Osteopontin promotes age-related adipose tissue remodeling through senescence-associated macrophage dysfunction.

Adipose tissue macrophages (ATMs) play an important role in obesity and inflammation, and they accumulate in adipose tissue (AT) with aging. Furthermore, increased ATM senescence has been shown in obesity-related AT remodeling and dysfunction. However, ATM senescence and its role are unclear in age-related AT dysfunction. Here, we show that ATMs (a) acquire a senescence-like phenotype during chronological aging; (b) display a global decline of basic macrophage functions such as efferocytosis, an essential process to preserve AT homeostasis by clearing dysfunctional or apoptotic cells; and (c) promote AT remodeling and dysfunction. Importantly, we uncover a major role for the age-associated accumulation of osteopontin (OPN) in these processes in visceral AT. Consistently, loss or pharmacologic inhibition of OPN and bone marrow transplantation of OPN-/- mice attenuate the ATM senescence-like phenotype, preserve efferocytosis, and finally restore healthy AT homeostasis in the context of aging. Collectively, our findings implicate pharmacologic OPN inhibition as a viable treatment modality to counter ATM senescence-mediated AT remodeling and dysfunction during aging.

Neutrophil Activation and Immune Thrombosis Profiles Persist in Convalescent COVID-19.

PURPOSE: Following a severe COVID-19 infection, a proportion of individuals develop prolonged symptoms. We investigated the immunological dysfunction that underlies the persistence of symptoms months after the resolution of acute COVID-19. METHODS: We analyzed cytokines, cell phenotypes, SARS-CoV-2 spike-specific and neutralizing antibodies, and whole blood gene expression profiles in convalescent severe COVID-19 patients 1, 3, and 6 months following hospital discharge. RESULTS: We observed persistent abnormalities until month 6 marked by (i) high serum levels of monocyte/macrophage and endothelial activation markers, chemotaxis, and hematopoietic cytokines; (ii) a high frequency of central memory CD4+ and effector CD8+ T cells; (iii) a decrease in anti-SARS-CoV-2 spike and neutralizing antibodies; and (iv) an upregulation of genes related to platelet, neutrophil activation, erythrocytes, myeloid cell differentiation, and RUNX1 signaling. We identified a "core gene signature" associated with a history of thrombotic events, with upregulation of a set of genes involved in neutrophil activation, platelet, hematopoiesis, and blood coagulation. CONCLUSION: The lack of restoration of gene expression to a normal profile after up to 6 months of follow-up, even in asymptomatic patients who experienced severe COVID-19, signals the need to carefully extend their clinical follow-up and propose preventive measures.

Profound Defect of Amphiregulin Secretion by Regulatory T Cells in the Gut of HIV-Treated Patients.

The persistence of a leaky gut in HIV-treated patients leads to chronic inflammation with increased rates of cardiovascular, liver, kidney, and neurological diseases. Tissue regulatory T (tTreg) cells are involved in the maintenance of intestinal homeostasis and wound repair through the IL-33 pathway. In this study, we investigated whether the persistence of gut mucosal injury during HIV infection might be explained in part by a flaw in the mechanisms involved in tissue repair. We observed an increased level of IL-33 in the gut of HIV-infected patients, which is associated with an increased level of fibrosis and a low peripheral reconstitution of CD4+ T cells. Our results showed that intestinal Treg cells from HIV-infected patients were enriched in tTreg cells prone to support tissue repair. However, we observed a functional defect in tTreg cells caused by the lack of amphiregulin secretion, which could contribute to the maintenance of intestinal damage. Our data suggest a mechanism by which the lack of amphiregulin secretion by tTreg may contribute to the lack of repair of the epithelial barrier.

Functional Ex Vivo Testing of Alveolar Monocytes in Patients with Pneumonia-Related ARDS.

Biomarkers of disease severity might help with individualizing the management of patients with acute respiratory distress syndrome (ARDS). During sepsis, a sustained decreased expression of the antigen-presenting molecule human leucocyte antigen-DR (HLA-DR) on circulating monocytes is used as a surrogate marker of immune failure. This study aimed at assessing whether HLA-DR expression on alveolar monocytes in the setting of a severe lung infection is associated with their functional alterations. BAL fluid and blood from immunocompetent patients with pneumonia-related ARDS admitted between 2016 and 2018 were isolated in a prospective monocentric study. Alveolar and blood monocytes were immunophenotyped using flow cytometry. Functional tests were performed on alveolar and blood monocytes after in vitro lipopolysaccharide (LPS) stimulation. Phagocytosis activity and intracellular tumor necrosis factor (TNF) production were quantified using fluorochrome-conjugated-specific antibodies. Ten ARDS and seven non-ARDS control patients were included. Patients with pneumonia-related ARDS exhibited significantly lower HLA-DR expression both on circulating (p < 0.0001) and alveolar (p = 0.0002) monocytes. There was no statistically significant difference observed between patient groups (ARDS vs. non-ARDS) regarding both alveolar and blood monocytes phagocytosis activity. After LPS stimulation, alveolar (p = 0.027) and blood (p = 0.005) monocytes from pneumonia-related ARDS patients had a significantly lower intracellular TNF expression than non-ARDS patients. Monocytes from pneumonia-related ARDS patients have a deactivated status and an impaired TNF production capacity but display potent phagocytic activity. HLA-DR level expression should not be used as a surrogate marker of the phagocytic activity or the TNF production capacity of alveolar monocytes.

Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques.

Achieving sufficient worldwide vaccination coverage against SARS-CoV-2 will require additional approaches to currently approved viral vector and mRNA vaccines. Subunit vaccines may have distinct advantages when immunizing vulnerable individuals, children and pregnant women. Here, we present a new generation of subunit vaccines targeting viral antigens to CD40-expressing antigen-presenting cells. We demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with long-term memory phenotypes, in a humanized mouse model. Additionally, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Vaccine-elicited antibodies cross-neutralize different SARS-CoV-2 variants, including D614G, B1.1.7 and to a lesser extent B1.351. Such vaccination significantly improves protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals.

Cigarette smoking induces human CCR6+Th17 lymphocytes senescence and VEGF-A secretion.

Chronic exposure to environmental pollutants is often associated with systemic inflammation. As such, cigarette smoking contributes to inflammation and lung diseases by inducing senescence of pulmonary cells such as pneumocytes, fibroblasts, and endothelial cells. Yet, how smoking worsens evolution of chronic inflammatory disorders associated with Th17 lymphocytes, such as rheumatoid arthritis, psoriasis, Crohn's disease, and multiple sclerosis, is largely unknown. Results from human studies show an increase in inflammatory CD4+ Th17 lymphocytes at blood- and pulmonary level in smokers. The aim of the study was to evaluate the sensitivity of CD4+ Th17 lymphocytes to cigarette smoke-induced senescence. Mucosa-homing CCR6+ Th17- were compared to CCR6neg -and regulatory T peripheral lymphocytes after exposure to cigarette smoke extract (CSE). Senescence sensitivity of CSE-exposed cells was assessed by determination of various senescence biomarkers (ß-galactosidase activity, p16Ink4a- and p21 expression) and cytokines production. CCR6+ Th17 cells showed a higher sensitivity to CSE-induced senescence compared to controls, which is associated to oxidative stress and higher VEGFα secretion. Pharmacological targeting of ROS- and ERK1/2 signalling pathways prevented CSE-induced senescence of CCR6+Th17 lymphocytes as well as VEGFα secretion. Altogether, these results identify mechanisms by which pro-oxidant environmental pollutants contribute to pro-angiogenic and pathogenic CCR6+Th17 cells, therefore potential targets for therapeutic purposes.

Overexpression of GILZ in macrophages limits systemic inflammation while increasing bacterial clearance in sepsis in mice.

Studies support the beneficial effects of glucocorticoids (GCs) during septic shock, steering research toward the potential role of GC-induced proteins in controlling excessive inflammatory responses. GILZ is a glucocorticoid-induced protein involved in the anti-inflammatory effects of GCs. We investigated whether the overexpression of GILZ specifically limited to monocytes and macrophages (M/M) alone could control inflammation, thus improving the outcome of septic shock in animal models. We also monitored the expression of GILZ in M/M from septic mice and septic-shock patients. M/M from patients and septic mice displayed significantly lower expression of GILZ than those isolated from controls. Furthermore, transgenic mice (Tg-mice) experiencing sepsis, with increased expression of GILZ restricted to M/M, showed lower frequencies of inflammatory monocytes than their littermates and lower plasma levels of inflammatory cytokines. Tg-mice also had lower blood bacterial counts. We further established that the upregulation of GILZ in M/M enhanced their phagocytic capacity in in vivo assays. The increase of GILZ in M/M was also sufficient to improve the survival rates of septic mice. These results provide evidence for a central role of both GILZ and M/M in the pathophysiology of septic shock and a possible clue for the modulation of inflammation in this disease.

Human CCR6+ Th17 Lymphocytes Are Highly Sensitive to Radiation-Induced Senescence and Are a Potential Target for Prevention of Radiation-Induced Toxicity.

PURPOSE: This study addresses the sensitivity of different peripheral CD4+ T-lymphocyte subsets to irradiation (IR) and identifies potential targets for the prevention or treatment of radiation-induced toxicity. METHODS: This study was performed on peripheral blood mononuclear cells or sorted peripheral memory lymphocytes of CCR6+ mucosa-homing Th17/CCR6negTh and regulatory T subtypes of healthy volunteers. Cells were irradiated with a 2 Gy with or without pharmacologic inhibitors of different signaling pathways. Senescence of irradiated cells was assessed by resistance to apoptosis and determination of various senescence-associated biomarkers (senescence associated b-galactosidase activity, p16Ink4a-, p21Cdkn1a-, gH2A.X-, H2A.J expression). Cytokine production was measured in supernatants of irradiated cells by Luminex technology. RESULTS: Not all CD4+ memory T lymphocyte subsets were equally radiosensitive. High sensitivity of CCR6+Th17 lymphocytes to IR-induced senescence was shown by expression of the histone variant H2A.J, higher SA-b-Gal activity, and upregulation of p16Ink4a and p21Cdkn1a expression. Lower Annexin V staining and cleaved caspase-3, and higher expression of antiapoptotic genes Bcl-2 and Bcl-xL LF, showed that CCR6+Th17 lymphocytes were more resistant to IR-induced apoptosis than CCR6neg memory Th and regulatory T lymphocytes. After a 2 Gy IR, both CCR6+Th17 and CCR6neg cells acquired a moderate senescence-associated secretory phenotype, but only CCR6+Th17 cells secreted interleukin 8 (IL-8) and vascular endothelial growth factor-A (VEGF-A). Pharmacologic targeting of reactive oxygen species (ROS), mitogen-activated protein kinases (MAPKs), and mammalian target of rapamycin (mTOR) signaling pathways prevented the expression of senescent markers and IL-8 and VEGF-A expression by CCR6+Th17 cells after IR. CONCLUSIONS: This study suggests that IR induces senescence of CCR6+Th17 lymphocytes associated with secretion of IL-8 and VEGF-A that may be detrimental to the irradiated tissue. ROS-MAPKs signaling pathways are candidate targets to prevent this CCR6+Th17-dependent radiation-induced potential toxicity. Finally, the ratio of circulating H2A.J+ senescent CCR6+ Th17/CD4+ T lymphocytes may be a candidate marker of individual intrinsic radiosensitivity.

Mechanisms of Thrombocytopenia During Septic Shock: A Multiplex Cluster Analysis of Endogenous Sepsis Mediators.

BACKGROUND: Thrombocytopenia is a common feature of sepsis and may involve various mechanisms often related to the inflammatory response. This study aimed at evaluating factors associated with thrombocytopenia during human septic shock. In particular, we used a multiplex analysis to assess the role of endogenous sepsis mediators. METHODS: Prospective, observational study. Thrombocytopenia was defined as an absolute platelet count <100 G/L or a 50% relative decrease in platelet count during the first week of septic shock. Plasma concentrations of 27 endogenous mediators involved in sepsis and platelet pathophysiology were assessed at day-1 using a multi-analyte Milliplex human cytokine kit. Patients with underlying diseases at risk of thrombocytopenia (hematological malignancies, chemotherapy, cirrhosis, and chronic heart failure) were excluded. RESULTS: Thrombocytopenia occurred in 33 (55%) of 60 patients assessed. Patients with thrombocytopenia were more prone to present with extrapulmonary infections and bacteremia. Disseminated intravascular coagulation was frequent (81%) in these patients. Unbiased hierarchical clustering identified five different clusters of sepsis mediators, including one with markers of platelet activation (e.g., thrombospondin-1) positively associated with platelet count, one with markers of inflammation (e.g., tumor necrosis factor alpha and heat shock protein 70), and endothelial dysfunction (e.g., intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) negatively associated with platelet count, and another involving growth factors of thrombopoiesis (e.g., thrombopoietin), also negatively associated with platelet count. Surrogates of hemodilution (e.g., hypoprotidemia and higher fluid balance) were also associated with thrombocytopenia. CONCLUSION: Multiple mechanisms seemed involved in thrombocytopenia during septic shock, including endothelial dysfunction/coagulopathy, hemodilution, and altered thrombopoiesis.

Serum suppression of tumorigenicity 2 level is an independent predictor of all-cause mortality in HIV-infected patients.

OBJECTIVE: To evaluate the predictive value of soluble suppression of tumorigenicity 2 (sST2), a decoy receptor of IL-33 involved in several inflammatory and immune diseases, for death in HIV infection. DESIGN: Patients enrolled in the ANRS CO3 Aquitaine Cohort, a prospective hospital-based cohort of HIV-1-infected patients, who had a plasma sample available in the biobank were systematically eligible. METHODS: sST2, soluble CD14 (sCD14) and IL-6 were measured using Luminex multiplex bead-based technology (R&D Systems) and a Bio-Plex 200 instrument (BioRad). Predictive capacities of sST2, sCD14, IL-6 and of the Veterans Aging Cohort Study clinical score at baseline on overall mortality were compared using multivariable Cox proportional hazards models. RESULTS: During a median follow-up of 7.2 years [interquartile range (IQR): 6.0; 7.9], 93 deaths from all causes (incidence rate 9.9 per 1000 patient-years; 95% confidence interval 7.9-11.9) were reported in 1414 patients. The median sST2 baseline concentration was 22.9 ng/ml (IQR: 17.7; 30.3) and was higher (30.8 ng/ml, IQR: 21.5; 42.1) in patients who died as compared with those who stayed alive (22.6 ng/ml; IQR: 17.5; 29.6) (P < 10). An increased risk of death of 21% for a concentration 10.0 ng/ml higher of sST2 remained after adjustment for sCD14, IL-6 and Veterans Aging Cohort Study score (adjusted hazard ratio: 1.21; P < 10). The predictive capacity of sST2 was confirmed in a validation cohort (n = 386, 31 deaths) with an improved area c-index from 0.804 without sST2 to 0.811 with sST2. CONCLUSION: sST2 is a new valuable biomarker to evaluate the risk of all-cause mortality in HIV disease.

Negative modulation of suppressive HIV-specific regulatory T cells by IL-2 adjuvanted therapeutic vaccine.

The potential benefit in using IL-2 in immunotherapy for cancer and autoimmunity has been linked to the modulation of immune responses, which partly relies on a direct effect on Tregs populations. Here, we revisited the role of IL-2 in HIV infection and investigated whether its use as an adjuvant with therapeutic vaccination, impacts on HIV-specific responses. Antiretroviral therapy treated-patients were randomized to receive 4 boosts of vaccination (ALVACHIV/Lipo-6T, weeks 0/4/8/12) followed by 3 cycles of IL-2 (weeks 16/24/32) before treatment interruption (TI) at week40. IL-2 administration increased significantly HIV-specific CD4+CD25+CD134+ T-cell responses, which inversely correlated with viral load after TI (r = -0.7, p <0.007) in the vaccine/IL-2 group. IL-2 increased global CD25+CD127lowFoxP3+Tregs (p <0.05) while it decreased HIV- but not CMV- specific CD39+FoxP3+CD25+CD134+Tregs (p <0.05). HIV-specific Tregs were inversely correlated with IFN-γ producing specific-effectors (p = 0.03) and positively correlated with viral load (r = 0.7, p = 0.01), revealing their undesired presence during chronic infection. Global Tregs, but not HIV-specific Tregs, inversely correlated with a decrease in exhausted PD1+CD95+ T-cells (p = 0.001). Altogether, our results underline the negative impact of HIV-specific Tregs on HIV-specific effectors and reveal the beneficial use of IL-2 as an adjuvant as its administration increases global Tregs that impact on T-cell exhaustion and decreases HIV-specific CD39+Tregs by shifting the balance towards effectors.

Nanotubular crosstalk with distressed cardiomyocytes stimulates the paracrine repair function of mesenchymal stem cells.

Mesenchymal stem cells (MSC) are known to repair broken heart tissues primarily through a paracrine fashion while emerging evidence indicate that MSC can communicate with cardiomyocytes (CM) through tunneling nanotubes (TNT). Nevertheless, no link has been so far established between these two processes. Here, we addressed whether cell-to-cell communication processes between MSC and suffering cardiomyocytes and more particularly those involving TNT control the MSC paracrine regenerative function. In the attempt to mimic in vitro an injured heart microenvironment, we developed a species mismatch coculture system consisting of terminally differentiated CM from mouse in a distressed state and human multipotent adipose derived stem cells (hMADS). In this setting, we found that crosstalk between hMADS and CM through TNT altered the secretion by hMADS of cardioprotective soluble factors such as VEGF, HGF, SDF-1α, and MCP-3 and thereby maximized the capacity of stem cells to promote angiogenesis and chemotaxis of bone marrow multipotent cells. Additionally, engraftment experiments into mouse infarcted hearts revealed that in vitro preconditioning of hMADS with cardiomyocytes increased the cell therapy efficacy of naïve stem cells. In particular, in comparison with hearts treated with stem cells alone, those treated with cocultured ones exhibited greater cardiac function recovery associated with higher angiogenesis and homing of bone marrow progenitor cells at the infarction site. In conclusion, our findings established the first relationship between the paracrine regenerative action of MSC and the nanotubular crosstalk with CM and emphasize that ex vivo manipulation of these communication processes might be of interest for optimizing current cardiac cell therapies.

IL-7 modulates in vitro and in vivo human memory T regulatory cell functions through the CD39/ATP axis.

The heterogeneity of human regulatory T cells (Tregs) may explain the discrepancies between studies on Tregs in physiology and pathology. Contrasting effects of IL-7 on the expansion and survival of human Tregs were reported. Therefore, we investigated the effects of IL-7 on the phenotype and function of well-characterized populations of human Tregs. We show that IL-7 signals via the CD127 receptor on naive, memory, and activated memory Tregs sorted from the blood of healthy donors, but it does not affect their proliferation. In contrast, IL-7 affects their suppressive capacities differently. This effect was modest on naive Tregs but was dramatic (90%) on memory Tregs. We provide evidence that IL-7 exerts a synergistic effect through downmodulation of the ectoenzyme CD39, which converts ATP to ADP/AMP, and an increase in ATP receptor P2X7. Both effects lead to an increase in the ATP-mediated effect, tipping the balance to favor Th17 conversion. Using an IL-7 therapeutic study, we show that IL-7 exerts the same effects in vitro and in vivo in HIV-infected individuals. Globally, our data show that IL-7 negatively regulates Tregs and contributes to increase the number of tools that may affect Treg function in pathology.

Cytokine and gene transcription profiles of immune responses elicited by HIV lipopeptide vaccine in HIV-negative volunteers.

OBJECTIVE: To dissect the biological mechanisms involved in the cellular responses to a candidate vaccine containing 5 HIV peptides coupled to a palmytoil tail (HIV-LIPO-5) in healthy volunteers, by using extensive immunogenicity assessments with different stimulation durations. DESIGN: Immunogenicity substudy of a randomized phase II prophylactic HIV vaccine trial (ANRS VAC 18). METHODS: HIV-LIPO-5 or placebo was administered at W0, W4, W12 and W24. Peripheral blood mononuclear cells from a subset of participants at W0 and W14 were stimulated with HIV-LIPO-5, Gag peptides contained in the vaccine and control peptides. ELISpot, lymphoproliferation, intracellular cytokine staining (ICS), cytokine multiplex and transcriptomic analyses were performed. Different time points and stimulation conditions were compared, controlling for test multiplicity. RESULTS: Cultured ELISpot and lymphoproliferation responses were detected at W14. Ex-vivo ICS showed mainly interleukin (IL)-2-producing cells. Secretion of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-5 and IL-13 increased significantly after culture and Gag stimulation at W14 compared to W0. Metallothionein genes were consistently overexpressed after HIV-LIPO-5 stimulation at W0 and W14. At W14, significant probes increased substantially, including IFN-γ, CXCL9, IL2RA, TNFAIP6, CCL3L1 and IL-6. Canonical pathway analyses indicated a role of interferon signalling genes in response to HIV-LIPO-5. CONCLUSION: HIV-LIPO-5 vaccination elicited Th1 and Th2 memory precursor responses and a consistent modulation in gene expression. The response profile before vaccination suggests an adjuvant effect of the lipid tail of HIV-LIPO-5. Our combined immunogenicity analyses allowed to identify a specific signature profile of HIV-LIPO-5 and indicate that HIV-LIPO-5 could be further developed as a prime in heterologous prime-boost strategies.

Immunogenicity and safety of an HIV-1 lipopeptide vaccine in healthy adults: a phase 2 placebo-controlled ANRS trial.

BACKGROUND: French National Agency for Research on AIDS and Viral Hepatitis's HIV-LIPO-5 vaccine includes five HIV-1 peptides, containing multiple CD8 and CD4 T-cell epitopes and coupled to a palmitoyl tail. Whether HIV-LIPO-5 immunogenicity varies with the dose is unknown. METHODS: HIV-negative volunteers were randomized to receive HIV-LIPO-5 vaccine at 50 microg/lipopeptide (N = 32), 150 microg/lipopeptide (N = 32), 500 microg/lipopeptide (N = 33) or placebo (N = 34) at weeks 0, 4, 12 and 24. HIV-1-specific CD8 (interferon-gamma ELISpot on peripheral blood mononuclear cells cultured for 12 days) and CD4 responses (peripheral blood mononuclear cell lymphoproliferation) were assessed at baseline, after each injection and at week 48. RESULTS: Local reactions were dose-dependent but no differences in systemic reactions appeared between groups. Sustained (at least on two separate occasions) CD8 response rates to at least one given HIV-1 pool were obtained in 22 of 32 (69%), 21 of 33 (64%) and 21 of 34 (62%) individuals for LIPO-5 50, 150 and 500 groups, respectively (P < or = 0.0001 for all comparisons to the placebo). Cumulative CD4 response rates were obtained in 15 of 32 (47%), 18 of 33 (55%) and 15 of 34 (44%) individuals (P < 0.0001 for all comparisons to placebo). At week 48, CD8 responses persisted in 47 of 91 (52%) HIV-LIPO-5 recipients. CONCLUSION: Doses of 50, 150 and 500 microg of French National Agency for Research on AIDS and Viral Hepatitis's HIV-LIPO-5 vaccine were able to elicit HIV-specific sustained CD8 and CD4 T-cell responses in healthy adults. Safety is good and all doses appear appropriate in further 'prime-boost' trials.

Cellular immune responses induced with dose-sparing intradermal administration of HIV vaccine to HIV-uninfected volunteers in the ANRS VAC16 trial.

OBJECTIVE: The objective was to compare the safety and cellular immunogenicity of intradermal versus intramuscular immunization with an HIV-lipopeptide candidate vaccine (LIPO-4) in healthy volunteers. METHODOLOGY: A randomized, open-label trial with 24 weeks of follow-up was conducted in France at six HIV-vaccine trial sites. Sixty-eight healthy 21- to 55-year-old HIV-uninfected subjects were randomized to receive the LIPO-4 vaccine (four HIV lipopeptides linked to a T-helper-stimulating epitope of tetanus-toxin protein) at weeks 0, 4 and 12, either intradermally (0.1 ml, 100 microg of each peptide) or intramuscularly (0.5 ml, 500 microg of each peptide). Comparative safety of both routes was evaluated. CD8+ T-cell immune responses to HIV epitopes (ELISpot interferon-gamma assay) and tetanus toxin-specific CD4+ T-cell responses (lymphoproliferation) were assessed at baseline, two weeks after each injection, and at week 24. RESULTS AND CONCLUSION: No severe, serious or life-threatening adverse events were observed. Local pain was significantly more frequent after intramuscular injection, but local inflammatory reactions were more frequent after intradermal immunization. At weeks 2, 6, 14 and 24, the respective cumulative percentages of induced CD8+ T-cell responses to at least one HIV peptide were 9, 33, 39 and 52 (intradermal group) or 14, 20, 26 and 37 (intramuscular group), and induced tetanus toxin-specific CD4+ T-cell responses were 6, 27, 33 and 39 (intradermal), or 9, 46, 54 and 63 (intramuscular). In conclusion, intradermal LIPO-4 immunization was well tolerated, required one-fifth of the intramuscular dose, and induced similar HIV-specific CD8+ T-cell responses. Moreover, the immunization route influenced which antigen-specific T-cells (CD4+ or CD8+) were induced. TRIAL REGISTRATION: ClinicalTrials.gov NCT00121121.