neutrophils was assessed by flow cytometry using AnnexinV and 7-AAD (18h) and correlated with early pro-inflammatory cytokine expression in the same cells (5h). ResultsStimulation with bacterial-derived products resulted in overexpression of pro-inflammatory cytokines in both neutrophils (IL-1β and TNF) and neutrophil-depleted leukocytes (IL-1β and IL-8) from heaves-affected horses. Neutrophil survival (18h) was associated with their early TNF expression, but not IL-8. Neutrophil-depleted leukocytes from these horses also had significantly increased basal TNF mRNA levels. Serum TNF concentration was also significantly higher in heaves-affected horses compared to healthy horses kept in similar environment. ConclusionsAltered innate immune response to bacterial products is observable ex vivo in peripheral blood leukocytes from asymptomatic heaves-susceptible horses and is associated with high serum TNF concentration. It remains to be determined if this phenomenon is caused by intrinsic differences in innate immune responses or to cellular priming caused by systemic inflammation.

Identification of haplotype tag single nucleotide polymorphisms within the receptor for advanced glycation end products gene and their clinical relevance in patients with major trauma.

IntroductionThe receptor for advanced glycation end products (RAGE) has been considered as one of the major pattern recognition receptors and plays an important role in the development of sepsis and multiple organ dysfunction in critical illnesses. Although genetic variants of the RAGE gene have been shown to be well associated with susceptibility to some inflammatory diseases, little is known about their clinical relevance in the development of sepsis in critical ill patients.MethodsFour genetic variants were selected from the entire RAGE gene and genotyped using pyrosequencing and polymerase chain reaction-length polymorphism methods. Association studies were performed in two independent Chinese Han populations.ResultsAmong the four genetic variants, only the rs1800625 polymorphism was significantly associated with sepsis morbidity rate and multiple organ dysfunction (MOD) scores in patients with major trauma both in Chongqing (n = 496) and Zhejiang (n = 232) districts, respectively. Results from ex vivo responsiveness of peripheral blood leukocytes indicated that the rs1800625 polymorphism was well associated with decreased production of TNFα. In addition, the rs1800625 polymorphism could significantly inhibit the promoter activities of the RAGE gene.ConclusionsThe rs1800625 polymorphism is a functional variant, which might be used as a relevant risk estimate for the development of sepsis and multiple organ dysfunction syndrome in patients with major trauma.

Identification of Haplotype Tag SNPs Within the Entire TLR2 Gene and Their Clinical Relevance in Patients With Major Trauma

Toll-like receptor 2 (TLR2) signaling plays a critical role in orchestrating the innate immune response and the development of sepsis and subsequent organ dysfunction after trauma. The objectives of this prospective study were to identify haplotype tag single-nucleotide polymorphisms (htSNPs) within the entire TLR2 gene and to investigate their clinical relevance in patients with major trauma. A total of 410 patients with major trauma were prospectively recruited. The htSNPs of the TLR2 gene was determined using HapMap database and linkage disequilibrium analysis. The htSNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism method. The whole peripheral blood samples obtained immediately after admission were stimulated with bacterial lipoprotein and then determined for production of tumor necrosis factor-α, interleukin 8, and interleukin 10. Sepsis morbidity rate and multiple organ dysfunction (MOD) scores were accessed. Three SNPs (rs1898830, rs3804099, and rs7656411) were identified as htSNPs for the TLR2 gene. All of them were shown to be high-frequency SNPs in this study cohort. Two of them (rs1898830 and rs3804099) and the haplotype ATT were significantly associated with cytokine production by peripheral blood leukocytes in response to bacterial lipoprotein stimulation. Only rs3804099, however, was significantly associated with higher sepsis morbidity rate and MOD scores in patients with major trauma. In addition, the patients with the haplotype ATT had lower sepsis morbidity rate than those without the haplotype ATT. Therefore, three SNPs might act as htSNPs for the entire TLR2 gene in the Chinese population. The rs3804099 and the haplotype ATT might be used as relevant risk estimates for the development of sepsis and MOD in patients with major trauma.

Functional significance of the Toll-like receptor 4 promoter gene polymorphisms in the Chinese Han population

Toll-like receptor 4 is an important signaling receptor for lipopolysaccharide in mammals, and the variation of the promoter may affect the activity of toll-like receptor 4 expression. Although 12 single nucleotide polymorphisms have been identified in the toll-like receptor 4 promoter, little is known about the functional significance of these single nucleotide polymorphisms. Genetic functional and association studies. National Key Laboratory of Trauma and Departments of Traumatic Surgery in two teaching hospitals. Three hundred seventy-nine healthy volunteers and 303 patients with major trauma. None. Five single nucleotide polymorphisms identified in the toll-like receptor 4 promoter in the Chinese Han population were selected. Three of them revealed a close relationship with transcription factor binding sites. Among the three single nucleotide polymorphisms, only the T-2242C polymorphism significantly increased transcriptional activities of the toll-like receptor 4 promoter, as shown by reporter gene assay. Results from flow cytometry and ex vivo responsiveness of peripheral blood leukocytes indicated that the T-2242C polymorphism was well-associated with increased expression of toll-like receptor 4 protein and production of tumor necrosis factor-alpha. The clinical relevance of these single nucleotide polymorphisms was then investigated in 303 patients with major trauma. The peripheral blood leukocytes of trauma patients with the variant C allele revealed greater capacity to produce tumor necrosis factor-alpha and interleukin-6 on the admission day. Furthermore, the toll-like receptor 4/2242 polymorphism was significantly associated with higher sepsis morbidity rates and multiple organ dysfunction scores in patients with major trauma. The toll-like receptor 4/2242 polymorphism is a functional variant and might be used as a relevant risk estimate for organ dysfunction and sepsis in trauma patients.

COLD-FX® Stimulates Cell Mediated Immune Response of Peripheral Leukocytes ex-vivo to Influenza Virus in National Hockey League Players

A proprietary extract from North American ginseng (Panax quinquefolium), COLD-FX ® (CVT-E002), has been found to be effective in the prevention of upper respiratory infections in a number of different groups including institutionalized seniors, community-dwelling seniors and healthy adults. These effects of COLD-FX are thought to be mediated by its ability to stimulate both the natural and acquired immune systems. Excessive exercise has been known to induce immune suppression and to increase the susceptibility for contracting recurrent upper respiratory tract infections. The purpose of the present study, therefore, was to determine if COLD-FX supplementation to players of National Hockey League caliber would be effective in altering the response of their leukocytes to influenza virus ex vivo. Periph- eral blood leukocytes (PBLs) were isolated from 2 groups of NHL players and immediately cultured for 2.5 days in the presence of 3 strains of influenza virus. One group (n=18) had been taking COLD-FX (400 mg/day) for 30 days, while the control group (n=19) was supplement free. Cultured PBLs isolated from those taking COLD-FX (400 mg/day) and stimulated with 3 strains of influenza virus released more TNF-α and IL-2, however, moderated granzyme-B release. This indicates that COLD-FX supplementation enhanced cell-mediated immune response of PBLs to influenza viruses and likely moderated the levels of cytotoxic T-cell effector molecules, sustained release of which may lead to autoimmune conditions. Additional studies on elite athletes, whose immune systems may be compromised, are warranted.

Acquisition of immunity to the protozoan parasite Eimeria adenoeides in turkey poults and the peripheral blood leukocyte response to a primary infection

A primary infection of 12.5 x 10(3) oocysts of Eimeria adenoeides , given to 20-d-old turkey poults, resulted in depression of weight gain, and the production of large numbers of oocysts in the feces, compared with uninfected controls. Poults were raised under conditions to prevent possible reinfection to determine the ability of the primary infection to confer protective immunity against a challenge infection of 5 x 10(4) oocysts given at 34 d of age. Using weight gain and oocyst production after challenge as criteria for protection, the results indicated that immunity had developed. The concentration and proportions among white blood cell (WBC) populations in peripheral blood were determined at different times after the primary infection. The WBC concentration of infected poults was elevated on d 7 and 11, primarily due to elevated levels of lymphocytes and monocytes on d 7 and eosinophils on d 11. There were no differences in heterophil and basophil concentrations between infected and uninfected poults at any of the time points examined. With the exception of increased percentages of eosinophils on d 11, infection was not associated with alterations in the proportions among WBC populations. Comparison of CD4+ and CD8+ defined lymphocyte subpopulations in the blood of infected versus uninfected poults revealed higher concentrations of CD4+ lymphocytes on d 11, lower concentrations of CD8+ cells on d 4, and higher concentrations of CD8+ cells on d 11 of infection, as well as elevated ratios of CD4+:CD8+ lymphocytes in infected birds on d 4 and 11. These alterations in WBC profiles after primary E. adenoeides infection in turkey poults suggest initiation of both innate and adaptive cellular immune activities designed to effectively cope with a parasitic, intracellular pathogen.

Procyanidin‐ and polysaccharide‐induced γδ T cell priming

We recently characterized the capacity of procyanidins and polysaccharides found in common dietary supplements to stimulate innate immunity. Our studies focused on γδ T cells, innate lymphocytes found in mucosal epithelium and well positioned to encounter and respond to agonists found in dietary supplements. Higher order oligomeric procyanidins, such as those found in unripe apple peel (APP) and polysaccharides from plant bark (Yamoa(tm)) primed γδ T cells. Priming of γδ T cells results in rapid increases in innate cytokine expression and enhanced responses to secondary stimuli. Intraperitoneal injection of either procyanidin or polysaccharide agonists induced rapid peritonitis in mice. Feeding Yamoa(tm) extract to calves altered peripheral blood leukocyte responses. Although both procyanidins and polysaccharides primed γδ T cells, they acted via different mechanisms. Whereas procyanidins directly primed γδ T cells, polysaccharides required accessory cells such as monocytes for optimal priming. Procyanidins induced GM‐CSF and IL‐8 production and mRNA stabilization. Our ongoing studies suggest that the responses of γδ T cells may be responsible, to some degree, for the innate immune benefit provided by these dietary supplements. These studies were supported by a PO1 grant from NIH‐NCCAM and an NIH‐NIAID Contract.

Experimental disseminated aspergillosis in mice: Histopathological study

Objective Systemic response to intravenous administration of Aspergillus fumigatus conidia was investigated in laboratory mice by survival/mortality, peripheral blood leukocyte response and histopathological evaluation of lung, liver, kidney and spleen. Methods Female C57Bl/6 mice were inoculated intravenously with 10 6–5 × 10 7 conidia of A. fumigatus (human isolate). Survival was monitored for two weeks. Histopathological analysis of organ was conducted postmortem and in survivors. Peripheral blood cells and leukocyte aggregation were determined in survivors. Results Administration of all doses of A. fumigatus conidia caused mortality, but the highest mortality rate and the shortest time of survival were noted at the highest doses applied. Increased peripheral blood lymphocyte counts and their propensity to aggregate were observed in survivors. Histological analysis revealed : (1) pulmonary inflammatory response proportional to the applied dose of conidia with detectable fungi in lungs of animals that received the highest applied dose; (2) pronounced kidney inflammatory response with variable intensity of tubular dilation in survivors versus tubular destruction in nonsurvivors and proteinuria as well as hemoglobinuria at the highest doses applied; (3) presence of microabscesses in the liver; (4) white pulp hyperplasia and dose-dependent increase in lymphoid follicles in the spleen of infected animals that survived. Conclusion Lymphocytosis and state of aggregation/adhesion in survivors and local histologically evident inflammatory response in all examined organs of mice characterize the response to systemic application of A. fumigatus conidia. The results obtained may contribute to our understanding of pathological mechanisms underlying disseminated aspergillosis and to the evaluation of therapeutic interventions that might improve survival or lessen pathology.

Blood culture and stimulation conditions for the diagnosis of tuberculosis in cervids by the Cervigam assay

Mitogen- and antigen-induced interferon-γ (ifn-γ) responses of peripheral blood leucocytes from cervids were evaluated by a commercial whole-blood assay. The assay was applied to Mycobacterium bovis-infected white-tailed deer and reindeer,...

Immunostimulating effects of the polyphenol‐rich fraction of sugar cane (Saccharum officinarum L.) extract in chickens

The phagocytic activity of peripheral blood leukocytes (PBL) in chickens orally administered sugar cane extracts (SCE) or polyphenol-rich fraction (PRF) of SCE (500 mg/kg/day) for 3 consecutive days increased significantly, when compared with that of saline-administered control chickens. Chickens orally administered SCE or PRF (500 mg/kg/day) for 3 consecutive days showed significantly higher antibody responses against sheep red blood cells and Brucella abortus than control chickens. In addition, oral administration of SCE or PRF also resulted in a significant increase in the number of IgM- and IgG-plaque forming cell responses of PBL, intestinal leukocytes and splenocytes, when compared with those of control chickens. Furthermore, delayed type hypersensitivity responses to human gamma globulin significantly increased in chickens orally administered SCE or PRF, compared with those of control chickens when evaluated on the basis of net increased wattle thickness at 24, 48 and 72 h after challenge. These results suggest that PRF of SCE has an immunostimulating effect in chickens.

Induction of Potentially Protective HIV-Specific T-Cell Responses In Vitro by Gag mRNA-Electroporated Dendritic Cells.

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by dysfunction of HIV-1-specific T-lymphocytes. In order to suppress the virus and delay evolution to AIDS, antigen-loaded antigen-presenting cells, including dendritic cells (DC) might be useful to boost and broaden HIV-1-specific T-cell responses. Monocyte-derived DC from 15 untreated (“naive”) and 15 highly active anti-retroviral therapy (HAART)-treated HIV-1-infected patients were electroporated with codon-optimized (“humanized”) mRNA encoding consensus HxB-2 (hHxB-2) Gag protein. These DC were co-cultured for 1 week with autologous peripheral blood leucocytes (PBL). Potential expansion of specific T-cells was measured by comparing ELISPOT responses of PBL before and after co-culture, using a pool of overlapping peptides, spanning the HxB-2 Gag. Expansion of specific PBL after co-culture was noted for T cells producing interferon (IFN)-gamma, interleukin (IL)-2 and perforin (Wilcoxon signed rank test p<0.05, except for IL-2 in naive patients). From all HIV-1-seropositive persons tested, 12 HAART-treated and 12 naive patients match in absolute number of CD4+ T-cells. A comparison of the increase of the response between day 0 and after 1 week of stimulation between those two groups showed that the response was higher in HAART-treated subjects for IFN-gamma and IL-2 but not for perforin in comparison to untreated subjects. Examining purified CD4+ and CD8+ T-cells after co-culture revealed that HxB-2 Gag peptides induced IFN-gamma in both subsets, that IL-2 was only secreted by CD4+ T-cells and that perforin was dominantly secreted by CD8+ T-cells. Remarkably, the perforin response in the treatment-naive persons was negatively correlated with the peripheral blood absolute CD4+ and CD8+ T-cell count (respectively R=0.618, p=0.014; and R=0.529, p=0.043). Furthermore, the nadir absolute CD4+ T-cell count in HAART-treated subjects was positively correlated with the IL-2 response (R=0.521, p=0.046) and negatively correlated with the perforin response (R=0.588, p=0.021). In conclusion, DC from HAART-treated and therapy-naive subjects, electroporated with hHxB-2 gag mRNA have the capacity to induce secondary T-cell responses. In an earlier study (Van Gulck ER et al. Blood 2006; 107:1818–1827), we already demonstrated ex vivo that CD4+ and CD8+ T-cells from non-treated HIV-1-infected subjects can be directly triggered by DC electroporated with autologous proviral-derived gag mRNA. Taken together, our results open the perspective for a DC immunotherapy for HIV disease.

CTLA-4 blockade augments human T lymphocyte-mediated suppression of lung tumor xenografts in SCID mice.

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patient's T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patient's PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.