Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

The Spleen CD4(+) T Cell Response to Blood-Stage Plasmodium chabaudi Malaria Develops in Two Phases Characterized by Different Properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-Ab(-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-alpha and IFN-gamma production depend mostly on conventional CD4(+) T cells. IFN-gamma is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-gamma and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.

Monocyte-derived dendritic cells from patients with severe forms of chromoblastomycosis induce CD4(+) T cell activation in vitro

Dendritic cells (DCs) have been described as initiators and modulators of the immune response. Recently we have shown a predominant production of interleukin-10 cytokine, low levels of interferon-gamma and inefficient T cell proliferation in patients with severe forms of chromoblastomycosis. Chromoblastomycosis starts with subcutaneous inoculation of Fonsecaea pedrosoi into tissue where DCs are the first line of defence against this microorganism. In the present study, the interaction of F. pedrosoi and DCs obtained from patients with chromoblastomycosis was investigated. Our results showed that DCs from patients exhibited an increased expression of human leucocyte antigen D-related (HLA-DR) and co-stimulatory molecules. In the presence of conidia, the expression of HLA-DR and CD86 was up-regulated by DCs from patients and controls. Finally, we demonstrate the reversal of antigen-specific anergy and a T helper type 1 response mediated by DCs incubated with F. pedrosoi conidea.

Better CD4+T Cell Recovery in Brazilian HIV-Infected Individuals Under HAART Due to Cumulative Carriage of SDF-1-3`A, CCR2-V64I, CCR5-D32 and CCR5-Promoter 59029A/G Polymorphisms

Polymorphisms of chemokines and chemokine-receptors genes have been shown to influence the rate of progression to AIDS; however, their influence on response to HAART remains unclear. We investigated the frequency of the SDF-1-3`A, CCR2-64I, CCR5-D32 and CCR5-Promoter-59029-A/G polymorphisms in Brazilian HIV-1-infected and uninfected individuals and their influence on CD4+ T-cell evolution HIV-1 infected individuals before and during HAART. Polymorphism detection was done in a transversal study of 200 HIV-1-infected and 82 uninfected individuals. The rate of CD4+ T cell increase or decrease was studied in a cohort of 155 HIV-1 infected individuals on pre and post-HAART. Polymorphisms were determined by PCR associated with RFLP. The rate of CD4+ T-cell decline or increase was also determined. HIV-1 infected and uninfected subjects showed, respectively, frequencies of 0.193 and 0.220 for SDF-1-3`A, of 0.140 and 0.110 for CCR2-V64I, of 0.038 and 0.055 for CCR5-D32, and of 0.442 and 0.390 for CCR5-P-59029-A/G. HIV-1-infected subjects carrying one, two or three of these four polymorphisms showed better CD4+ T-cell recovery than HIV-1-infected subjects carrying the four wild-type alleles (+2.7, +1.6, +3.5, and -0.9 lymphocytes/mu l/month, respectively). Regression logistic analysis showed that the CCR5-D32/CCR2-V64I association was predictor of positive CD4+ T cell slope after HAART. The distribution of polymorphisms did not differ between HIV-1-infected and uninfected individuals, but differed from more homogenous ethnic groups probably reflecting the miscegenation of the Brazilian population. We add further evidence of the role of these polymorphisms by showing that the CD4 gain was influenced by carriage of one or more of the polymorphisms studied here. These results highlight the possibility that these genetic traits can be useful to identify patients at risk for faster progression to AIDS or therapeutic failure.

A novel human CD4(+) T-cell inducer subset with potent immunostimulatory properties

The complexity of immunoregulation has focused attention on the CD4(+) T ""suppressor"" regulatory cell (T(reg)), which helps maintain balance between immunity and tolerance. An immunoregulatory T-cell population that upon activation amplifies cellular immune responses was described in murine models more than 30 years ago; however, no study has yet identified a naturally occurring T ""inducer"" cell type. Here, we report that the ectoenzyme CD39/NTPDase1 (ecto-nucleoside triphosphate diphosphohydrolase 1) helps to delineate a novel population of human ""inducer"" CD4(+) T cells (T(ind)) that significantly increases the proliferation and cytokine production of responder T cells in a dose-dependent manner. Furthermore, this unique T(ind) subset produces a distinct repertoire of cytokines in comparison to the other CD4(+) T-cell subsets. We propose that this novel CD4(+) T-cell population counterbalances the suppressive activity of suppressor T(reg) in peripheral blood and serves as a calibrator of immunoregulation.

Dynamics of HIV latency and reactivation in a primary CD4+ T cell model.

HIV latency is a major obstacle to curing infection. Current strategies to eradicate HIV aim at increasing transcription of the latent provirus. In the present study we observed that latently infected CD4+ T cells from HIV-infected individuals failed to produce viral particles upon ex vivo exposure to SAHA (vorinostat), despite effective inhibition of histone deacetylases. To identify steps that were not susceptible to the action of SAHA or other latency reverting agents, we used a primary CD4+ T cell model, joint host and viral RNA sequencing, and a viral-encoded reporter. This model served to investigate the characteristics of latently infected cells, the dynamics of HIV latency, and the process of reactivation induced by various stimuli. During latency, we observed persistence of viral transcripts but only limited viral translation. Similarly, the reactivating agents SAHA and disulfiram successfully increased viral transcription, but failed to effectively enhance viral translation, mirroring the ex vivo data. This study highlights the importance of post-transcriptional blocks as one mechanism leading to HIV latency that needs to be relieved in order to purge the viral reservoir.

CD4+ T cell count recovery in HIV type 1-infected patients is independent of class of antiretroviral therapy.

BACKGROUND: In recent years, treatment options for human immunodeficiency virus type 1 (HIV-1) infection have changed from nonboosted protease inhibitors (PIs) to nonnucleoside reverse-transcriptase inhibitors (NNRTIs) and boosted PI-based antiretroviral drug regimens, but the impact on immunological recovery remains uncertain. METHODS: During January 1996 through December 2004 [corrected] all patients in the Swiss HIV Cohort were included if they received the first combination antiretroviral therapy (cART) and had known baseline CD4(+) T cell counts and HIV-1 RNA values (n = 3293). For follow-up, we used the Swiss HIV Cohort Study database update of May 2007 [corrected] The mean (+/-SD) duration of follow-up was 26.8 +/- 20.5 months. The follow-up time was limited to the duration of the first cART. CD4(+) T cell recovery was analyzed in 3 different treatment groups: nonboosted PI, NNRTI, or boosted PI. The end point was the absolute increase of CD4(+) T cell count in the 3 treatment groups after the initiation of cART. RESULTS: Two thousand five hundred ninety individuals (78.7%) initiated a nonboosted-PI regimen, 452 (13.7%) initiated an NNRTI regimen, and 251 (7.6%) initiated a boosted-PI regimen. Absolute CD4(+) T cell count increases at 48 months were as follows: in the nonboosted-PI group, from 210 to 520 cells/muL; in the NNRTI group, from 220 to 475 cells/muL; and in the boosted-PI group, from 168 to 511 cells/muL. In a multivariate analysis, the treatment group did not affect the response of CD4(+) T cells; however, increased age, pretreatment with nucleoside reverse-transcriptase inhibitors, serological tests positive for hepatitis C virus, Centers for Disease Control and Prevention stage C infection, lower baseline CD4(+) T cell count, and lower baseline HIV-1 RNA level were risk factors for smaller increases in CD4(+) T cell count. CONCLUSION: CD4(+) T cell recovery was similar in patients receiving nonboosted PI-, NNRTI-, and boosted PI-based cART.

Biomedical nanoparticles modulate specific CD4(+) T cell stimulation by inhibition of antigen processing in dendritic cells

Understanding how nanoparticles may affect immune responses is an essential prerequisite to developing novel clinical applications. To investigate nanoparticle-dependent outcomes on immune responses, dendritic cells (DCs) were treated with model biomedical poly(vinylalcohol)-coated super-paramagnetic iron oxide nanoparticles (PVA-SPIONs). PVA-SPIONs uptake by human monocyte-derived DCs (MDDCs) was analyzed by flow cytometry (FACS) and advanced imaging techniques. Viability, activation, function, and stimulatory capacity of MDDCs were assessed by FACS and an in vitro CD4(+) T cell assay. PVA-SPION uptake was dose-dependent, decreased by lipopolysaccharide (LPS)-induced MDDC maturation at higher particle concentrations, and was inhibited by cytochalasin D pre-treatment. PVA-SPIONs did not alter surface marker expression (CD80, CD83, CD86, myeloid/plasmacytoid DC markers) or antigen-uptake, but decreased the capacity of MDDCs to process antigen, stimulate CD4(+) T cells, and induce cytokines. The decreased antigen processing and CD4(+) T cell stimulation capability of MDDCs following PVA-SPION treatment suggests that MDDCs may revert to a more functionally immature state following particle exposure.

Toward Protein Biomarkers for Allergy: CD4+ T Cell Proteomics in Allergic and Nonallergic Subjects Sampled in and out of Pollen Season.

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (± antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4+ T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase ω 1, and 2,4-dienoyl-CoA reductase. The results were partly validated by Western blotting.

Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence.

Memory CD4 T cell responses are functionally and phenotypically heterogeneous. In the present study, memory CD4 T cell responses were analyzed in different models of Ag-specific immune responses differing on Ag exposure and/or persistence. Ag-specific CD4 T cell responses for tetanus toxoid, HSV, EBV, CMV, and HIV-1 were compared. Three distinct patterns of T cell response were observed. A dominant single IL-2 CD4 T cell response was associated with the model in which the Ag can be cleared. Polyfunctional (single IL-2 plus IL-2/IFN-gamma plus single IFN-gamma) CD4 T cell responses were associated with Ag persistence and low Ag levels. A dominant single IFN-gamma CD4 T cell response was associated with the model of Ag persistence and high Ag levels. The results obtained supported the hypothesis that the different patterns observed were substantially influenced by different conditions of Ag exposure and persistence.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Monitoring of human CD4 T cell responses in metastatic melanoma and arthritic patients

SUMMARY : Detailed knowledge of the different components of the immune system is required for the development of new immunotherapeutic strategies. CD4 T lymphocytes represent a highly heterogeneous group of cells characterized by various profiles of cytokine production and effector vs. regulatory functions. They are central players in orchestrating adaptive immune responses: unbalances between the different subtypes can lead either to aggressive autoimmune disorders or can favour the uncontrolled growth of malignancies. In this study we focused on the characterization of human CD4 T cells in advanced stage melanoma patients as well as in patients affected by various forms of autoimmune inflammatory spondyloarthropathies. In melanoma patients we report that a population of FOXP3 CD4 T cells, known as regulatory T cells, is overrepresented in peripheral blood, and even more in tumor-infitrated lymph nodes as well as at tumor sites, as compared to healthy donors. In tumor-infiltrated lymph nodes, but not in normal lymph nodes or in peripheral blood, FOXP3 CD4 T cells feature a highly differentiated phenotype (CD45RA-CCR7+/-), which suggests for a recent encounter with their cognate antigen. FOXP3 CD4 T cells have been described to be an important component of the several known immune escape mechanisms. We demonstrated that FOXP3 CD4 T cells isolated from melanoma patients exert an in vitro suppressive action on autologous CD4 T cells, thus possibly inhibiting an efficient anti-tumor response. Next, we aimed to analyse CD4 T cells at antigen-specific level. In advanced stage melanoma patients, we identified for the first time, using pMHCII multimers, circulating CD4 T cells specific for the melanoma antigen Melan-A, presented by HLA-DQB1 *0602. Interestingly, in a cohort of melanoma patients enrolled in an immunotherapy trails consisting of injection of a Melan-A derived peptide, we did not observe signif cant variations in the ex vivo frequencies of Melan-A specific CD4 T cells, but important differences in the quality of the specific CD4 T cells. In fact, up to 50% of the ex vivo Melan-A/DQ6 specific CD4 T cells displayed a regulatory phenotype and were hypoproliferative before vaccination, while more effector, cytokine-secreting Melan-A/DQ6 specific CD4 T cells were observed after immunization. These observations suggest that peptide vaccination may favourably modify the balance between regulatory and effector tumor-specific CD4 T cells. Finally, we identified another subset of CD4 T cells as possible mediator of pathology in a group of human autoimmune spondyloarthropathies, namely Th17 cells. These cells were recently described to play a critical role in the pathogenesis of some marine models of autommunity. We document an elevated presence of circulating Th17 cells in two members of seronegative spondyloarthropathies, e.g. psoriatic arthritis and ankylosing spondylitis, while we do not observe increased frequencies of Th17 cells in peripheral blood of rheumatoid arthritic patients. In addition, Th17 cells with a more advanced differentiation state (CD45RA-CCR7-CD27-) and polyfunctionality (concomitant secretion of IL-17, IL-2 and TNFα) were observed exclusively in patients with seronegative spondylarthropathies. Together, our observations emphasize the importance of CD4 T cells in various diseases and suggest that immunotherapeutic approaches considering CD4 T cells as targets should be evaluated in the future.

Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production.

In the present study, we have investigated the distribution of HIV-specific and HIV-infected CD4 T cells within different populations of memory CD4 T cells isolated from lymph nodes of viremic HIV-infected subjects. Four memory CD4 T cell populations were identified on the basis of the expression of CXCR5, PD-1, and Bcl-6: CXCR5(-)PD-1(-)Bcl-6(-), CXCR5(+)PD-1(-)Bcl-6(-), CXCR5(-)PD-1(+)Bcl-6(-), and CXCR5(+)PD-1(+)Bcl-6(+). On the basis of Bcl-6 expression and functional properties (IL-21 production and B cell help), the CXCR5(+)PD-1(+)Bcl-6(+) cell population was considered to correspond to the T follicular helper (Tfh) cell population. We show that Tfh and CXCR5(-)PD-1(+) cell populations are enriched in HIV-specific CD4 T cells, and these populations are significantly increased in viremic HIV-infected subjects as compared with healthy subjects. The Tfh cell population contained the highest percentage of CD4 T cells harboring HIV DNA and was the most efficient in supporting productive infection in vitro. Replication competent HIV was also readily isolated from Tfh cells in subjects with nonprogressive infection and low viremia (<1,000 HIV RNA copies). However, only the percentage of Tfh cells correlated with the levels of plasma viremia. These results demonstrate that Tfh cells serve as the major CD4 T cell compartment for HIV infection, replication, and production.

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1.

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.