Impaired Thymic Output Can Be Related to the Low Immune Reconstitution and T Cell Repertoire Disturbances in Relapsing Visceral Leishmaniasis Associated HIV/AIDS Patients.

Background: Visceral leishmaniasis/HIV-co-infected patients (VL/HIV) accounts for around 8% of VL reported cases in Brazil. Relapses of Leishmania infection after anti-leishmanial treatment constitute a great challenge in the clinical practice because of the disease severity and drug resistance. We have shown that non-relapsing-VL/HIV (NR-) evolved with increase of CD4+ T-cell counts and reduction of activated CD4+ and CD8+ T cells after anti-leishmanial treatment. This immune profile was not observed in relapsing-VL/HIV patients (R-), indicating a more severe immunological compromising degree. Elevated activation status may be related to a deficient immune reconstitution and could help to explain the frequent relapses in VL/HIV co-infection. Our aim was to evaluate if this gain of T cells was related to changes in the peripheral TCRVß repertoire and inflammatory status, as well as the possible thymus involvement in the replenishment of these newly formed T lymphocytes. Methods: VL/HIV patients, grouped into non-relapsing (NR- = 6) and relapsing (R- = 12) were evaluated from the active phase up to 12 months post-treatment (mpt). HIV-infected patients (non-VL) and healthy subjects (HS) were included. The TCRVß repertoire was evaluated ex vivo by flow cytometry, whereas the plasmatic cytokine levels were assessed by Luminex assay. To evaluate the thymic output, DNA was extracted from PBMCs for TCR rearrangement excision circles (TREC) quantification by qPCR. Results: VL/HIV cases presented an altered mobilization profile (expansions or retractions) of the TCRVß families when compared to HS independent of the follow-up phase (p < 0.05). TCRVß repertoire on CD4+ T-cells was more homogeneous in the NR-VL/HIV cases, but heterogeneous on CD8+ T-cells, since different Vß-families were mobilized. NR-VL/HIV had the inflammatory pattern reduced after 6 mpt. Importantly, VL/HIV patients showed number of TREC copies lower than controls during all follow-up. An increase of recent thymic emigrants was observed in NR-VL/HIV individuals at 10 mpt compared to R- patients (p < 0.01), who maintained lower TREC contents than the HIV controls. Conclusions: VL/HIV patients that maintain the thymic function, thus generating new T-cells, seem able to replenish the T lymphocyte compartment with effector cells, then enabling parasite control.

Analysis of Swine Conventional Dendritic Cells, DEC205+CD172a+/-CADM1+, from Blood and Spleen in Response to PRRSV and PEDV.

Conventional dendritic cells (cDCs) cannot be infected by porcine reproductive and respiratory syndrome virus (PRRSV) but respond to infection via cytokine production, indicating a possible role in initiation/regulation of the immune response against PRRSV. In this work, we evaluated the responses of splenic and blood cDCs, with DEC205+CADM1+CD172a+/- phenotype, as well as those of CD163+ cells against PRRSV and porcine epidemic diarrhea virus (PEDV). Both populations were incubated in the presence of PRRSV or PEDV with and without naïve CD3+ T cells, and cytokine responses were evaluated by qPCR and ELISA. Our results showed that cDCs, but not CD163+ cells, produced IL-12 in response to PRRSV. PEDV did not induce IL-12 production. Cocultures of cDCs and autologous naïve CD3+ cells resulted in decreased IL-12 production and low expression of IFN-γ transcripts in response to PRRSV. Interestingly, cDCs increased the proliferation of naïve T cells in the presence of PRRSV compared with that achieved with monocytes and peripheral blood mononuclear cells (PBMCs). Cocultures of CD163+ cells induced IL-10 and IL-4 expression in the presence of PRRSV and PEDV, respectively. In conclusion, cDCs can selectively produce IL-12 in response to PRRSV but poorly participate in the activation of naïve T cells.

Cytotoxic CD4+ T-cells during HIV infection: Targets or weapons?

Classically, CD4+ T-cells have been referred as cytokine-producing cells and important players in immune responses by providing soluble factors that potentiate several effector immune functions. However, it is now evident that CD4+ T-cells can also elaborate cytotoxic responses, inducing apoptosis of target cells. Cytotoxic CD4+ T cells (CD4+ CTLs), exhibit cytolytic functions that resemble those of CD8+ T-cells; in fact, there is evidence suggesting that they may have a role in the control of viral infections. In this article, we discuss the role of CD4+ CTLs during HIV infection, where CD4+ CTLs have been associated with viral control and slow disease progression. In addition, we address the implication of CD4+ CTLs in the context of antiretroviral therapy and the partial reconstitution of CD8+ T-cells effector function.

Diminished effector and memory CD8+ circulating T lymphocytes in patients with severe influenza caused by the AH1N1 pdm09 virus.

The T cell immune response to viral infection includes the expansion of naïve T cells, effector cell differentiation and the induction of long-lived memory cells. We compared the differentiation of CD8+ T cells in patients with severe or mild pneumonia induced by influenza infection occurring during the 2009 influenza outbreak and compared their T cell subsets with those in blood samples obtained from healthy volunteers before the AH1N1 influenza outbreak in Mexico. Patients with severe influenza exhibited significantly lower numbers of effector memory CD8+CD26 high CD45RO+CCR7+ phenotype and lower numbers of central memory CD8+CD26high CD62L+CCR7+, CD26 high CD62L+CD127+ or CD26 high CD45RO+CD57 low phenotypes than patients with mild influenza or unexposed healthy subjects. Effector T cells with CD8+CD26CD62L low CD57+ phenotype were significantly diminished in severe influenza patients compared to those in patients with mild influenza or unexposed healthy subjects. These results suggest that low levels of circulating CD8+ T effector and central memory cells are associated with influenza severity.

A transcriptome-based model of central memory CD4 T cell death in HIV infection.

BACKGROUND: Human central memory CD4 T cells are characterized by their capacity of proliferation and differentiation into effector memory CD4 T cells. Homeostasis of central memory CD4 T cells is considered a key factor sustaining the asymptomatic stage of Human Immunodeficiency Virus type 1 (HIV-1) infection, while progression to acquired immunodeficiency syndrome is imputed to central memory CD4 T cells homeostatic failure. We investigated if central memory CD4 T cells from patients with HIV-1 infection have a gene expression profile impeding proliferation and survival, despite their activated state. METHODS: Using gene expression microarrays, we analyzed mRNA expression patterns in naive, central memory, and effector memory CD4 T cells from healthy controls, and naive and central memory CD4 T cells from patients with HIV-1 infection. Differentially expressed genes, defined by Log2 Fold Change (FC) ≥ |0.5| and Log (odds) > 0, were used in pathway enrichment analyses. RESULTS: Central memory CD4 T cells from patients and controls showed comparable expression of differentiation-related genes, ruling out an effector-like differentiation of central memory CD4 T cells in HIV infection. However, 210 genes were differentially expressed in central memory CD4 T cells from patients compared with those from controls. Expression of 75 of these genes was validated by semi quantitative RT-PCR, and independently reproduced enrichment results from this gene expression signature. The results of functional enrichment analysis indicated movement to cell cycle phases G1 and S (increased CCNE1, MKI67, IL12RB2, ADAM9, decreased FGF9, etc.), but also arrest in G2/M (increased CHK1, RBBP8, KIF11, etc.). Unexpectedly, the results also suggested decreased apoptosis (increased CSTA, NFKBIA, decreased RNASEL, etc.). Results also suggested increased IL-1ß, IFN-γ, TNF, and RANTES (CCR5) activity upstream of the central memory CD4 T cells signature, consistent with the demonstrated milieu in HIV infection. CONCLUSIONS: Our findings support a model where progressive loss of central memory CD4 T cells in chronic HIV-1 infection is driven by increased cell cycle entry followed by mitotic arrest, leading to a non-apoptotic death pathway without actual proliferation, possibly contributing to increased turnover.

Age-associated Epstein-Barr virus-specific T cell responses in seropositive healthy adults.

Epstein-Barr virus (EBV) is present in 95% of the world's adult population. The immune response participates in immune vigilance and persistent infection control, and this condition is maintained by both a good quality (functionality) and quantity of specific T cells throughout life. In the present study, we evaluated EBV-specific CD4(+) and CD8(+) T lymphocyte responses in seropositive healthy individuals younger and older than 50 years of age. The assessment comprised the frequency, phenotype, functionality and clonotypic distribution of T lymphocytes. We found that in both age groups a similar EBV-specific T cell response was found, with overlapping numbers of tumour necrosis factor (TNF)-α(+) T lymphocytes (CD4(+) and CD8(+)) within the memory and effector cell compartments, in addition to monofunctional and multi-functional T cells producing interleukin (IL)-2 and/or interferon (IFN)-γ. However, individuals aged more than 50 years showed significantly higher frequencies of IL-2-producing CD4(+) T lymphocytes in association with greater production of soluble IFN-γ, TNF-α and IL-6 than subjects younger than 50 years. A polyclonal T cell receptor (TCR)-variable beta region (Vß) repertoire exists in both age groups under basal conditions and in response to EBV; the major TCR families found in TNF-α(+) /CD4(+) T lymphocytes were Vß1, Vß2, Vß17 and Vß22 in both age groups, and the major TCR family in TNF-α(+) /CD8(+) T cells was Vß13·1 for individuals younger than 50 years and Vß9 for individuals aged more than 50 years. Our findings suggest that the EBV-specific T cell response (using a polyclonal stimulation model) is distributed throughout several T cell differentiation compartments in an age-independent manner and includes both monofunctional and multi-functional T lymphocytes.

Epstein-Barr virus presence in pediatric diffuse large B-cell lymphoma reveals a particular association and latency patterns: analysis of viral role in tumor microenvironment.

Non-Hodgkin's lymphoma represents 6-10% of pediatric malignancies, and diffuse large B-cell lymphoma (DLBCL) is one of the three major subtypes. The 2008 WHO classification included a new entity, Epstein-Barr virus (EBV)-positive DLBCL of the elderly, affecting patients >50 years. It has been demonstrated that EBV may play a role in tumor microenvironment composition, disturbing antitumor immune response and disease progression. As most studies were performed in adults, our aim was to assess EBV presence and latency pattern, as well as T-cell microenvironment in a pediatric DLBCL series of Argentina. The study was conducted on formalin-fixed paraffin-embedded biopsies from 25 DLBCL patients. EBV-encoded small nuclear early regions (EBERs) expression was performed by in situ hybridization, whereas EBV gene expression was analyzed using real-time PCR. Epstein-Barr virus latent membrane proteins (LMP)1, LMP2A, CD3, CD4, CD8 and Foxp3 expression were assessed by immunohistochemistry (IHC). Forty percent of cases showed EBV expression, with a significantly higher incidence among patients <10 years (p = 0.018), and with immunosuppressed (p = 0.023). T-cell subsets were not altered by EBV presence. Full EBV latency antigen expression (latency type III) was the most frequently pattern observed, together with BZLF1 lytic gene expression. One patient showed II-like pattern (LMP1 without LMP2A expression). Based exclusively on IHC, some patients showed latency II/III (EBERs and LMP1 expression) or I (EBERs only). These findings suggest that EBV association in our series was higher than the previously demonstrated for elderly DLBCL and that EBV latency pattern could be more complex from those previously observed. Therefore, EBV could be an important cofactor in pediatric DLBCL lymphomagenesis.

Association between magnitude of the virus-specific plasmablast response and disease severity in dengue patients.

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated, and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 d of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average >70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes, but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum-neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil, which is associated with increasing disease severity.

The impact of pregnancy on the HIV-1-specific T cell function in infected pregnant women.

Evidences indicate that pregnancy can alter the Ag-specific T-cell responses. This work aims to evaluate the impact of pregnancy on the in vitro HIV-1-specific immune response. As compared with non-pregnant patients, lower T-cell proliferation and higher IL-10 production were observed in T-cell cultures from pregnant patients following addition of either mitogens or HIV-1 antigens. In our system, the main T lymphocyte subset involved in producing IL-10 was CD4(+)FoxP3(-). Depletion of CD4(+) cells elevated TNF-α and IFN-γ production. Interestingly, the in vitro HIV-1 replication was lower in cell cultures from pregnant patients, and it was inversely related to IL-10 production. In these cultures, the neutralization of IL-10 by anti-IL-10 mAb elevated TNF-α release and HIV-1 replication. In conclusion, our results reveal that pregnancy-related events should favor the expansion of HIV-1-specific IL-10-secreting CD4(+) T-cells in HIV-1-infected women, which should, in the scenario of pregnancy, help to reduce the risk of vertical HIV-1 transmission.

Inflammatory profiles in severe pneumonia associated with the pandemic influenza A/H1N1 virus isolated in Mexico City.

The immune mechanisms underlying the pathogenesis of severe pneumonia associated with the A/H1N1 virus are not well known. The objective of this study was to determine whether severe A/H1N1-associated pneumonia can be explained by the emergence of particular T-cell subsets and the cytokines/chemokines they produced, as well as distinct responses to infection. T-cell subset distribution and cytokine/chemokine levels in peripheral blood and bronchoalveolar lavage (BAL) were determined in patients with severe A/H1N1 infection, asymptomatic household contacts, and healthy controls. Cytokine and chemokine production was also evaluated after in vitro infection with seasonal H1N1 and pandemic A/H1N1 strains. We found an increase in the frequency of peripheral Th2 and Tc2 cells in A/H1N1 patients. A trend toward increased Tc1 cells was observed in household contacts. Elevated serum levels of IL-6, CXCL8, and CCL2 were found in patients and a similar cytokine/chemokine profile was observed in BAL, in which CCL5 was also increased. Infection assays revealed that both strains induce the production of several cytokines/chemokines at 24 and 72 h, however, IL-6, CCL3, and CXCL8 were strongly up-regulated in 72-h cultures in presence of the A/H1N1 virus. Several inflammatory mediators are up-regulated in peripheral and lung samples from A/H1N1-infected patients who developed severe pneumonia. In addition, the A/H1N1 strain induces higher levels of pro-inflammatory cytokines and chemokines than the seasonal H1N1 strain. These findings suggest that it is possible to identify biomarkers of severe pneumonia and also suggest the therapeutic use of immunomodulatory drugs in patients with severe pneumonia associated with A/H1N1 infection.

Impairment of T cell immunity by the respiratory syncytial virus: targeting virulence mechanisms for therapy and prophylaxis.

Worldwide, Respiratory Syncytial Virus (RSV) causes severe bronchiolitis and pneumonia in children, the elderly and immuno-compromised individuals. Moreover, RSV is the mayor cause of infant hospitalization due to lower respiratory infection, regardless socioeconomic status. Accumulating data support the notion that immune responses elicited against naturally acquired RSV infections are non-lasting and inappropriate for efficient virus clearance. Although there is consensus over the capacity of RSV to impair the development of an effective and protective adaptive immune response, very little is known about specific viral determinants involved in these processes as well as the molecular mechanisms developed by this virus to inhibit T cell function. Recent studies have provided evidence supporting an important role for dendritic cells in RSV-induced suppression of immunity. Although recognized for over 50 years as an important respiratory pathogen and healthcare problem, to date there are no available vaccines against this virus, which highlights the complexity of RSV-induced immunopathology. The development of new prophylactic and therapeutic tools against RSV requires the unveiling of molecular mechanisms and virulence factors responsible for the pathogenesis caused by this virus. In this review, we discuss recent findings describing virulence mechanisms evolved by RSV to negatively modulate the adaptive immune response in the host. Furthermore, novel strategies aimed to induce efficient T cell immunity against RSV are reviewed.

Association between high serum prolactin levels and concomitant infections in HIV-infected patients.

Although prolactin (PRL) is now recognized as a cytokine and persistent immune activation is a common immunopathogenic feature of the human immunodeficiency virus infection (HIV), the circumstances associated with the onset of hyperprolactinemia during the course of this infection remain controversial. Given that PRL is able to exert not only endocrinologic effects but also immunologic influences, a study was conducted to investigate whether raised serum levels of PRL were more likely to prevail when HIV-infected patients developed concomitant infections. Serum PRL concentrations, as well as immunoglobulin isotypes, plasmatic viral burden, CD3+, CD4+, CD8+, CD19+, and natural killer (NK) cell counts were measured in 46 nonselected HIV-infected patients stratified on the basis of the presence or absence of clinically active concomitant infections. Serum PRL levels were significantly higher in patients presenting secondary infections as compared with the asymptomatic ones, with hyperprolactinemia being detected in 10/18 (55%) and 2/28 (7%) of these patient groups, respectively. Hyperprolactinemia was not related with viral burden, antiretroviral treatment, gender differences, or CD4+ cell counts. CD3+, CD4+, CD8+, and CD19+ cells were significantly lower in the group presenting active infections, whereas comparisons in NK cell counts, immunoglobulin levels and HIV viral burden revealed no differences between groups. These results provide evidence that hyperprolactinemia is more prevalent during the onset of secondary infections, which might have diagnostic and therapeutic consequences.