CD4+ regulatory T cells (Tregs) are essential for the maintenance of the immune system's equilibrium, by dampening the activation of potential auto-reactive T cells and avoiding excessive immune activation. To correctly perform their function, Tregs must be maintained at the right proportion with respect to effector T cells. Since this equilibrium is frequently disrupted in individuals infected with the human immunodeficiency virus (HIV), we hypothesize that its deregulation could hamper immune reconstitution in patients with poor CD4+ T cell recovery under highly active antiretroviral therapy (HAART). We analysed Tregs percentages amongst CD4+ T cells in 53 HIV-infected patients under HAART, with suppression of viral replication and distinct levels of immune reconstitution. As controls, 51 healthy individuals were also analysed. We observed that amongst the patients with Nadir values (the lowest CD4+ T cell counts achieved) <200 cells/µL, the individuals with high Tregs percentages (≥10% of total CD4+ T cells) had the worse CD4+ T cell reconstitution. In accordance, the well-described direct correlation between the Nadir value and CD4+ T cell reconstitution is clearly more evident in individuals with high Tregs proportions. Furthermore, we observed a strong negative correlation between Tregs percentages and CD4+ T cell recovery among immunological non-responder HIV+ individuals. All together, this work shows that high Tregs frequency is an important factor associated with sub-optimal CD4+ T cell recovery. This is particularly relevant for immunological non-responders with low Nadir values. Our results suggest that the Tregs proportion might be of clinical relevance to define cut-offs for HAART initiation.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of myeloid cells that suppress T cell immunity in tumor-bearing hosts. In patients with colon cancer, MDSCs have recently been described as Lin−/lowHLA-DR−CD11b+CD33+ cells correlating with cancer stage, metastasis and chemotherapy response. To learn in more detail the dynamic change and clinical relevance of circulating and tumor-infiltrating Lin−/lowHLA-DR−CD11b+CD33+ MDSC in colorectal cancer, we harvested the blood from 64 patients with varying stage of colorectal cancer and tumor and matched paraneoplastic tissues from 5 patients with advanced colorectal cancer, subjected them to multicolor flow cytometric analysis of percentage, absolute number and phenotype of MDSC and finally characterized their immunosuppressive functions. Our results demonstrate that peripheral blood from colorectal cancer patients contains markedly increased percentage and absolute number of Lin−/lowHLA-DR−CD11b+CD33+ MDSCs compared with healthy individuals, and this increase is closely correlated with clinical cancer stage and tumor metastasis but not primary tumor size and serum concentrations of cancer biomarker. A similar increase of MDSCs was also observed in the tumor tissues. Phenotyping MDSCs shows that they express high CD13 and CD39, low CD115, CD117, CD124 and PD-L1, and devoid of CD14, CD15 and CD66b, reminiscent of precursor myeloid cells. MDSCs from cancer patients but not healthy donors have the immunosuppressive activity and were able to inhibit in vitro autologous T-cell proliferation. Collectively, this study substantiates the presence of increased immunosuppressive circulating and tumor-resident Lin−/lowHLA-DR−CD11b+CD33+ MDSCs in patients with colorectal cancers correlating with cancer stage and metastasis, and suggests that pharmacologic blockade of MDSCs should be considered in future clinical trials.
Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression.
In a recent phase II clinical trial for HNSCC patients, IRX-2, a cell-derived biologic, promoted T-cell infiltration into the tumor and prolonged overall survival. Mechanisms responsible for these IRX-2-mediated effects are unknown. We hypothesized that IRX-2 enhanced tumor antigen-(TA)-specific immunity by up-regulating functions of dendritic cells (DC).
Monocyte-derived DC obtained from 18 HNSCC patients and 12 healthy donors were matured using IRX-2 or a mix of TNF-α, IL-1β and IL-6 (“conv. mix”). Multicolor flow cytometry was used to study the DC phenotype and antigen processing machinery (APM) component expression. ELISPOT and cytotoxicity assays were used to evaluate tumor-reactive cytotoxic T lymphocytes (CTL). IL-12p70 and IL-10 production by DC was measured by Luminex® and DC migration toward CCL21 was tested in transwell migration assays. IRX-2-matured DC functions were compared with those of conv. mix-matured DC. IRX-2-matured DC expressed higher levels (p<0.05) of CD11c, CD40, CCR7 as well as LMP2, TAP1, TAP2 and tapasin than conv. mix-matured DC. IRX-2-matured DC migrated significantly better towards CCL21, produced more IL-12p70 and had a higher IL12p70/IL-10 ratio than conv. mix-matured DC (p<0.05 for all). IRX-2-matured DC carried a higher density of tumor antigen-derived peptides, and CTL primed with these DC mediated higher cytotoxicity against tumor targets (p<0.05) compared to the conv. mix-matured DC.
Excellent ability of IRX-2 to induce ex vivo DC maturation in HNSCC patients explains, in part, its clinical benefits and emphasizes its utility in ex vivo maturation of DC generated for therapy.
Activation of complement is one of the earliest immune responses to exogenous threats, resulting in various cleavage products including anaphylatoxin C3a. In addition to its contribution to host defense, C3a has been shown to mediate Th2 responses in animal models of asthma. However, the role of C3a on pulmonary Th17 responses during allergic inflammation remains unclear. Here, we show that mice deficient in C3a receptor (C3aR) exhibited (i) higher percentages of endogenous IL-17-producing CD4+ T cells in the lungs, (ii) higher amounts of IL-17 in the bronchoalveolar lavage fluid, and (iii) more neutrophils in the lungs than wild-type mice when challenged with intranasal allergens. Moreover, adoptive transfer experiments showed that the frequencies of antigen-specific IL-17-producing CD4+ T cells were significantly higher in the lungs and bronchial lymph nodes of C3aR-deficient recipients than those of wild-types recipients. Bone-marrow reconstitution study indicated that C3aR-deficiency on hematopoietic cells was required for the increased Th17 responses. Furthermore, C3aR-deficient mice exhibited increased percentages of Foxp3+ regulatory T cells; however, depletion of these cells minimally affected the induction of antigen-specific Th17 cell population in the lungs. Neutralization of IL-17 significantly reduced the number of neutrophils in bronchoalveolar lavage fluid of C3aR-deficient mice. Our findings demonstrate that C3a signals negatively regulate antigen-specific Th17 responses during allergic lung inflammation and the size of Foxp3+ regulatory T cell population in the periphery.
Recently, it was shown that peripheral blood FOXP3+CD4+ T cells are composed of three phenotypic and functionally distinct subpopulations. Two of them having in vitro suppressive effects were characterized as resting Treg cells (rTregs) and activated Treg cells (aTregs). A third subset, identified as FOXP3+ non-Tregs, does not display any suppressor activity and produce high levels of Th1 and Th17 cytokines upon stimulation. In the present study we focus on the characteristics of these three subsets of FOXP3+CD4+ T cells in untreated HIV-1-infected patients. We found that the absolute counts of rTregs, aTregs and FOXP3+ non-Tregs were reduced in HIV-1 patients compared with healthy donors. The relative frequency of rTregs and aTregs was similar in HIV-1 patients and healthy donors, while the frequency of FOXP3+ non-Tregs was significantly higher in HIV-1 patients, reaching a maximum in those patients with the lower values of CD4 counts. Contrasting with the observations made in FOXP3- CD4+ T cells, we did not find a negative correlation between the number of rTregs, aTregs or FOXP3+ non-Tregs and virus load. Studies performed with either whole PBMCs or sorted aTregs and FOXP3+ non-Tregs cells showed that these two populations of FOXP3+ T cells were highly permissive to HIV-1 infection. Upon infection, FOXP3+ non-Tregs markedly down-regulates its capacity to produce Th1 and Th17 cytokines, however, they retain the ability to produce substantial amounts of Th2 cytokines. This suggests that FOXP3+ non-Tregs might contribute to the polarization of CD4+ T cells into a Th2 profile, predictive of a poor outcome of HIV-1-infected patients.
Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that is characterized by decreased platelet count. Regulatory T (Treg) cells and T helper type 17 (Th17) cells are two subtypes of CD4+ T helper (Th) cells. They play opposite roles in immune tolerance and autoimmune diseases, while they share a common differentiation pathway. The imbalance of Treg/Th17 has been demonstrated in several autoimmune diseases. In this study, we aimed to investigate the ratio of the number of Treg cells to the number of Th17 cells in ITP patients and evaluate the clinical implications of the alterations in this ratio.
Thirty adult patients with newly diagnosed ITP enrolled in this study. Twelve patients had been clinically followed up for 12 months. The percentages of CD4+CD25hiFoxp3+ Treg cells and CD3+CD4+IL-17-producing Th17 cells in these patients and healthy controls (n = 17) were longitudinally analyzed by flow cytometry.
The percentage of Treg cells in ITP patients was significantly lower than that of healthy controls, and the percentage of Th17 cells increased significantly at disease onset. The ratio of Treg/Th17 correlated with the disease activity.
The ratio of Treg/Th17 might be relevant to the clinical diversity of ITP patients, and this Treg/Th17 ratio might have prognostic role in ITP patients.
Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4+CD25+Foxp3+ Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-β. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4+CD25+Foxp3+ Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4+ and CD8+ T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25+ cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.
Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 105 TCID50 of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.
Considerable evidence supports the prediction that CD25 is directly regulated by the forkhead transcription factor FOXP3. However, given that CD25 is normally upregulated in activated T cells, regardless of whether they express FOXP3, this issue has still to be definitively demonstrated. Here we describe that FOXP3, induced by CD28 signals in human CD4+CD25− T lymphocytes, synergizes with RelA on a regulatory region of Cd25 promoter to mediate the transcriptional activation of Cd25 gene. We found that a striking feature of this regulatory region is the presence of a κB site and of two tandem copies of a non-consensus FOXP3 binding site separated at 5′ ends by 19 nucleotides that allow FOXP3 and RelA binding to DNA and their physical interaction. The occupancy of the two FOXP3 binding sites in conjunction with RelA binding site occupancy allows FOXP3 to function as a positive activator of Cd25 gene. Indeed mutations of both FOXP3 binding sites such as mutation of κB site on Cd25 promoter abolished FOXP3 activatory functions. Moreover, FOXP3 mutation ΔE251, that compromises FOXP3 homotypic interactions, failed to trans activate Cd25 promoter, suggesting that both FOXP3 DNA binding and dimerization are required to trans activate Cd25 promoter. These findings identify a novel mechanism by which RelA and FOXP3 cooperate to mediate transcriptional regulation of target genes and characterize a region on Cd25 promoter where FOXP3 dimer could bridge intramolecularly two DNA sites and trans activate Cd25 gene.
Cancer vaccines are designed to expand tumor antigen-specific T cells with effector function. However, they may also inadvertently expand regulatory T cells (Treg), which could seriously hamper clinical efficacy. To address this possibility, we developed a novel assay to detect antigen-specific Treg based on down-regulation of surface CD3 following TCR engagement, and used this approach to screen for Treg specific to the NY-ESO-1 tumor antigen in melanoma patients treated with the NY-ESO-1/ISCOMATRIXTM cancer vaccine. All patients tested had Treg (CD25bright FoxP3+ CD127neg) specific for at least one NY-ESO-1 epitope in the blood. Strikingly, comparison with pre-treatment samples revealed that many of these responses were induced or boosted by vaccination. The most frequently detected response was toward the HLA-DP4-restricted NY-ESO-1157–170 epitope, which is also recognized by effector T cells. Notably, functional Treg specific for an HLA-DR-restricted epitope within the NY-ESO-1115–132 peptide were also identified at high frequency in tumor tissue, suggesting that NY-ESO-1-specific Treg may suppress local anti-tumor immune responses. Together, our data provide compelling evidence for the ability of a cancer vaccine to expand tumor antigen-specific Treg in the setting of advanced cancer, a finding which should be given serious consideration in the design of future cancer vaccine clinical trials.
The antiviral factor CPSF6-358 interferes with the nuclear entry of human immunodeficiency virus type 1 (HIV-1). HIV-1 acquires resistance to CPSF6-358 through the N74D mutation of the capsid (CA), which alters its nuclear entry pathway. Here we show that compared to wild-type (WT) HIV-1, N74D HIV-1 is more sensitive to cyclosporine, has increased sensitivity to nevirapine, and is impaired in macrophage infection prior to reverse transcription. These phenotypes suggest a difference in the N74D reverse transcription complex that manifests early after infection and prior to interaction with the nuclear pore. Overall, our data indicate that N74D HIV-1 replication in transformed cells requires cyclophilin A but is dependent on other interactions in macrophages.
Complex interactions between effector T cells and Foxp3+ regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4+Foxp3− T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression.
CD4+CD25+Foxp3+ Regulatory T cells (Treg) and programmed death-1 (PD-1) molecules have emerged as pivotal players in immune suppression of chronic diseases. However, their impact on the disease severity, therapeutic response and restoration of immune response in human tuberculosis remains unclear. Here, we describe the possible role of Treg cells, their M. tuberculosis driven expansion and contribution of PD-1 pathway to the suppressive function of Treg cells among pulmonary tuberculosis (PTB) patients. Multicolor flow cytometry, cell culture, cells sorting and ELISA were employed to execute the study. Our results showed significant increase in frequency of antigen-reactive Treg cells, which gradually declined during successful therapy and paralleled with decline of M. tuberculosis–specific IL-10 along with elevation of IFN-γ production, and raising the IFN-γ/IL-4 ratio. Interestingly, persistence of Treg cells tightly correlated with MDR tuberculosis. Also, we show that blocking PD-1/PD-L1 pathway abrogates Treg-mediated suppression, suggesting that the PD-1/PD-L1 pathway is required for Treg-mediated suppression of the antigen-specific T cells. Treg cells possibly play a role in dampening the effector immune response and abrogating PD-1 pathway on Treg cells significantly rescued protective T cell response, suggesting its importance in immune restoration among tuberculosis patients.
The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear.
Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur.
These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein.
MSC display potent suppressive properties initially described a decade ago. More recently, MSC suppressive activities on T-cell effector pathways have been investigated. MSC modulate CD4 differentiation through different mechanisms depending on culture conditions and display disparate activities on T cells according to their differentiation status. A significant amount of evidence for MSC effects on Th17 cells revealed that MSC could be suppressive under diverse circumstances but also enhance Th17 cell activity under other conditions. In the present study, we investigated the suppressive effects of MSC on Th1 and Th17 subsets of T cells using T cells undergoing Th1 and Th17 polarization or mature Th1 and Th17 cells. MSC inhibited the proliferation of T cells during their differentiation toward Th1 cells and mature Th1 cells. This suppressive effect was maintained in a transwell cell culture insert demonstrating the major role played by soluble factors. Using the transwell cell separation barrier, we observed that MSC decrease the number of T cells undergoing Th17 differentiation whereas they did not affect IL-17 production by mature Th17, demonstrating the need for cell contact for suppressing Th17 cell function. Moreover, we reported that PD-L1 is highly expressed on MSC co-cultured with differentiating or polarized Th1 and Th17 cells. Using neutralizing antibodies specific for PD-L1 and PD-1 we showed that the mechanisms by which MSC mediate Th17 cell repolarization depend on PD-L1 expression on MSC. Taken together our results demonstrated a cell-to-cell contact depend mechanism in the selective immunosuppression of MSC on mature Th17 cells through up-regulation of PD-L1.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4+ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.
Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam. Milk was analyzed for activity in vitro against both cell-free and cell-associated HIV-1. Potent inhibition of cell-free R5 and X4 HIV-1 occurred in the presence of milk from all donors regardless of HIV-1 serostatus. Inhibition of cell-free HIV-1 infection positively correlated with milk levels of sialyl-LewisX from HIV-positive donors. In contrast, milk from 8 of 16 subjects enhanced infection with cell-associated HIV-1 regardless of donor serostatus. Milk from two of these subjects contained high levels of multiple pro-inflammatory cytokines including TNFα, IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, MCP-1 and IP-10, and enhanced cell-associated HIV-1 infection at dilutions as high as 1∶500. These findings indicate that breast milk contains innate factors with divergent activity against cell-free and cell-associated HIV-1 in vitro. Enhancement of cell-associated HIV-1 infection by breast milk may be associated with inflammatory conditions in the mother and may contribute to infant infection during breastfeeding.
Mitochondrial function influences T cell dynamics and is affected by
mitochondrial DNA (mtDNA) variation. We previously reported an association
between African mtDNA haplogroup L2 and less robust CD4 cell recovery on
antiretroviral therapy (ART) in non-Hispanic black ACTG 384 subjects. We
explored whether additional T cell parameters in this cohort differed by
ACTG 384 randomized ART-naïve subjects to two different nucleoside
regimens with efavirenz, nelfinavir, or both. CD4 and CD8 memory and
activation markers were available at baseline and week 48 on most subjects.
mtDNA sequencing was performed on whole blood DNA, and haplogroups were
determined. We studied non-Hispanic black subjects with HIV RNA <400
copies/mL at week 48. Analyses included Wilcoxon ranksum test and linear
Data from 104 subjects were included. Major African mtDNA haplogroups
included L1 (N = 25), L2 (N = 31),
and L3 (N = 32). Baseline age, HIV RNA, and CD4 cells
did not differ between L2 and non-L2 haplogroups. Compared to non-L2
haplogroups, L2 subjects had lower baseline activated CD4 cells (median
12% vs. 17%; p = 0.03) and tended toward
lower activated CD8 cells (41% vs. 47%;
p = 0.06). At 48 weeks of ART, L2 subjects had smaller
decreases in activated CD4 cells (−4% vs. −11%;
p = 0.01), and smaller CD4 cell increases (+95 vs.
+178; p = 0.002). In models adjusting for baseline
age, CD4 cells, HIV RNA, and naïve-to-memory CD4 cell ratio, haplogroup
L2 was associated with lower baseline (p = 0.04) and
48-week change in (p = 0.01) activated CD4 cells.
Among ART-naïve non-Hispanic blacks, mtDNA haplogroup L2 was associated
with baseline and 48-week change in T cell activation, and poorer CD4 cell
recovery. These data suggest mtDNA variation may influence CD4 T cell
dynamics by modulating T cell activation. Further study is needed to
replicate these associations and identify mechanisms.
Myeloid dendritic cells (mDCs) are the antigen-presenting cells best capable of promoting peripheral induction of regulatory T cells (Tregs), and are among the first targets of HIV. It is thus important to understand whether HIV alters their capacity to promote Treg conversion. Monocyte-derived DCs (moDCs) from uninfected donors induced a Treg phenotype (CD25+FOXP3+) in autologous conventional T cells. These converted FOXP3+ cells suppressed the proliferation of responder T cells similarly to circulating Tregs. In contrast, the capacity of moDCs to induce CD25 or FOXP3 was severely impaired by their in vitro infection with CCR5-utilizing virus. MoDC exposure to inactivated HIV was sufficient to impair FOXP3 induction. This DC defect was not dependent on IL-10, TGF-β or other soluble factors, but was due to preferential killing of Tregs by HIV-exposed/infected moDCs, through a caspase-dependent pathway. Importantly, similar results were obtained with circulating primary myeloid DCs. Upon infection in vitro, these mDCs also killed Treg through mechanisms at least partially caspase-dependent, leading to a significantly lower proportion of induced Tregs. Taken together, our data suggest that Treg induction may be defective when DCs are exposed to high levels of virus, such as during the acute phase of infection or in AIDS patients.
Elevated levels of the immunoregulatory cytokine TGF-β1 in cancer and HIV infection have been linked to the suppression of protective immune responses. The transcriptional regulation of TGF-β1 is complex and still not completely understood. We report here for the first time that the transcription factor GLI2 regulates the expression of TGF-β1 in human CD4+ T cells. In silico screening revealed five novel putative GLI binding sites in the human TGF-β1 promoter. At least two of these sites within the human TGF-β1 promoter are regulated by the GLI2 activator as knockdown of GLI2 in regulatory CD4+CD25hi T cells, high producers of TGF-β1, significantly decreased TGF-β1 transcription. Additionally, naïve CD4+ T cells, low producers of TGF-β1, increased their basal level of TGF-β1 mRNA following lentiviral infection with GLI2. The transcriptional regulation of TGF-β1 by GLI2 is a new extension to Sonic Hedgehog (SHH) and TGF-β1 cross-regulation and may provide insight into the detrimental elevation of TGF-β1 leading to pathogenesis in cancer and HIV infection.
The role of the high affinity IgE receptor, FcεRI, in IgE-mediated immune responses of the gastrointestinal (GI) mucosa is poorly understood. Currently, a detailed characterization of FcεRI expression throughout the human gut is lacking. The aim of this study was to define the expression pattern of FcεRI in the GI tract.
We compared FcεRI expression in children with gastritis/esophagitis (n = 10), celiac disease (n = 10), inflammatory bowel disease (IBD) (n = 9), and normal mucosa (n = 5). The α–subunit of FcεRI (FcεRIα), detected by immunohistochemistry, was found on cells infiltrating the mucosa of the esophagus, the stomach, and the duodenum, but was rarely detected in more distal sections of the GI tract. Accordingly, quantitative RT-PCR analysis on esophagus, stomach, duodenum, colon, and rectum biopsies revealed that FcεRIα and -β expression levels decreased towards the distal intestine. mRNA transcripts of the common Fc-receptor-γ chain were present in the entire GI mucosa. Double-immunofluorescence staining of esophageal specimens confirmed that FcεRIα was expressed on intraepithelial mast cells and Langerhans cells. The mRNA expression levels of the α, β, and γ subunits of FcεRI did not correlate with total serum IgE but were associated with mucosal inflammation.
Our data define the upper GI tract as the main site for IgE-mediated immune activation via FcεRI. Tissue mRNA levels of FcεRIα are regulated by inflammatory conditions rather than serum IgE, indicating that FcεRI might also play a role in pathologies other than allergy.
The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA+ and their memory/effector CD45RO+ T cell counterparts in the presence and absence of low dose hydrogen peroxide (H2O2) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA+ and CD45RO+ T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress.
The emergence of X4 tropic viral strains throughout the course of HIV infection is associated with poorer prognostic outcomes and faster progressions to AIDS than for patients in whom R5 viral strains predominate. Here we investigate a stochastic model to account for the emergence of X4 virus via mutational intermediates of lower fitness that exhibit dual/mixed (D/M) tropism, and employ the model to investigate whether the administration of CCR5 blockers in-vivo is likely to promote a shift towards X4 tropism. We show that the proposed stochastic model can account for X4 emergence with a median time of approximately 4 years post-infection as a result of: 1.) random stochastic mutations in the V3 region of env during the reverse transcription step of infection; 2.) increasing numbers of CXCR4-expressing activated naive CD4+ T cells with declining total CD4+ T cell counts, thereby providing increased numbers of activated target cells for productive infection by X4 virus. Our model indicates that administration of the CCR5 blocker maraviroc does not promote a shift towards X4 tropism, assuming sufficient efficacy of background therapy (BT). However our modelling also indicates that administration of maraviroc as a monotherapy or with BT of suboptimal efficacy can promote emergence of X4 tropic virus, resulting in accelerated progression to AIDS. Taken together, our results demonstrate that maraviroc is safe and effective if co-administered with sufficiently potent BT, but that suboptimal BT may promote X4 emergence and accelerated progression to AIDS. These results underscore the clinical importance for careful selection of BT when CCR5 blockers are administered in-vivo.
Regulatory T cells (Tregs) are essential for controlling peripheral tolerance by the active suppression of various immune cells including conventional T effector cells (Teffs). Downstream of the T cell receptor (TCR), more than 500 protein kinases encoded by the human genome have to be considered in signaling cascades regulating the activation of Tregs and Teffs, respectively. Following TCR engagement, Tregs posses a number of unique attributes, such as constitutive expression of Foxp3, hyporesponsiveness and poor cytokine production. Furthermore, recent studies showed that altered regulation of protein kinases is important for Treg function. These data indicate that signaling pathways in Tregs are distinctly organized and alterations at the level of protein kinases contribute to the unique Treg phenotype. However, kinase-based signaling networks in Tregs are poorly understood and necessitate further systematic characterization. In this study, we analyzed the differential expression of kinases in Tregs and Teffs by using a kinase-selective proteome strategy. In total, we revealed quantitative information on 185 kinases expressed in the human CD4+ T cell subsets. The majority of kinases was equally abundant in both T cell subsets, but 11 kinases were differentially expressed in Tregs. Most strikingly, Tregs showed an altered expression of cell cycle kinases including CDK6. Quantitative proteomics generates first comparative insight into the kinase complements of the CD4+ Teff and Treg subset. Treg-specific expression pattern of 11 protein kinases substantiate the current opinion that TCR-mediated signaling cascades are altered in Tregs and further suggests that Tregs exhibit significant specificities in cell-cycle control and progression.