CD8 IFN-gamma Research Articles

Diphencyprone reduces the CD8+ lymphocytes and IL-4 and enhences IgG2a/IgG1 ratio in pathogenicity of acute leishmania major infection in BALB/c mice

BackgroundThe exact role of different immune cells and cytokines in control or promotion of intracellular growth of leishmania has still remained a controversial topic. The aim of the present study was to study effects of cellular changes and relevant cytokines in cell mediated immunity by diphencyprone (DCP) in pathogenicity of acute L.major infection in BALB/c mice. Methods45 healthy female BALB/c mice were injected with L. major promastigotes under the base of tail. The mice were randomly divided to three groups of 15 mice: (1) control group without any treatment. (2) acetone group: Acetone was applied topically on the cutaneous lesions weekly and (3) DCP group: DCP was applied topically on the cutaneous lesions with increasing concentrations to induce local allergy. The mice were followed by the end of eighth week, and then macroscopic changes, histopathology, immunology studies, and organ parasite burden were determined. ResultsIn DCP group, in comparison to other groups the ulcer size and parasite burden in ulcer site and spleen increased, significantly. There was a deep lymphohistiocytic infiltration in the ulcer site. Total IgG, IgG1, and IgG2a levels as well as IgG2a/IgG1 ratio and intracellular IFN-gamma in CD8+ lymphocytes were significantly higher. IL4 and T CD8+ lymphocytes were significantly lower in DCP group. The IgG2a/IgG1 ratio was more than 1 in all groups. ConclusionOur findings demonstrated that DCP reduced the CD8+ lymphocytes and IL-4 production. In spite of increased IgG2a/IgG1 ratio, the parasite burden and inflammation severity increased in infected mice. The results can show the pivotal role of CD8+ lymphocytes in conjunction with Th1 lymphocytes in the control of acute leishmania infection in mice.

Identification of novel Mycobacterium tuberculosis CD4 T-cell antigens via high throughput proteome screening

Elicitation of CD4 IFN-gamma T cell responses to Mycobacterium tuberculosis (MTB) is a rational vaccine strategy to prevent clinical tuberculosis. Diagnosis of MTB infection is based on T-cell immune memory to MTB antigens. The MTB proteome contains over four thousand open reading frames (ORFs). We conducted a pilot antigen identification study using 164 MTB proteins and MTB-specific T-cells expanded in vitro from 12 persons with latent MTB infection. Enrichment of MTB-reactive T-cells from PBMC used cell sorting or an alternate system compatible with limited resources. MTB proteins were used as single antigens or combinatorial matrices in proliferation and cytokine secretion readouts. Overall, our study found that 44 MTB proteins were antigenic, including 27 not previously characterized as CD4 T-cell antigens. Antigen truncation, peptide, NTM homology, and HLA class II tetramer studies confirmed malate synthase G (encoded by gene Rv1837) as a CD4 T-cell antigen. This simple, scalable system has potential utility for the identification of candidate MTB vaccine and biomarker antigens.

LFA-3/CD2 Pathway, Potential Target For Immunosuppressive Therapy In Aplastic Anemia: A Phase I/II Trial Of Alefacept In Patients With Relapsed/Refractory Aplastic Anemia

Aplastic Anemia (AA) is a rare bone marrow failure disorder characterized by pancytopenia, and an empty bone marrow. The disease can develop from unknown causes (idiopathic) or can be secondary to drugs, toxins, chemotherapy (acquired). Hematopoietic bone marrow transplant is a potential cure for idiopathic AA patients. Pharmacologic therapies remain the foremost therapy for patients who lack suitable donors or ineligible for bone marrow transplant. Antithymocyte globulin (ATG) is a first line treatment for these patients. However, relapsed and refractory cases of AA can occur even after ATG treatment. Patients with relapsed/ refractory AA may benefit from alternative immunosuppressive therapies. Alefacept is a novel immunosuppressive agent that targets the CD2-LFA3 pathway important in various T cell functions especially T cell activation. The occurrence of relapsed/ refractory cases of AA coupled with the side effect profile of ATG prompted us to test alefacept in refractory AA. This Phase I/II study was fully approved by the Institutional Review Board of the Cleveland Clinic and conducted in patients with relapsed/ refractory AA. This trial was registered under ClinicalTrial.Gov Identifier Number: NCT01267643. A total of four patients were successfully enrolled in this trial. All four patients had refractory AA. Antecedent treatment for three patients was immunosuppressive therapy: ATG in one case, cyclosporine in one, and both plus daclizumab in one case. All patients are female. Patient ages at the beginning of the study were 39, 57, 83, and 90 years. Three of the four patients were ECOG performance status 1 and one patient was ECOG 2. Three patients received dose level 1 (7.5 mg/ week) while one patient was treated at dose level 2 (10 mg/week). All patients received once weekly treatments for a total of 12 weeks. All 4 patients have been followed for 15, 13, 12, and 11 months, respectively. None of the patients had a PNH clone at presentation. Blood parameters at the beginning of the alefacept treatment indicated that two patients had three cytopenias (Hgb<10 g/dL, platelets<100 K/mL, ANC<1.5 K/mL) and two patients had two (platelets and ANC). Three of the four patients achieved a partial response. Side effects were very mild and included a grade 2 cough and grade 1 sore throat and nasal congestion in one patient and grade 1 muscle aches in a second. All side effects were transient and resolved after 2 weeks of treatment. No severe infections or grade 3 or 4 adverse events were reported. Complete blood cell count of two representative patients are shown below. Routine test values (absolute reticulocytes, ANC, Hgb, and platelets) are shown one per month for pre- and post-treatment while one per week during the treatment. We performed a flow cytometric measurement of intracellular cytokine profile before, during and after Alefacept treatment. Peripheral blood mononuclear cells were stimulated with PMA/ ionomycin for 6 hours in culture. We found a decrease in IFN-gamma, a cytokine implicated in AA pathogenesis post-treatment (Patient A: CD4 IFN-gamma 11.1 vs 1.7%,CD8 IFN-gamma 60.8% vs 16.9%; Patient B: CD4 IFN-gamma 32% vs 7% vs 5.7% ,CD8 IFN-gamma 76.7% vs 30.6% vs. 25.8%). Conversely we found an increase in IL-17, post-treatment (Patient A: CD4 IL-17 0.7 vs 56%,CD8 IL-17 61% vs 57.4%; Patient B: CD4 IL-17 1.6% vs 1.9% vs 29.4% ,CD8 IL-17 4.2% vs 6% vs. 31.9%) To date, three out of four patients who achieved a PR continue to be transfusion independent and none are on any other immunosuppressive therapies such as cyclosporine. All four patients remain progression-free and none have developed any PNH clone. On December, 2011 Astellas Pharma US announced that the company has voluntarily discontinued the promotion, manufacturing, distribution and sales of alefacept because of business needs but not related to safety issues. In conclusion, this pilot study even if conducted in a limited number of patients suggests targeting CD2-LFA-3 pathway using alefacept is a viable and safe option for treating patients with relapsed/ refractory AA. Disclosures:No relevant conflicts of interest to declare.

Comparison of the Effects of Desflurane and Bupivacaine on Th1 and Th2 Responses

Anesthesia and surgical trauma are known to affect various functions of the immune system. Alterations reported in the immune system, such as imbalance of Th1 (IFN-gamma) and Th2 (IL-4, 5, 10) cytokines, may result from a number of factors, including pre-medication, type of anesthetic drug, and modality of anesthesia. In this study, we investigated the effects of spinal and general anesthesia with desflurane and bupivakain, respecttively, on Th1 (IFN-gamma) and Th2 (IL-10) cytokines, absolute lymphocyte and natural regulatory T cell numbers (Treg). Peripheral Blood Mononuclear cells from 24 patients with Benign Prostate Hyperplasia (BPH), undergoing transuretheral prostatectomy under spinal (n = 12) and general (n = 12) anesthesia were analyzed before and 24 hours after surgery. Intracellular cytokine production in response to mitogen stimulation and absolute numbers of Tregs and lymphocytes were determined by using flow cytometry. In patients who received spinal anesthesia, while the frequency of IFN-gamma (1.68% +/- 0.74 vs. 1.03% +/- 0.74) and IL-10 producing CD4+ T cells decreased (2.62% +/- 2.24 vs. 1.04% +/- 1.06; p < 0.05), the ratio of Th1/Th2 remained similar (1.14 +/- 0.7 vs. 1.52 +/- 1.02; p > 0.05) after surgery. In contrast, in the general anesthesia group the frequency of CD4 IFN-gamma+ T cells increased (1.3% +/- 0.7 vs. 2.5% +/- 1.2; p < 0.05) and the frequency of CD4+ IL-10+ T cells decreased (1.1% +/- 0.68 vs. 0.67% +/- 0.47), resulting in an increased Th1/Th2 ratio (1.61 +/- 1.1 vs. 4.77 +/- 3.95; p < 0.05). Absolute lymphocyte and Treg numbers did not change significantly in both groups following surgery. Our results support the notion that general anesthesia, rather than spinal anesthesia, alters the balance of Th1 and Th2 in favor of Th1 responses. However, whether this has any effect on the susceptibility to postsurgery-related infections remains to be determined.

Functional state of CD4+ and CD8+ T lymphocytes and their role in the slow progression of HIV infection in pediatric patients

To evaluate simultaneously the functional state of CD4+ and CD8+ T lymphocytes from Venezuelan HIV-1-infected pediatric patients. Children were assigned to subgroups of rapid progressors (RPs) and slow progressors (SPs), based on clinical features. To determine the degree of CD4+ and CD8+ T-lymphocyte functionality, flow cytometry techniques were used, and diverse parameters of the functionality of these cells were characterized by ex vivo tests, such as expression of CD95/Fas and CD127, and frequency of apoptosis. In addition, we determined, in cultured peripheral blood mononuclear cells, HIV-specific proliferation and the production of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), besides measuring intracellular IFN-gamma in CD4+ T cells. Our results indicate that several molecular and cellular mechanisms of CD4+ and CD8+ T lymphocytes are deteriorated in RPs in comparison with SPs and controls. Indeed, both types of T lymphocytes from RPs exhibited an increased expression of CD95/Fas (p < 0.01), a significantly reduced expression of CD127 (p < 0.01), and an augmented frequency of apoptosis (p < 0.01). Furthermore, T cells from these patients displayed a diminished capacity of mitogen proliferation (p < 0.05), a reduced percentage of IFN-gamma producing CD4+ T lymphocytes (p < 0.05), and a smaller capacity of IL-10, TNF-alpha and IFN-gamma production (p < 0.01) in comparison with SP and control patients. Our findings indicate that the decline of the normal T lymphocyte molecular and cellular responses is related to a rapid progression and a decreased resistance to HIV-1 infection in children.

HTLV-1 bZIP factor perturbs immune response to the pathogens in vivo by inhibiting IFN-gamma production

HTLV-1 carriers and the patients with adult T-cell leukemia (ATL) frequently suffer from the opportunistic infections. Although it has been known that HTLV-1 infection evokes cell-mediated immune deficiency, its mechanism remains unknown. HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1, which is constitutively expressed and involved in the proliferation of HTLV-1-infected cells. In this study, we investigated the role of HBZ in immunodeficiency in a mice model. HBZ transgenic (HBZ-Tg) mice were infected with HSV-2 and Listeria monocytogenes and evaluated for cellular immunity to those pathogens. The clearance of both pathogens was impaired in HBZ-Tg mice compared with the non-transgenic littermate. In the both infection, production of IFN-gamma in CD4+ T cells was significantly reduced in HBZ-Tg mice. In addition, ectopic expression of HBZ in primary human CD4+ T cells impaired IFN-gamma production in vitro. As the molecular mechanisms, we found that HBZ suppressed transcription of IFNG promoter. Our results suggest that HBZ plays a crucial role for cellular immunodeficiency in HTLV-1-infected subjects.

Kinetics of cytomegalovirus (CMV) pp65 and IE‐1‐specific IFNγ CD8+ and CD4+ T cells during episodes of viral DNAemia in allogeneic stem cell transplant recipients: Potential implications for the management of active CMV infection

The dynamics of CMV pp65 and IE-1-specific IFNgamma-producing CD8(+) (IFNgamma CD8(+)) and CD4(+) (IFNgamma CD4(+)) T cells and CMV DNAemia were assessed in 19 pre-emptively treated episodes of active CMV infection. Peripheral counts of IFNgamma CD8(+) and IFNgamma CD4(+) T cells inversely correlated with CMV DNAemia levels (P = <0.001 and P = 0.003, respectively). A threshold value of 1.3 cells/microl predicting CMV DNAemia clearance was established for IFNgamma CD8(+) T cells (PPV, 100%; NPV, 93%) and for IFNgamma CD4(+) T cells (PPV, 100%; NPV, 75%). Undetectable T-cell responses were usually observed at the time of initiation of pre-emptive therapy. Either a rapid (within 7 days) or a delayed (median 31 days) expansion of both T-cell populations concomitant with CMV DNAemia clearance was observed in 5 and 8 episodes, respectively. An inconsistent or a lack of expansion of both T-cell subsets was related to a persistent CMV DNAemia. Robust and maintained CMV-specific T-cell responses after CMV DNAemia clearance and cessation of antiviral therapy were associated with a null incidence of relapsing infections at least during the following month. Data obtained in the present study may be helpful in the design of therapeutic strategies for the management of active CMV infections in the allo-SCT recipient.

An Assessment of the Effect of Human Herpesvirus-6 Replication on Active Cytomegalovirus Infection after Allogeneic Stem Cell Transplantation

Human herpesvirus-6 (HHV-6) may enhance cytomegalovirus (CMV) replication in allogeneic stem cell transplant (allo-SCT) recipients either through direct or indirect mechanisms. Definitive evidence supporting this hypothesis are lacking. We investigated the effect of HHV-6 replication on active CMV infection in 68 allo-SCT recipients. Analysis of plasma HHV-6 and CMV DNAemia was performed by real-time PCR. Enumeration of pp65 and IE-1 CMV-specific IFNgamma CD8(+) and CD4(+)T cells was performed by intracellular cytokine staining. HHV-6 DNAemia occurred in 39.8% of patients, and was significantly associated with subsequent CMV DNAemia in univariate (P=.01), but not in multivariate analysis (P=.65). The peak of HHV-6 DNAemia was not predictive of the development of CMV DNAemia. Timing and kinetics of active CMV infection were comparable in patients either with or without a preceding episode of HHV-6 DNAemia. The occurrence of HHV-6 DNAemia had no impact on CMV-specific T cell immunity reconstitution early after transplant. The receipt of a graft from an HLA-mismatched donor was independently associated with HHV-6 (P=.009) and CMV reactivation (P=.04). The data favor the hypothesis that a state of severe immunosuppression leads to HHV-6 and CMV coactivation, but argue against a role of HHV-6 in predisposing to the development of CMV DNAemia or influencing the course of active CMV infection.

Lack of prompt expansion of cytomegalovirus pp65 and IE-1-specific IFNγ CD8+ and CD4+ T cells is associated with rising levels of pp65 antigenemia and DNAemia during pre-emptive therapy in allogeneic hematopoietic stem cell transplant recipients

Rising levels of cytomegalovirus (CMV) DNAemia and/or pp65 antigenemia have been observed during pre-emptive ganciclovir therapy in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT). We assessed the incidence of this event in our series, and investigated whether its occurrence was associated with an impairment in the CMV-specific T-cell response. A total of 36 allo-SCT recipients experienced one or more episodes of active CMV infection (n=68) that were pre-emptively treated with val(ganciclovir). Rising levels of antigenemia and DNAemia, and an isolated increase in antigenemia, were observed in 39.7 and 2.9% of all episodes, respectively. Receipt of corticosteroids was associated with rising levels of antigenemia and DNAemia. Median increases of 12- and 6.8-fold of IFNgamma CD8(+) T and IFNgamma CD4(+) T cells, respectively, were observed at a median of 16.5 days after initiation of therapy in episodes with decreasing levels in antigenemia and DNAemia. In contrast, the numbers of both T-cell subsets at a median of 13.5 days after initiation of therapy did not differ significantly from those of pre-treatment samples in episodes with rising levels of antigenemia and DNAemia. Lack of prompt expansion of CMV pp65 and IE-1-specific IFNgamma CD8(+) and CD4(+) T cells is associated with rising levels in antigenemia and DNAemia during pre-emptive therapy.

Cellular responses to MPT-51, GlcB and ESAT-6 among MDR-TB and active tuberculosis patients in Brazil

Multi-drug resistant pulmonary tuberculosis (MDR-TB) may result from either insufficiency of the host cellular immune response or mycobacterial mechanisms of resistance. Mycobacterium tuberculosis-specific CD8+ and CD4+ T lymphocytes from MDR-TB patients are poorly studied. The aim of this study was to evaluate CD4+IFN-gamma+, CD4+IL-10+, CD8(+)IFN-gamma+ and CD8+IL-10+ cell populations by flow cytometry in non-resistant TB and multi-drug resistant tuberculosis (MDR-TB) patients from mid-central Brazil after stimulation with MPT-51, GlcB and ESAT-6 recombinant antigens from M. tuberculosis in comparison to tuberculin skin test negative (TST) healthy individuals. Non-resistant TB patients present specific cellular responses (CD4 and CD8, both IFN-gamma and IL-10) to GlcB, MPT-51 and ESAT-6; while MDR-TB patients present only CD8+IFN-gamma+ responses to ESAT-6 and CD8+IL-10+ responses to GlcB and ESAT-6. The results show that MDR-TB patients present impaired specific CD4 IFN-gamma and IL-10 responses and increased/normal specific CD8 IFN-gamma and IL-10 responses. This suggests an important role for CD8 function in these patients.

Prospective analysis of effector and regulatory CD4+ T cells in chronic HCV patients undergoing combination antiviral therapy

The role of HCV-specific CD4(+) T cells and regulatory T cells in influencing the outcome of antiviral therapy is incompletely defined. CD4(+) IFN-gamma ELISPOT assays (n=58) and flow cytometric analysis of FoxP3-expressing T regulatory cells (n=62) were performed on patients from the Virahep-C study at baseline, during and after cessation of antiviral therapy. Total HCV-specific IFN-gamma CD4(+) T cell ELISPOT responses did not increase with therapy, but rather decreased by 8 weeks and remained below baseline 24 weeks after cessation of therapy. There were no statistically significant differences with respect to viral kinetics, race and virologic outcome. In contrast, viral relapse after treatment was associated with a three-fold increase in HCV-specific responses. The frequency and phenotype of regulatory T cells during therapy were not significantly different in terms of race, viral kinetic groups or virologic outcome. A contraction of HCV-specific CD4(+) T cell responses was found during treatment with recovery of responses in patients experiencing virologic relapse after treatment. The levels of FoxP3-expressing regulatory T cells did not vary by race and were not predictive of virologic outcome. Work is ongoing to explore the contribution of mechanisms independent of CD4(+) T cells in therapy-induced viral clearance.

Full-Length Characterization of A1/D Intersubtype Recombinant Genomes from a Therapy-Induced HIV Type 1 Controller during Acute Infection and His Noncontrolling Partner

To increase the understanding of mechanisms of HIV control we have genetically and immunologically characterized a full-length HIV-1 isolated from an acute infection in a rare case of undetectable viremia. The subject, a 43-year-old Danish white male (DK1), was diagnosed with acute HIV-1 infection after 1 year in Uganda. Following transient antiretroviral therapy DK1 maintained undetectable viral load for more than 10 years. His Ugandan wife (UG1) developed high viral load. HIV-1 sequences from both individuals were compared by bootscanning for recombination break points. Diversity plots and phylogenic trees were constructed and diversity and evolutionary distances were calculated. Intracellular IFN-gamma in CD8(+)CD3(+) T-lymphocyte reactions was investigated by intracellular flow cytometry (IC-FACS). Virus isolates from both patients were A1D intersubtype recombinants showing 98% sequence homology in shared regions. Four of seven crossover points were identical; however, the env gene from UG1 was subtype D, but A1 in DK1. Both viruses encoded proteins of the expected length and replicated equally well in vitro. DK1 and UG1 shared the HLA-A02 tissue type. HLA-A02-restricted CD8(+) T cell IFN-gamma IC-FACS response in DK1 was detected against only one (Pol(476)) of 23 conserved epitopes. Neutralizing antibodies were induced only to the homologous isolate. These results indicate an A1D intersubtype recombination or transmission of a minor variant. Transient early antiretroviral therapy may have induced full HIV-1 control in this individual mediated by a narrow specific cytotoxic T lymphocyte and neutralizing antibody response and/or other factors yet to be characterized.

Analysis of splenic Gr‐1int immature myeloid cells in tumor‐bearing mice

It is known that the number of ImC, expressing myeloid markers, CD11b and Gr-1, increase with tumor growth and ImC play a role in the escape of tumor cells from immunosurveillance in tumor-bearing mice and cancer patients. However, the mechanisms by which ImC suppress immune responses in tumor-bearing mice have not been completely elucidated. In the present study, we investigated the function of splenic ImC freshly isolated from tumor-bearing mice and splenic ImC differentiated in vitro by GM-CSF. Freshly isolated splenic ImC were divided into two groups depending on Gr-1 expression, Gr-1 high (Gr-1hi) and intermediate (Gr-1int). Freshly isolated splenic Gr-1int ImC, but not Gr-1hi ImC, from tumor-bearing mice reduced production of IFN-gamma in CD8+ T cells, but neither splenic Gr-1int ImC nor Gr-1hi ImC isolated from naive mice did. Both Gr-1int and Gr-1hi ImC differentiated in vitro by GM-CSF inhibited production of IFN-gamma in both CD8+ and CD4+ T cells. In addition, the differentiated Gr-1int ImC, one-third of which were CD11c+F4/80+ cells, and their culture supernatants suppressed proliferative responses of T cells stimulated by CD3 ligation, but the differentiated Gr-1hi ImC and their culture supernatants did not. These results suggest that Gr-1int ImC are altered to immune-suppressive cells in tumor circumstances and that they are differentiated by GM-CSF progressively into CD11c+F4/80+ cells with further suppressive activity against T cells.

Role of IFN-γ responsiveness in CD8 T cell-mediated viral clearance and demyelination in coronavirus-infected mice

Immunocompetent, but not RAG1−/− mice infected with MHV–JHM develop demyelination. Transferred CD8 T cell-enriched splenocytes reconstitute demyelination, and this ability is dependent on donor IFN-γ. We used IFN-γR1−/− mice to examine the target of IFN-γ in CD8 T cell-mediated demyelination. In IFN-γR1−/−RAG1−/− recipients, demyelination is decreased, but not eliminated, while viral titers are significantly increased when compared to IFN-γR1+/+RAG1−/− recipients. IFN-γR1−/− CD8 T cells retain virus-specific effector function regardless of IFN-γR1 expression. Although IFN-γR1 responsiveness is critical for maximal demyelination, increased levels of infectious virus coupled with adoptive transfer of CD8 T cells may result in myelin destruction independent of IFN-γR1 expression.

Allogeneic tumor lysate can serve as both antigen source and protein supplementation for dendritic cell culture.

Recent preclinical and clinical evidence suggests the use of allogeneic tumor as a source of antigen for DC-based immunotherapy against cancer. We hypothesized that addition of allogeneic tumor lysate to monocyte-derived DC culture could serve a dual purpose: (1) antigen source and (2) protein supplementation of DC culture media. Protein supplementation whether of known origin (human serum/plasma, fetal bovine serum, human serum albumin) or undeclared origin ("serum-free" media) is a source of variability and bias. We addressed the question whether protein supplementation can be omitted in the presence of allogeneic tumor lysate. Human DC cultured in the presence of lysate from medullary thyroid carcinoma (MTC) cell line SHER-I (TuLy-DC) and DC pulsed with the same lysate but cultured in the presence of FBS (FBS-DC) were assessed for morphology, phenotype, maturation and functional properties. In comparison of FBS-DC/TuLy-DC no significant differences in morphology, phenotype and maturation could be detected. Both culture conditions produced CD1a(high), CD14(low) DC with high expression of costimulatory molecules and CD83 upon stimulation. TuLy-DC gave significantly better yields and produced more IL12p70. DC showed high (allo)stimulatory capacity toward T-cells. TuLy-DC induced more intracellular IFNgamma in CD8+T-cells of vaccinated MTC patients. Both types of DC induced killing of SHER-I after short in vitro restimulation. Tumor lysate from SHER-I can substitute for further protein supplementation in DC culture. Allogeneic tumor lysates should be taken into consideration as both source of antigen and protein supplementation in monocyte-derived DC culture.

The adjuvant effects of co‐stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination

Because DNA vaccines on their own tend to induce weak immune responses in humans, adjuvant methods are needed in order to improve their efficacy. The co-stimulatory molecules 4-1BBL, OX40L, and CD70 have been shown to induce strong T cell activities; therefore, in this study, we investigated whether they may be used as molecular adjuvants for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcDS2) in eliciting strong cellular and memory responses. Compared to mice immunized with pcDS2 alone, addition of the co-stimulatory molecules increased T cell proliferation and an HBsAg-specific antibody response that was marked with a higher ratio of IgG2a/IgG1. Importantly, pcDS2 plus these co-stimulatory molecules elicited a higher level of IFN-gamma and IL-4 in CD4(+) T cells and a higher level of IFN-gamma in CD8(+) T cells. In addition, a significantly robust antigen-specific cytotoxic T lymphocyte (CTL) response and the production of long-term memory CD8(+) T cells were also observed in the groups immunized with pcDS2 plus 4-1BBL, OX40L, or CD70. Consistently, as late as 100 days after immunization, upregulated expressions of BCL-2, Spi2A, IL-7Ra, and IL-15Ra were still observed in mice immunized with pcDS2 plus these co-stimulatory molecules, suggesting the generation of memory T cells in these groups. Together, these results suggest that the co-stimulatory molecules 4-1BBL, OX40L, or CD70 can enhance the immunogenicity of HBsAg DNA vaccines, resulting in strong humoral, cellular, and memory responses. This approach may lead to an effective therapeutic vaccine for chronic hepatitis B virus (HBV) infection.

Functional patterns of HIV-1-specific CD4 T-cell responses in children are influenced by the extent of virus suppression and exposure

Virus-specific CD4 T cells play a critical role in antiviral immunity. HIV-1-specific CD4 T cells in chronically infected adults are mostly composed of IFN-gamma-secreting cells, whereas a selective defect in IL-2-secreting CD4 T cells has been demonstrated. HIV-1-specific IL-2-secreting CD4 T cells are key components of effective immunity. To determine the function of HIV-1-specific CD4 T cells in HIV-1 vertically infected children after antiretroviral treatment (ART). Twenty-three vertically HIV-infected children treated with ART for an extended period (mean 7 years) were retrospectively studied. The function of HIV-1-specific CD4 T cells was determined by their ability to secrete IL-2 and IFN-gamma after stimulation with HIV-1 p55 gag protein using polychromatic flow cytometry. : Substantial differences in the patterns of CD4 T-cell responses were associated with different conditions of response to ART. Interestingly, children with suppression of viraemia below 50 HIV-1-RNA copies/ml of plasma for at least 2 years showed dominant IL-2 CD4 T-cell responses; children with viraemia below 50 copies but experiencing transient blips of viraemia showed polyfunctional (IL-2 plus IFN-gamma) CD4 T-cell responses; children with uncontrolled high viraemia levels had dominant IFN-gamma CD4 T-cell responses. Furthermore, the total frequency of HIV-1-specific CD4 T cells including IL-2 and IFN-gamma-secreting cells was significantly higher compared with HIV-infected adults with chronic infection. The higher frequency of HIV-1-specific CD4 T cells in children compared with adults and the recovery of IL-2-secreting CD4 T cells after successful ART-mediated suppression of virus replication indicate a greater capacity of immune restoration in children than adults.

Cross‐presentation of oral antigens by liver sinusoidal endothelial cells leads to CD8 T cell tolerance

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood-borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross-presented on H-2K(b) by LSEC to naive CD8 T cells. Cross-presentation efficiency in LSEC but not dendritic cells was increased by antigen-exposure to heat or low pH. Mechanistically, cross-presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H-2K(b)-restricted cross-presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2-/- knockout mice, previously reconstituted with naive ovalbumin-specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN-gamma in CD8 T cells. Using a new transgenic mouse line expressing H-2K(b) only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H-2K(b)-restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens.

The implantation of Taenia solium metacestodes in mice induces down-modulation of T-cell proliferation and cytokine production

The purpose of this study was to determine the effect of the implantation of Taenia solium metacestodes and the treatment with suppressive metacestode factor (F1) on the ability of spleen cells from Balb/c mice to produce cytokines. Cytokine production was estimated 12 days following the implantation or 4 days after the last dose of F1 (five doses) by RT-PCR and flow cytometry analyses. Spleen cells were obtained from metacestode-implanted, F1-treated and control mice. They were stimulated with concanavalin A (ConA) ex vivo and used for RT-PCR studies and for CD25 expression and intracellular cytokine production estimations using specific monoclonal antibodies labeled with phycoerithrin or fluorescein. Results of the RT-PCR showed that all cells expressed IFN-gamma, IL-2 and IL-4 mRNAs. IL-10 mRNA was not expressed in any case. Flow cytometry analyses showed that both spleen CD4+ and CD8+ cells from metacestode-implanted or treated-F1 mice expressed significantly diminished percentages of CD25 when compared with control cells (P<0.05). The estimation of intracellular cytokines showed that the production of IL-2 and IL-4 in CD8+ cells, and of IFN-gamma in CD4+ cells from mice implanted with metacestodes was significantly impaired when compared with the values from control cells (P<0.05).

HIV viral shedding in semen

Semen is a major transmission vector for HIV. Virus-specific CD8 T cells are critical in HIV control, but their relationship with semen viral load is unknown. We therefore examined the association between systemic HIV-specific IFN-gamma CD8 responses and viral load in the semen and blood of HIV-infected men. No correlation was observed between viral load in either semen or blood and systemic CD8 T-cell responses. Further studies of immune correlates of semen HIV shedding are needed.