CD4+CD25+ regulatory T cell-mediated changes in the expression of endocytic receptors and endocytosis process of human dendritic cells

CD4⁺CD25⁺ regulatory T cells (Tregs) are known to inhibit immune responses to antigens. Since, the process of antigen uptake by dendritic cells (DC) is central to induction of immune responses, we analyzed the effect of Tregs on the expression of endocytic receptors on DC and its repercussion on antigen uptake. Our results demonstrate that Tregs down-regulate the expression and uptake of antigens via C-type lectin-like receptors CD206 and DC-SIGN, restrain the pinocytosis process of DC and augment the expression of FcγRIIB, an inhibitory Fcγ receptor the engagement of which by IgG-bound antigens leads to inhibition of DC activation. Our results thus provide an additional insight on the pertinence of strategies aimed at blocking Treg functions towards improved vaccination protocols.     

Effects of Vitamin D Supplementation on CD4+ T Cell Subsets and mTOR Signaling Pathway in High-Fat-Diet-Induced Obese Mice

Obesity is associated with an impaired balance of CD4⁺ T cell subsets. Both vitamin D and obesity have been reported to affect the mTOR pathway. In this study, we investigated the effects of vitamin D on CD4⁺ T cell subsets and the mTOR pathway. Ten-week-old male C57BL/6 mice were divided into four groups and fed diets with different fat (control or high-fat diets: CON or HFD) and vitamin D contents (vitamin D control or supplemented diets: vDC or vDS) for 12 weeks. T cells purified by negative selection were stimulated with anti-CD3/anti-CD28 mAbs and cultured for 48 h. The percentage of CD4⁺IL-17⁺ T cells was higher in the vDS than vDC groups. The CD4⁺CD25⁺Foxp3⁺ T cells percentage was higher in HFD than CON groups. The phospho-p70S6K/total-p70S6K ratio was lower in vDS than vDC, but the phospho-AKT/total-AKT ratio was higher in vDS than vDC groups. Hif1α mRNA levels were lower in vDS than vDC groups. These findings suggest HIF1α plays an important role in vitamin-D-mediated regulation of glucose metabolism in T cells, and dietary vitamin D supplementation may contribute to the maintenance of immune homeostasis by regulating the mTOR pathway in T cells.     

An HIVgp41 vaccine protects CD4 central memory T cells in SHIV-infected macaques

Background

The immunodeficiency defining AIDS results from a progressive decline in the number of CD4⁺ T cells. Although no single viral protein is likely to be the sole effector of immune impairment, the gp41 envelope protein is believed to contribute significantly to AIDS pathogenesis. We have shown that 3S, a unique motif of gp41, is highly conserved in HIV-1 strains and specifically induces NKp44L, a ligand of the natural cytotoxicity receptor NKp44, on CD4⁺ T cells, rendering them sensitive to NK lysis. We therefore hypothesized that a 3S/gp41 vaccine strategy designed to modulate the NK cell compartment might improve CD4⁺ T cell resistance, independently of any effect on viral load.

Methods

Nine macaques were chronically infected with the SHIV163P3; four were then immunized with the 3S/gp41 peptide and five with the carrier alone. Frequency of CD4⁺ T cell subsets, proliferation, cell activation and apoptosis were analyzed in the periphery and the lymph nodes, spleen and rectum by flow cytometry.

Results

The anti-3S antibodies prevented NKp44L expression on CD4⁺ T cells in vivo and subsequently preserved the CD4⁺ central memory T cells in 3S/gp41-vaccinated animals. They also limited the NK cytotoxic activity against autologous CD4⁺ T cells, the cell activation, the proliferation, and the apoptosis in peripheral blood and secondary lymphoid tissues remained intact.

Conclusion

These data suggest a new paradigm for AIDS vaccine development, aimed at generating specific responses to protect a specific subset of CD4⁺ T cells from attack by activated NK cells.     

Characteristics of human CD34+ cells exposed to ionizing radiation under cytokine-free conditions

To clarify the mechanisms underlying radiation-induced hematopoietic stem cell death, we investigated the effects of excessive ionizing radiation on the clonogenic potential of CD34⁺ cells obtained from human umbilical cord blood under cytokine-free conditions. The CD34⁺ cells were X-ray–irradiated (up to 2 Gy) and were cultured for 0–48 h under cytokine-free conditions. At various time-points, the CD34⁺ cells were investigated for survival, clonogenic potential and the generation of mitochondrial superoxide. At 12 h after X-ray irradiation, the number of viable cells had decreased to ∼70–80% compared with the 0-h non-irradiated control, whereas the clonogenic potential in the X-ray–irradiated cells had decreased to ∼50%–60% compared with the 0-h non-irradiated control. Furthermore, significant generation of mitochondrial superoxide was observed at 6 h, and reached a maximum value between 12 and 24 h after X-ray irradiation. However, no significant differences were observed between non-irradiated and X-ray–irradiated cells in terms of the generation of reactive oxygen species or in the intracellular mitochondrial contents. In addition, a cDNA microarray analysis showed that the majority of the altered genes in the CD34⁺ cells at 6 h after X-ray irradiation were apoptosis-related genes. These results suggest the possibility that the elimination of the clonogenic potentials of CD34⁺ cells involves the generation of mitochondrial superoxide induced by ionizing radiation.     

Effects of occupational metallic mercury vapour exposure on suppressor-inducer (CD4+CD45RA+) T lymphocytes and CD57+CD16+ natural killer cells

Objectives: To examine the effects of metallic mercury vapour on the cellular and humoral immune system. Methods: We measured T lymphocyte and natural killer (NK) cell subpopulations, B lymphocytes, and serum immunoglobulins (i.e. IgG, IgA and IgM) together with total T (CD3+) lymphocytes and total lymphocytes in blood samples from 20 male, fluorescent-lamp makers (mercury workers) and the same number of gender-, age- and smoking-matched controls. Urinary concentrations of inorganic mercury (UHg) in the 20 workers ranged from 1.8 to 163.5 (mean 44.8) μg/l. They had been exposed to mercury vapour for 4 to 62 (mean 31) months. Results: Numbers of CD4+CD45RA+ (suppressor-inducer) T lymphocytes and total CD4+ T lymphocytes in the mercury workers were significantly smaller than those in the controls (paired-sample t-test, P < 0.01). The number of CD57+CD16+ NK cells was inversely correlated with UHg. Conclusion: It is suggested that numbers of CD4+CD45RA+ T lymphocytes and CD57+CD16+ NK cells are inversely affected by exposure to metallic mercury vapour in workers, with an average urinary inorganic mercury concentration of 45 μg/l being found. Received: 7 September 1999 / Accepted: 6 May 2000     

Phenotype of the anti-Rickettsia CD8 T cell response suggests cellular correlates of protection for the assessment of novel antigens

The obligately intracellular bacteria Rickettsia infect endothelial cells and cause systemic febrile diseases that are potentially lethal. No vaccines are currently available and current knowledge of the effective immune response is limited. Natural and experimental rickettsial infections provide strong and cross-protective cellular immunity if the infected individual survives the acute infection. Although resistance to rickettsial infections is attributed to the induction of antigen-specific T cells, particularly CD8⁺ T cells, the identification and validation of correlates of protective cellular immunity against rickettsial infections, an important step toward vaccine validation, remains a gap in this field. Here, we show that after a primary challenge with Rickettsia typhi in the C3H mouse model, the peak of anti-Rickettsia CD8⁺ T cell-mediated responses occurs 7 days post-infection (dpi), which coincides with the beginning of rickettsial clearance. At this time point, both effector-type and memory-type CD8⁺ T cells are present, suggesting that 7 dpi is a valid time point for the assessment of CD8⁺ T cell responses of mice previously immunized with protective antigens. Based on our results, we suggest four correlates of cellular protection for the assessment of protective rickettsial antigens: (1) production of IFN-γ by antigen-experienced CD3⁺CD8⁺CD44^high cells, (2) production of Granzyme B by CD27^lowCD43^low antigen-experienced CD8⁺ T cells, (3) generation of memory-type CD8⁺ T cells [Memory Precursor Effector Cells (MPECs), as well as CD127^highCD43^low, and CD27^highCD43^low CD8⁺ T cells], and (4) generation of effector-like memory CD8⁺ T cells (CD27^lowCD43^low). We propose that these correlates could be useful for the general assessment of the quality of the CD8⁺ T cell immune response induced by novel antigens with potential use in a vaccine against Rickettsia.     

Mucosal immunization in macaques upregulates the innate APOBEC 3G anti-viral factor in CD4 memory T cells

APOBEC3G is an innate intracellular anti-viral factor which deaminates retroviral cytidine to uridine. In vivo studies of APOBEC3G (A3G) were carried out in rhesus macaques, following mucosal immunization with SIV antigens and CCR5 peptides, linked to the 70 kDa heat shock protein. A progressive increase in A3G mRNA was elicited in PBMC after each immunization (p < 0.0002 to p ≤ 0.02), which was maintained for at least 17 weeks. Analysis of memory T cells showed a significant increase in A3G mRNA and protein in CD4⁺CCR5⁺ memory T cells in circulating (p = 0.0001), splenic (p = 0.0001), iliac lymph nodes (p = 0.002) and rectal (p = 0.01) cells of the immunized compared with unimmunized macaques. Mucosal challenge with SIVmac 251 showed a significant increase in A3G mRNA in the CD4⁺CCR5⁺ circulating cells (p < 0.01) and the draining iliac lymph node cells (p < 0.05) in the immunized uninfected macaques, consistent with a protective effect exerted by A3G. The results suggest that mucosal immunization in a non-human primate can induce features of a memory response to an innate anti-viral factor in CCR5⁺CD4⁺ memory and CD4⁺CD95⁺CCR7⁻ effector memory T cells.     

Humoral and cellular immune response generated by different vaccine programs before and after Salmonella Enteritidis challenge in chickens

The poultry industry has a high demand for Salmonella vaccines in order to generate safer Salmonella-free food for consumers around the world. Vaccination against S. Enteritidis (SE) is vastly undertaken in many countries, although the criteria for the use of live vaccine (LV) or killed vaccine (KV) should also depend on the immune mechanisms triggered by each. In this study, a commercial bacterin (KV) and an attenuated SG mutant (LV) were used in four different vaccine programs (LV; LV + LV; KV; LV + KV). At 1 day before (dbi) and 1, 6 and 9 days after SE challenge (dpi), humoral (IgM, IgG and secretory IgA) and cellular (CD8⁺ T cells) immune responses were evaluated along with the production of IL-10, IL-12 and IFN-γ. Although after challenge, all birds from each group had an influx of CD8⁺ T cells, birds which received KV had lower levels of these cells in organs and significantly higher levels of immunoglobulins. The expression of the cytokines was up-regulated in all groups post-vaccination, although, after challenge, cytokine expression decreased in the vaccinated groups, and increased in the unvaccinated group A. IL-10 levels were significantly higher at 1 day post-infection in the group that received KV, which may be involved in the weak cellular immune response observed within this group. In caecal tonsils, IFN-γ expression at 1 dbi was higher in birds which received two vaccine doses, and after challenge, the population of CD8⁺ T cells constantly increased in birds that were only vaccinated with the LV. This study demonstrated that the development of a mature immune response by CD8⁺ T cells, provided by the use of the LV, had better efficacy in comparison to the high antibody levels in the serum stimulated by the KV. However, high secretory IgA levels in the intestinal lumen associated with influx CD8⁺ T cells may be indicative of protection as noticed in group E (LV + KV).     

GP120-specific exosome-targeted T cell-based vaccine capable of stimulating DC- and CD4(+) T-independent CTL responses

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutics. In this study, we generated ovalbumin (OVA)-pulsed and pcDNAgp120-transfected dendritic cell (DC)-released exosomes (EXOova and EXOgp120) and ConA-stimulated C57BL/6 CD8⁺ T cells. OVA- and Gp120-Texo vaccines were generated from CD8⁺ T cells with uptake of EXOova and EXOgp120, respectively. We demonstrate that OVA-Texo stimulates in vitro and in vivo OVA-specific CD4⁺ and CD8⁺ cytotoxic T lymphocyte (CTL) responses leading to long-term immunity against OVA-expressing BL6-10_OVA melanoma. Interestingly, CD8⁺ T cell responses are DC and CD4⁺ T cell independent. Importantly, Gp120-Texo also stimulates Gp120-specific CTL responses and long-term immunity against Gp120-expressing B16 melanoma. Therefore, this novel HIV-1-specific EXO-targeted Gp120-Texo vaccine may be useful in induction of efficient CTL responses in AIDS patients with DC dysfunction and CD4⁺ T cell deficiency.     

CD207 cells recruitment to the vaccination site and draining lymph nodes after the administration of DC-Apo/Nec vaccine in mice

De novo ectopic lymphoid tissue formation is known to occur in certain disease and inflammatory settings. After an effective vaccination with dendritic cells (DC) charged with melanoma apoptotic/necrotic cells (Apo/Nec), a subcutaneous tertiary lymphoid structure was organized, where retained vaccine cells interacted with recruited inflammatory and T cells. In this work we report for the first time the recruitment of two morphologically different CD207⁺ cells to vaccination site. The time-course behavior of CD207⁺ cells was reciprocal between vaccination site and draining lymph nodes (DLNs). After 6–10 days, CD207⁺ cells localized at the paracortical region of DLNs, in close contact with T cell population. DLNs were enriched in a peculiar MHCII⁺ CD11c⁽⁻⁾ CD207⁺ population, whose role remains to be determined. Whether CD207⁺ cells migration to the vaccination site can be associated with a differential anti-tumoral response remains as an open and exciting question.     

CD4 T-cell responses among adults and young children in response to Streptococcus pneumoniae and Haemophilus influenzae vaccine candidate protein antigens

We characterized cytokine profiles of CD4⁺ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4⁺ T cells producing IL-2, (p = 0.004). Vaccine antigen-specific CD4⁺ T-cell populations in adults were largely of effector (T_EM) and/or central memory (T_CM) phenotypes as defined by CD45RA⁻CCR7⁺ or CD45RA⁻CCR7⁻ respectively; however among young children antigen-specific IL-2 producing CD4⁺ T cells demonstrated CD45RA⁺ expression (non-memory cells). We conclude that adults have circulating memory CD4⁺ T cells (CD45RA⁻) that can be stimulated by all the tested Spn and NTHi protein vaccine candidate antigens, whereas young children have a more limited response.     

Memory CD8 T cells elicited by HIV-1 lipopeptide vaccines display similar phenotypic profiles but differences in term of magnitude and multifunctionality compared with FLU- or EBV-specific memory T cells in humans

Differentiation marker, multifunctionality and magnitude analyses of specific-CD8⁺ memory T cells are crucial to improve development of HIV vaccines designed to generate cell-mediated immunity. Therefore, we fully characterized the HIV-specific CD8⁺ T cell responses induced in volunteers vaccinated with HIV lipopeptide vaccines for phenotypic markers, tetramer staining, cytokine secretion, and cytotoxic activities. The frequency of ex vivo CD8⁺ T cells elicited by lipopeptide vaccines is very rare and central-memory phenotype and functions of these cells were been shown to be important in AIDS immunity. So, we expanded them using specific peptides to compare the memory T cell responses induced in volunteers by HIV vaccines with responses to influenza (FLU) or Epstein Barr virus (EBV). By analyzing the differentiation state of IFN-γ-secreting CD8⁺ T cells, we found a CCR7⁻CD45RA⁻CD28^+int/CD28⁻ profile (>85%) belonging to a subset of intermediate-differentiated effector T cells for HIV, FLU, and EBV. We then assessed the quality of the response by measuring various T cell functions. The percentage of single IFN-γ T cell producers in response to HIV was 62% of the total of secreting T cells compared with 35% for FLU and EBV, dual and triple (IFN-γ/IL-2/CD107a) T cell producers could also be detected but at lower levels (8% compared with 37%). Finally, HIV-specific T cells secreted IFN-γ and TNF-α, but not the dual combination like FLU- and EBV-specific T cells. Thus, we found that the functional profile and magnitude of expanded HIV-specific CD8⁺ T precursors were more limited than those of to FLU- and EBV-specific CD8⁺ T cells. These data show that CD8⁺ T cells induced by these HIV vaccines have a similar differentiation profile to FLU and EBV CD8⁺ T cells, but that the vaccine potency to induce multifunctional T cells needs to be increased in order to improve vaccination strategies.     

CD4 T lymphocytes mediated protection against invasive pneumococcal infection induced by mucosal immunization with ClpP and CbpA

Intranasal delivery of pneumococcal protein vaccines would be a promising way to prevent invasive pneumococcal infection. Using an invasive infection model by intranasal inoculation of pneumococci, we demonstrated that immunizing mice intranasally with a mixture of ClpP (the caseinolytic protease) and CbpA (Choline binding protein A) elicited better protection than that of immunizing either single ClpP or CbpA. Anti-ClpP or anti-CbpA hyperimmune sera from intranasal-immunized mice significantly inhibited the adhesion of Streptococcus pneumoniae to A549 cells and combination of two antisera resulted in an additive effect. Both of two antisera could also kill S. pneumoniae by polymorphonuclear leukocytes in a complement-dependent way. The anti-infection activity and production of hyperimmune antibodies induced by mucosal immunization with ClpP and CbpA could be abrogated by the depletion of CD4⁺ T lymphocytes. Our data therefore indicated a critical role for CD4⁺ T lymphocytes in developing mucosal protein-based vaccines against invasive pneumococcal infection.     

Identification and Validation of Nutrient State-Dependent Serum Protein Mediators of Human CD4+ T Cell Responsiveness

Intermittent fasting and fasting mimetic diets ameliorate inflammation. Similarly, serum extracted from fasted healthy and asthmatic subjects’ blunt inflammation in vitro, implicating serum components in this immunomodulation. To identify the proteins orchestrating these effects, SOMAScan technology was employed to evaluate serum protein levels in healthy subjects following an overnight, 24-h fast and 3 h after refeeding. Partial least square discriminant analysis identified several serum proteins as potential candidates to confer feeding status immunomodulation. The characterization of recombinant IGFBP1 (elevated following 24 h of fasting) and PYY (elevated following refeeding) in primary human CD4⁺ T cells found that they blunted and induced immune activation, respectively. Furthermore, integrated univariate serum protein analysis compared to RNA-seq analysis from peripheral blood mononuclear cells identified the induction of IL1RL1 and MFGE8 levels in refeeding compared to the 24-h fasting in the same study. Subsequent quantitation of these candidate proteins in lean versus obese individuals identified an inverse regulation of serum levels in the fasted subjects compared to the obese subjects. In parallel, IL1RL1 and MFGE8 supplementation promoted increased CD4⁺ T responsiveness to T cell receptor activation. Together, these data show that caloric load-linked conditions evoke serological protein changes, which in turn confer biological effects on circulating CD4⁺ T cell immune responsiveness.     

Modulation of HIV peptide antigen specific cellular immune response by synthetic α- and β-defensin peptides

Defensin peptides have their direct role in host defense against microbial infection as innate molecules and also thought to contribute to adaptive immunity by recruiting naïve T-cells and immature dendritic cells at the site of infection through CCR6 receptor. The main aim of the present study is to investigate the efficacy of defensins for the induction of cell mediated immune response against the peptide antigen of HIV-1 encapsulated in PLG microparticles through intranasal (IN) route in mice model. To characterized, we have analyzed T-cell proliferation, Th1/Th2 cytokines, β-chemokines production and IFN-γ/perforin secretion from CD4⁺/CD8⁺ T-cells in response to HIV immunogen alone and with defensins at different mucosal site i.e. lamina propria (LP), spleen (SP) and peyer's patches (PP). The cellular immunogenicity of HIV peptide with defensin formulations showed a significantly higher (p < 0.001) proliferation response as compared to individual HIV peptide. The enhanced cytokines measurement profile showed mixed Th1 and Th2 type of peptide specific immune response by the incorporation of defensins. In the continuation, enhancement in MIP-1α and RANTES level was also observed in HIV peptide–defensin formulations. The FACS data had revealed that CD4⁺/CD8⁺ T-cells showed significantly (p < 0.001) higher IFN-γ and perforin secretion in HIV with defensin peptide formulations than HIV antigen alone group. Thus, the study emphasized here that defensin peptides have a potential role as mucosal adjuvant, might be responsible for the induction of cell mediated immunity when administered in mice through IN route with HIV peptide antigen.     

An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8 T cells and antibody when expressed from modified vaccinia Ankara

Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8⁺ T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8⁺ T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8⁺ T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8⁺ T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8⁺ T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.     

Frequencies of CD4+ T Regulatory Cells and their CD25high and FoxP3high Subsets Augment in Peripheral Blood of Patients with Acute and Chronic Brucellosis

Objectives

Brucellosis remains one of the most common zoonotic diseases worldwide. In humans, brucellosis can be a serious, debilitating, and sometimes chronic disease. Different mechanisms can be postulated as to the basis for the induction of the chronic status of infectious diseases that T regulatory cells are one of the most important related mechanisms. The current study was designed to determine whether percentage of CD4+Treg cells and their CD25^high and FoxP3^high subpopulations in peripheral blood are changed in human brucellosis samples in comparison to a control group.

Methods

In total, 68 brucellosis patients (acute form: n = 43, chronic form: n = 25) and 36 healthy volunteers entered our study. After isolating of peripheral blood mononuclear cells, heparinized venous blood samples were obtained from both patients and healthy donors, CD4, CD25, and FoxP3 molecules were evaluated by two- and three-color flow cytometric methods.

Results

The results revealed a new finding in relation to Treg cells and human brucellosis. The numbers of CD4⁺Treg cells and their CD25^high and FoxP3^high subsets increase significantly in the peripheral blood of acute and chronic forms of brucellosis samples compared with healthy groups, with this increase being greater in the chronic group.

Conclusion

There seems to be a correlation between increase of CD4+Treg cells and their subsets and the disease progress from healthy state to acute and chronic brucellosis.     

Antibody and memory CD4 T-cell responses after meningococcal disease

Currently, Neisseria meningitidis serogroup C (MenC) is the major cause of bacterial meningitis in Brazil, affecting mainly teenagers and adults due to the lack of routine public vaccination of these age groups. The goal of this study was to investigate the bactericidal antibody response and the development of CD4⁺ T cell memory during the convalescent phase of patients infected with N. menigitidis. Most (85.7%) of the patients developed a protective antibody response against MenC and 57% also responded to N. meningitidis serogroup B. We detected a significant CD4⁺ T central memory (T_CM) response to meningococcal outer membrane proteins.