Temporal Dynamics of T Helper Populations in the Proximal Small Intestine after Oral Bovine Lactoferrin Administration in BALB/c Mice

Bovine lactoferrin (bLf), a component of milk and a dietary supplement, modulates intestinal immunity at effector and inductor sites. Considering the regional difference in intestinal compartments and the dynamics of local cytokine-producing cells in the gut across time, the aim of this work was to characterize the effects of bLf on the proximal small intestine in a BALB/c murine model of oral administration. Male BALB/c mice were treated with oral bLf vs. saline control as mock by buccal deposition for 28 days. Intestinal secretions were obtained at different time points and cells were isolated from Peyer’s patches (PP) and lamina propria (LP) of the proximal small intestine as representative inductor and effector sites, respectively. Total and specific anti-bLF IgA and IgM were determined by enzyme-immuno assay; the percentages of IgA⁺ and IgM⁺ plasma cells (PC) and cytokine-producing CD4⁺ T cells of PP and LP were analyzed by flow cytometry. We found that total and bLf-specific IgA and IgM levels were increased in the intestinal secretions of the bLf group in comparison to mock group and day 0. LP IgA⁺ PC and IgM⁺ PC presented an initial elevation on day 7 and day 21, respectively, followed by a decrease on day 28 in comparison to mock. Higher percentages of CD4⁺ T cells in LP were found in the bLf group. Cytokines-producing CD4⁺ T cells populations presented a pattern of increases and decreases in the bLf group in both LP and PP. Transforming growth factor beta (TGF-β)⁺ CD4⁺ T cells showed higher percentages after bLf administration with a marked peak at day 21 in both LP and PP in comparison to mock-treated mice. Oral bLf exhibits complex immune properties in the proximal small intestine, where temporal monitoring of the inductor and effector compartments reveals patterns of rises and falls of different cell populations. Exceptionally, TGF-β⁺ CD4⁺ T cells show consistent higher numbers after bLf intervention across time. Our work suggests that isolated measurements do not show the complete picture of the modulatory effects of oral bLf in immunological sites as dynamic as the proximal small intestine.     

Altered or impaired immune response upon vaccination in WNIN/Ob rats

The present study was aimed to study the immune response in three months old male and female naïve obese animals and upon hepatitis B vaccination in three months old female WNIN/Ob obese mutant rats, established at our institute in comparison with its lean litter mates. Altered immune profile was seen in naïve obese mutant rats in terms of percentage of splenic CD8⁺ cytotoxic cells in males and percentages of splenic CD3⁺ T lymphocytes and CD4⁺ T helper cells in females respectively. Furthermore these obese mutant rats also exhibited impaired immune response to hepatitis B vaccine with low specific Hepatitis B surface antigen (HBsAg) specific IgG response and splenic lymphocyte proliferative response to HBsAg compared to the lean counterpart. The loss of immunological memory following vaccination could be attributed to the metabolic and hormonal changes associated with obesity. This observation has implication in public health policies related to vaccination in developed as well as developing countries.     

Intranasal immunization with vaccine vector Streptococcus gordonii elicits primed CD4 and CD8 T cells in the genital and intestinal tracts

Generation of primed T cells is crucial for the development of optimal vaccination strategies. Using a TCR-transgenic CD4⁺ and CD8⁺ T cell adoptive transfer model, we demonstrate that a single nasal immunization with recombinant Streptococcus gordonii induces antigen-specific primed T cells in lymph nodes draining the genital and intestinal tracts with about 80% of CD4⁺ and 50% of CD8⁺ proliferating cells. T cell clonal expansion was also observed in cervical lymph nodes, draining the immunization site, and in the spleen. The modulation of CD44 and CD45RB marker expression indicated that proliferating T cells were activated. Proliferation in distal mesenteric and iliac lymph nodes and in the spleen was observed 5 days after nasal immunization, while in draining cervical lymph nodes proliferation peaked already at day 3. The division profile of transgenic T cells observed in iliac and mesenteric lymph nodes was discontinuous, showing the lack of early cell divisions. The kinetics of T cell clonal expansion, the discontinuous division profile and the modulation of migration markers such as CD62L suggest that activated antigen-specific T cells disseminate from the immunization site to distal intestinal and genital tracts.     

Glutamine modulates CD8αα+ TCRαβ+ intestinal intraepithelial lymphocyte expression in mice with polymicrobial sepsis

ObjectivesCD8αα⁺ T-cell receptor (TCR) αβ⁺ intestinal intraepithelial lymphocytes (IELs) were found to have a regulatory function in the mucosal immune system. Glutamine (GLN) is an amino acid with immunomodulatory effects. The aim of this study was to investigate the influences of GLN on the proportion of CD8αα⁺ TCRαβ⁺ IELs and associated inflammatory mediator gene expression in polymicrobial sepsis.MethodsMice were randomly assigned to a normal (NC) group, a sepsis with saline (SS) group, or a sepsis with GLN (SG) group. The NC group was fed a chow diet. Sepsis was induced by cecal ligation and puncture (CLP). The SS group was administered saline, and the SG group was given 0.75 g GLN/kg body weight via a tail vein after CLP. Mice were sacrificed 12 h after CLP, and CD8αα⁺ TCRαβ⁺ IELs were isolated for further analysis.ResultsSepsis resulted in a lower percentage of CD8αα⁺ TCRαβ⁺ IELs, and higher messenger (m)RNA expression of complement 5a receptor, interleukin (IL)-2 receptor β, IL-15 receptor α, and interferon-γ by CD8αα⁺ TCRαβ⁺ IELs. These immunomodulatory mediator genes decreased, whereas IL-7 receptor and transforming growth factor-β expressions increased in CD8αα⁺ TCRαβ⁺ IELs in septic mice with GLN administration. Annexin V?7-AAD staining revealed significantly lower apoptotic rates of CD8αα⁺ TCRαβ⁺ IELs in the SG group.ConclusionA single dose of GLN administered after the initiation of sepsis increased the percentage of CD8αα⁺ TCRαβ⁺ IELs, prevented apoptosis of CD8αα⁺ TCRαβ⁺ IELs, and downregulated CD8αα⁺ TCRαβ⁺ IEL-expressed inflammatory mediators. These results suggest that GLN influenced the distribution and cytokine secretion of the CD8αα⁺ TCRαβ⁺ IEL subset, which may ameliorate sepsis-induced inflammatory reactions and thus mitigate the severity of intestinal epithelial injury.     

Alterations in serotype-specific B cell responses to the 13-valent pneumococcal conjugate vaccine in aging HIV-infected adults

BackgroundAdvanced age and human immunodeficiency virus (HIV) infection are associated with increased pneumococcal disease risk. The impact of these factors on cellular responses to vaccination is unknown.MethodsHIV-infected (HIV+) individuals 50–65 years old with CD4⁺ T cells/μl (CD4) >200 on antiretroviral therapy (ART) ≥1 year received either the 13-valent pneumococcal conjugate vaccine followed by the 23-valent pneumococcal polysaccharide vaccine (PCV/PPV) or PPV only. HIV-uninfected (HIV−) controls received PCV/PPV. Phenotype distribution and surface expression of complement receptor CD21 and tumor necrosis factor superfamily receptors (TNFRs) were compared on serotype-specific B cells postvaccination.ResultsPostvaccination serotype-specific B cell percentages were significantly lower in HIV+ PCV/PPV compared to PPV groups, but similar between HIV+ or HIV− PCV/PPV groups. Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI)⁺ serotype-specific B cell percentages were significantly decreased in HIV+ PCV/PPV compared to PPV groups. CD21⁺ serotype-specific B cells were significantly higher in HIV− compared to HIV+ PCV/PPV groups.ConclusionsAn initial dose of PCV reduced the frequency, but not phenotype distribution, of serotype-specific B cells and also lowered TACI expression in aging HIV+ subjects postvaccination with PPV. These findings suggest that PCV does not enhance cellular responses to revaccination with PPV.     

The temperature-sensitive mutants of Toxoplasma gondii and ocular toxoplasmosis

The risk of blindness caused by ocular toxoplasmosis supports efforts to improve our understanding for control of this disease. In this study, the involvement of CD8⁺, CD4⁺, B cell, and IL-10 gene in the immune response of primary ocular infection with the temperature-sensitive mutant (ts-4) of the RH Toxoplasma gondii strain, and in the protective immunity of ocular ts-4 vaccination and challenge with RH strain was investigated in murine models utilizing inbred C57BL/6 mice-deficient in CD4⁺, CD8⁺, B cells (μMT), or IL-10 gene. Compared to naive mice, all WT and mutant mice had different degree of ocular pathological changes after ts-4 ocular infection, in which both CD8 KO and IL-10 KO mice showed the most severe ocular lesions. Immunized by ts-4 intracameral (i.c.) inoculation, all mutant mice had partially decreased vaccine-induced resistance associated with increased ocular parasite burdens after RH strain challenge. A significant increase of the percentages of B cells and CD8⁺ T cells in the draining lymph nodes were observed in WT and IL-10 KO mice after either infection or challenge. The levels of specific anti-toxoplasma IgG in both eye fluid and serum from all the mice were significantly increased after ts-4 i.c. immunization, except μMT mice. These results suggest that the avirulent ts-4 of T. gondii inoculated intracamerally can induce both ocular pathology and ocular protective immunity; CD4⁺, CD8⁺, B cell, and IL-10 gene are all necessary to the vaccine-induced resistance to ocular challenge by virulent RH strain, in which CD8⁺ T cells are the most important component.     

Transcriptional Profiling of the Small Intestine and the Colon Reveals Modulation of Gut Infection with Citrobacter rodentium According to the Vitamin A Status

Vitamin A (VA) deficiency and diarrheal diseases are both serious public health issues worldwide. VA deficiency is associated with impaired intestinal barrier function and increased risk of mucosal infection-related mortality. The bioactive form of VA, retinoic acid, is a well-known regulator of mucosal integrity. Using Citrobacter rodentium-infected mice as a model for diarrheal diseases in humans, previous studies showed that VA-deficient (VAD) mice failed to clear C. rodentium as compared to their VA-sufficient (VAS) counterparts. However, the distinct intestinal gene responses that are dependent on the host’s VA status still need to be discovered. The mRNAs extracted from the small intestine (SI) and the colon were sequenced and analyzed on three levels: differential gene expression, enrichment, and co-expression. C. rodentium infection interacted differentially with VA status to alter colon gene expression. Novel functional categories downregulated by this pathogen were identified, highlighted by genes related to the metabolism of VA, vitamin D, and ion transport, including improper upregulation of Cl⁻ secretion and disrupted HCO₃⁻ metabolism. Our results suggest that derangement of micronutrient metabolism and ion transport, together with the compromised immune responses in VAD hosts, may be responsible for the higher mortality to C. rodentium under conditions of inadequate VA.     

Continuous versus discontinuous drinking of an ethanol liquid diet in peripubertal rats: effect on 24-h variation of lymph node and splenic mitogenic responses and lymphocyte subset populations

Excessive alcohol consumption continues to be a major public health problem, particularly in the adolescent and young adult populations. Generally, such a behavior tends to be confined to the weekends, to attain frequently binge drinking. This study in peripubertal male rats compares the effect of the discontinuous feeding of a liquid diet containing a moderate amount of ethanol (6.2% wt/vol) to that of continuous ethanol administration or a control diet, taking as end points the 24-h variations of plasma prolactin levels and mitogenic responses and lymphocyte subset populations in submaxillary lymph nodes and spleen. Animals received the ethanol liquid diet starting on day 35 of life, the diet being similar to that given to controls except for that maltose was isocalorically replaced by ethanol. Ethanol provided 36% of the total caloric content. Every week, the discontinuous ethanol group received the ethanol diet for 3 days and the control liquid diet for the remaining 4 days. After 4 weeks, rats were killed at six time intervals, beginning at 0900 h. A significant decrease of splenic cells’ response to concanavalin A, and of lymph node and splenic cells’ response to lipopolysaccharide was found in rats under the discontinuous ethanol regime, when compared with control- or ethanol-chronic rats. Under discontinuous ethanol feeding, mean values of lymph node and splenic CD8⁺ and CD4⁺-CD8⁺cells decreased, whereas those of lymph node and splenic T cells, and splenic B cells, augmented. In rats chronically fed with ethanol, splenic mean levels of CD8⁺ and CD4⁺-CD8⁺cells augmented. Both modalities of ethanol administration disrupted the 24 h variation in immune function seen in controls. Mean plasma prolactin levels increased by 3.6-fold and 8.5-fold in rats chronically or discontinuously fed with alcohol, respectively. The immune parameters examined in an additional group of rats fed regular chow and water ad libitum did not differ significantly from control liquid diet. The results support the view that the discontinuous drinking of a moderate amount of ethanol can be more harmful for the immune system than a continuous ethanol intake, presumably by inducing a greater stress as indicated by the augmented plasma prolactin levels observed.     

β-carotene regulates cancer stemness in colon cancer in vivo and in vitro

BACKGROUND/OBJECTIVESColorectal cancer (CRC) is the third most common cancer worldwide and has a high recurrence rate, which is associated with cancer stem cells (CSCs). β-carotene (BC) possesses antioxidant activity and several anticancer mechanisms. However, no investigation has examined its effect on colon cancer stemness.MATERIALS/METHODSCD133⁺CD44⁺ HCT116 and CD133⁺CD44⁺ HT-29 cells were isolated and analyzed their self-renewal capacity by clonogenic and sphere formation assays. Expressions of several CSCs markers and Wnt/β-catenin signaling were examined. In addition, CD133⁺CD44⁺ HCT116 cells were subcutaneously injected in xenograft mice and analyzed the effect of BC on tumor formation, tumor volume, and CSCs markers in tumors.RESULTSBC inhibited self-renewal capacity and CSC markers, including CD44, CD133, ALDH1A1, NOTCH1, Sox2, and β-catenin in vitro. The effects of BC on CSC markers were confirmed in primary cells isolated from human CRC tumors. BC supplementation decreased the number and size of tumors and delayed the tumor-onset time in xenograft mice injected with CD133⁺CD44⁺ HCT116 cells. The inhibitory effect of BC on CSC markers and the Wnt/β-catenin signaling pathway in tumors was confirmed in vivo as well.CONCLUSIONSThese results suggest that BC may be a potential therapeutic agent for colon cancer by targeting colon CSCs.     

FlaC supplemented with VAA,OmpK or OmpR as bivalent subunit vaccine candidates induce immune responses against Vibrio anguillarum in flounder (Paralichthys olivaceus)

In our previous study, ten candidate proteins have been identified with immunogenicity and protection against Vibrio anguillarum in flounder (Paralichthys olivaceus). Among them, FlaC is the important outer protein in the flagellum with immunogenicity; VAA, OmpK and OmpR are protection proteins against V. anguillarum. In this paper, FlaC supplemented with VAA, OmpK or OmpR as bivalent subunit vaccine candidates, and their immune response of flounder and protective effects were evaluated, respectively. Recombinant(r) proteins of FlaC were mixed with rVAA, rOmpK and rOmpR, respectively, rVAA + rFlaC (AF), rOmpK + rFlaC (KF) and rOmpR + rFlaC (RF); formalin-killed cells (FKC) or PBS were injected to flounder, respectively. After immunization, the percentages of CD3⁺ T lymphocytes and surface membrane immunoglobulin-positive (sIg⁺) B lymphocytes in peripheral blood lymphocytes (PBLs), total antibodies (TA), specific antibodies against V. anguillarum (VA), specific antibodies against bivalent recombinant proteins (PA), the expression of immune-related genes and relative percent survivals (RPS) were measured, respectively. The results showed that three bivalent vaccines candidates and FKC could induce the proliferation of sIg⁺ B lymphocytes and CD3⁺ T lymphocytes in PBLs. The TA, VA and PA induced in bivalent vaccines candidates and FKC groups were significantly higher than that of the control group. CD3, IgM, CD4-1, CD4-2, CD8α and CD8β genes were up-regulated. After challenge with V. anguillarum, RPS in AF, KF, RF and FKC groups exhibited 62.6 ± 2.33%, 78.95 ± 3.01%, 75.45 ± 0.97%, and 56.71 ± 2.15% respectively. The results revealed that three bivalent vaccines candidates and FKC could induce the immune response in flounder, and have good protection against V. anguillarum, and KF can be an efficient bivalent subunit vaccine candidate.     

Mice immunized with bone marrow-derived dendritic cells stimulated with recombinant Coxiella burnetii Com1 and Mip demonstrate enhanced bacterial clearance in association with a Th1 immune response

The recombinant membrane-associated proteins of Coxiella burnetii, Com1, Mip and GroEL, were used in vitro to stimulate BALB/c mouse bone marrow-derived dendritic cells (BMDCs). The antigen-activated BMDCs were transferred into naïve BALB/c mice. Seven days after challenge of C. burnetii, the bacterial loads of mice receiving BMDCs activated with Com1 or Mip, but not GroEL, were significantly lower than that of mice receiving BMDCs pulsed with TrxA (Esherichia coli thioredoxin) in a quantitative polymerase chain reaction assay. After in vitro interaction with cognate antigen-pulsed BMDCs, the percentages of CD69-positive cells and TNF-α-positive cells in CD4⁺ and CD8⁺ T cells isolated from the spleens of mice receiving Com1-, Mip-, or GroEL-pulsed BMDCs were significantly higher than that of mice receiving mock-pulsed BMDCs in flow cytometric analysis. The percentages of IFN-γ-positive cells in CD4⁺ and CD8⁺ T cells from mice receiving Com1- or Mip-pulsed BMDCs were significantly greater than that of mice receiving GroEL-pulsed BMDCs. However, the percentage of IL-4-positive cells in CD4⁺ T cells of mice receiving GroEL-pulsed BMDCs was obviously higher than that of mice receiving Com1- or Mip-pulsed BMDCs. Our results demonstrate that Com1 and Mip are protective antigens and strongly indicate that they favor to induce IFN-γ-producing Th1 and Tc1 cells, whereas the non-protective antigen GroEL is biased to induce a Th2 response. Therefore, Com1 and Mip are key antigens to induce a protective immune response against C. burnetii infection.     

Vaccination of pigs with attenuated Lawsonia intracellularis induced acute phase protein responses and primed cell-mediated immunity without reduction in bacterial shedding after challenge

BackgroundLawsonia intracellularis causes porcine proliferative enteropathy and is one of the most economically important diseases in modern pig production worldwide. The Enterisol^® Ileitis vaccine have been shown to reduce clinical disease and to increase weight gain, however, while the natural infection with L. intracellularis can provide complete protection against re-infection, this has not been achieved by this vaccine. We therefore undertook a detailed characterization of immune responses to L. intracellularis infection in vaccinated pigs (VAC) compared to previously infected pigs (RE) in order to pinpoint immunological determinants of protection.ResultsThe VAC pigs shed L. intracellularis to the same extent as non-vaccinated pigs after challenge, however less L. intracellularis in ileum and lymph nodes was seen post mortem. In the RE group, challenge did not lead to L. intracellularis shedding and no challenge bacteria were found post mortem. In both VAC and RE the acute phase haptoglobin response was diminished and L. intracellularis specific IgG responses were delayed and reduced compared to non-vaccinated pigs. On the other hand L. intracellularis specific IFN-γ responses tended to develop faster in the VAC group compared to controls.ConclusionAlthough vaccinated and non-vaccinated pigs shed L. intracellularis at similar levels after challenge, a lower number of intestinal L. intracellularis was observed in the vaccinated pigs at post mortem inspection. This might be due to the observed faster CMI responses upon challenge in vaccinated pigs. Complete protection against infection without L. intracellularis shedding, however, was only seen after a previous infection resulting in IFN-γ production predominantly by CD8⁺ and CD4⁺ CD8⁺ cells. Improved protective vaccines against L. intracellularis should therefore target stimulation of these T cell subsets.     

Reciprocal changes in CD11c+CD11b+ and CD11c+CD8α+ dendritic cell subsets determine protective or permissive immune response in murine experimental VL

CD11c⁺CD8α⁺ and CD11c⁺CD11b⁺ dendritic cells are two major subsets of murine splenic CD11c⁺ DCs which play a crucial role in T cell priming and shaping Th1/Th2 responses, but their role in the context of experimental visceral leishmaniasis (VL) is poorly understood. Herein, we showed that L. donovani infection in Balb/c mice preferentially decreased the population abundance of CD11c⁺CD11b⁺ DCs and increased relative abundance of splenic CD11c⁺CD8α ⁺DCs. During infection, splenic CD11c⁺CD11b⁺ DCs induced Th1 differentiation whereas CD11c⁺CD8α⁺ DCs promoted Th2 differentiation. Additionally, treatment of infected mice with miltefosine as experimental control exhibited host defense allowing the restoration of CD11c⁺CD11b⁺ population and decrease in CD11c⁺CD8α⁺ subset. Furthermore, reciprocal regulation of immune accessory surface molecules, Sema4A and OX40L critically determined Th1/Th2 response induced by these DC subsets during VL. L. donovani infection significantly induced OX40L expression and slightly downregulated SEMA 4A expression in CD11c⁺CD8α⁺ DCs whereas miltefosine treatment significantly downregulated OX40L expression along with pronounced upregulation of SEMA 4A expression in CD11c⁺CD11b⁺ DCs. SiRNA mediated knockdown of SEMA 4A markedly reduced CD11c⁺CD11b⁺ driven IFN-γ, TNF-α and IL-12 synthesis in miltefosine treated mice whereas functional blocking of OX40L decreased CD11c⁺CD8α⁺ induced IL-10, IL-4 and TGF-β synthesis in L. donovani infected group. Vaccination of Balb/c mice with antigen-pulsed + CpG-ODN-activated DC subsets revealed that only antigen-pulsed CD11c⁺CD11b⁺ DCs eliminated parasite load in visceral organ and restored protective Th1 cytokine response. Collectively, our results suggest that differential regulation of splenic CD11c⁺ subsets by L. donovani is essential for disease progression and specific subtypes may be exploited as prophylactic measures against visceral leishmaniasis.     

Characterization of the protective immune response to Yersinia pseudotuberculosis infection in mice vaccinated with an LcrV-secreting strain of Lactococcus lactis

BackgroundPseudotuberculosis is an infection caused by the bacterial enteropathogen Yersinia pseudotuberculosis and is considered to be a significant problem in veterinary medicine. We previously found that intranasal administration of a recombinant Lactococcus lactis strain that secretes the low-calcium response V (LcrV) antigen from Y. pseudotuberculosis (Ll-LcrV) confers protection against a lethal Y. pseudotuberculosis infection. Here, we aimed at characterizing the immunological basis of this LcrV-elicited protective response and at determining the duration of vaccine-induced immunity.MethodsSplenocytes from BALB/c mice intranasally immunized with Ll-LcrV or Ll as control were immunostained then analyzed by flow cytometry. Protection against a lethal intravenous injection of Y. pseudotuberculosis was also determined (i) in immunized BALB/c mice depleted or not of CD4⁺, CD8⁺ or CD25⁺ cells and (ii) in naïve BALB/c mice receiving serum from immunized mice by counting the number of bacteria in liver and spleen. Lastly, survival rate of immunized BALB/c mice following a lethal intravenous injection of Y. pseudotuberculosis was followed up to 9-months.ResultsWe found that T and B lymphocytes but not non-conventional lymphoid cells were affected by Ll-LcrV immunization. We also observed that depletion of CD4⁺ and CD25⁺ but not CD8⁺ cells in immunized mice eradicated protection against a lethal systemic Y. pseudotuberculosis infection, suggesting that activated CD4⁺ T lymphocytes are required for vaccine-induced protection. Adoptive transfer of LcrV-specific antibodies from Ll-LcrV-immunized animals significantly reduced the bacterial counts in the liver compared to non-vaccinated mice. Lastly, the protective immunity conferred by Ll-LcrV decreased slightly over time; nevertheless almost 60% of the mice survived a lethal bacterial challenge at 9 months post-vaccination.ConclusionMucosal vaccination of mice with Ll-LcrV induced cell- and antibody-mediated protective immunity against Y. pseudotuberculosis infection in the mouse and the protection is long-lasting.     

Study of lymphocyte subpopulations in bone marrow in a model of protein–energy malnutrition

ObjectiveProtein–energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. Hematopoietic tissue requires a high nutrient supply, and a reduction in leukocytes, especially lymphocytes, suggests that some nutritional deficiencies might be altering bone marrow function and decreasing its ability to produce lymphocytes. In this study, we evaluated the effect that PEM has on lymphocyte subtypes and the cell cycle of CD5⁺ cells.MethodsSwiss mice were subjected to PEM using a low-protein diet containing 4% protein. When the experimental group had lost about 20% of their original body weight, we collected blood and bone marrow cells and evaluated the hemogram, the myelogram, bone marrow lymphoid markers using flow cytometry, and the cell cycle in CD5⁺ bone marrow.ResultsMalnourished animals presented anemia, reticulocytopenia, and leukopenia with lymphopenia. The bone marrow was hypocellular, and flow cytometric analyses of bone marrow cells showed cells that were CD45⁺ (91.2%), CD2⁺ (84.9%), CD5⁺ (37.3%), CD3⁺ (23.5%), CD19⁺ (43.3%), CD22⁺ (34.7%), CD19⁺/CD2⁺ (51.2%), CD19⁺/CD3⁺ (24.0%), CD19⁺/CD5⁺ (13.2%), CD22⁺/CD2⁺ (40.1%), CD22⁺/CD3⁺ (30.3%), and CD22⁺/CD5⁺ (1.1%) in malnourished animals and CD45⁺ (97.5%), CD2⁺ (42.9%), CD5⁺ (91.5%), CD3⁺ (92.0%), CD19⁺ (52.0%), CD22⁺ (75.6%), CD19⁺/CD2⁺ (62.0%), CD19⁺/CD3⁺ (55.4%), CD19⁺/CD5⁺ (6.7%), CD22⁺/CD2⁺ (70.3%), CD22⁺/CD3⁺ (55.9%), and CD22⁺/CD5⁺ (8.4%) in control animals. Malnourished animals also presented more CD5⁺ cells in the G0 phase of cell cycle development.ConclusionMalnourished animals presented bone marrow hypoplasia, maturation interruption, prominent lymphopenia with depletion in the lymphoid lineage, and changes in cellular development. We suggest that these changes are some of the primary causes of lymphopenia in cases of PEM and partly explain the increase in susceptibility to infections found in malnourished individuals.     

CD11a and CD49d enhance the detection of antigen-specific T cells following human vaccination

BackgroundDetermining the efficacy of human vaccines that induce antigen-specific protective CD4 T cell responses against pathogens can be particularly challenging to evaluate. Surface expression of CD11a and CD49d has been shown to identify antigen-specific CD4 T cells against viral pathogens in mice. We hypothesized that CD11a and CD49d would also serve as markers of human antigen-specific T cells responding to vaccination.MethodsA phase I vaccine trial enabled us to evaluate a novel gating strategy based on surface expression of CD11a and CD49d as a means of detecting antigen-specific, cytokine producing CD4 and CD8 T cells induced after vaccination of naïve individuals against leishmaniasis. Three study groups received LEISH-F3 recombinant protein combined with either squalene oil-in-water emulsion (SE) alone, SE with the synthetic TLR-4 ligand glucopyranosyl lipid adjuvant (GLA-SE), or SE with Salmonella minnesota-derived monophosphoryl lipid A (MPL-SE). Individuals were given 3 vaccine doses, on days 0, 28 and 168.ResultsStarting after the first vaccine dose, the frequency of both CD11a^hiCD49d⁺ CD4 and CD11a^hiCD49d⁺ CD8 T cells significantly increased over time throughout the 24-week trial. To confirm the role of CD11a^hiCD49d⁺ expression in the identification of the antigen-specific T cells, cytokine production was measured following LEISH-F3 stimulation. All of the IFN-γ, TNF-α, and IL-2 producing cells were found within the CD11a^hiCD49d⁺ population.ConclusionsOur results suggest that the change in the frequency of CD11a^hiCD49d⁺ T cells can be used to track antigen-specific CD4 and CD8 T cell responses following T cell-targeted vaccination.     

Adoptive transfer of protective immunity from Cryptosporidium parvum-infected interferon-γ and interleukin-12-deficient mice to naive recipients

We investigated the possibility of transfer immunity from Cryptosporidium parvum-infected interferon-γ (GKO) and interleukin-12p40 (IL-12KO) deficient C57BL/6 mice to naive mice by transfer of intraepithelial lymphocytes (IELs) and CD4⁺ T cells from spleen and mesenteric lymph nodes (MLNs). Three days after the transfer recipients were infected with C. parvum. IELs isolated from GKO donor mice after resolution of infection (day 15) but not at the peak of infection (day 8) significantly reduced the parasite load in recipient mice. In IL-12KO mice, IELs and also CD4⁺ T cells isolated from the spleen and MLNs of donor mice at the peak of infection (day 5) and after resolution (day 15) significantly reduced the parasite excretion, emphasizing the role of interferon-γ in the host–parasite interaction. However, after resolution of infection, interferon-γ-independent mechanisms have evolved that render GKO IELs capable of protecting mice from severe infection.     

CD8+ T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity

CD8⁺ T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8⁺ T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8⁺ T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8⁺ T cells are generated and provide limited protection against challenge with virulent OVA⁺ Y. pseudotuberculosis. Perforin expression by OVA-specific CD8⁺ T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8⁺ T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8⁺ T cell status. These studies indicate that CD8⁺ T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death.     

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).