Immunotherapies inducing immunogenic cell death in cancer: insight of the innate immune system.

Cancer immunotherapies include monoclonal antibodies, cytokines, oncolytic viruses, cellular therapies, and other biological and synthetic immunomodulators. These are traditionally studied for their effect on the immune system's role in eliminating cancer cells. However, some of these therapies have the unique ability to directly induce cytotoxicity in cancer cells by inducing immunogenic cell death (ICD). Unlike general immune stimulation, ICD triggers specific therapy-induced cell death pathways, based on the release of damage-associated molecular patterns (DAMPs) from dying tumour cells. These activate innate pattern recognition receptors (PRRs) and subsequent adaptive immune responses, offering the promise of sustained anticancer drug efficacy and durable antitumour immune memory. Exploring how onco-immunotherapies can trigger ICD, enhances our understanding of their mechanisms and potential for combination strategies. This review explores the complexities of these immunotherapeutic approaches that induce ICD, highlighting their implications for the innate immune system, addressing challenges in cancer treatment, and emphasising the pivotal role of ICD in contemporary cancer research.

Natural Killer Repertoire Restoration in TB/HIV Co-Infected Individuals Experienced an Immune Reconstitution Syndrome (CAMELIA Trial, ANRS 12153).

IRIS is a common complication in HIV-infected patients treated for tuberculosis (TB) and cART. Our aim was to evaluate NK cell reconstitution in HIV-infected patients with TB-IRIS compared to those without IRIS. 147 HIV-infected patients with TB from the CAMELIA trial were enrolled. HIV+TB+ patients were followed for 32 weeks. The NK cell repertoire was assessed in whole blood at different time points. As CAMELIA has two arms (early and late cART initiation), we analysed them separately. At enrolment, individuals had low CD4 cell counts (27 cells/mm3) and high plasma viral loads (5.76 and 5.50 log/mL for IRIS and non-IRIS individuals, respectively). Thirty-seven people developed IRIS (in the early and late arms). In the early and late arms, we observed similar proportions of total NK and NK cell subsets in TB-IRIS and non-IRIS individuals during follow-up, except for the CD56dimCD16pos (both arms) and CD56dimCD16neg (late arm only) subsets, which were higher in TB-IRIS and non-IRIS individuals, respectively, after cART. Regarding the repertoire and markers of NK cells, significant differences (lower expression of NKp30, NKG2A (CD159a), NKG2D (CD314) were observed in TB-IRIS compared to non-IRIS individuals after the start of cART. In the late arm, some changes (increased expression of CD69, NKG2C, CD158i) were observed in TB-IRIS compared to non-IRIS individuals, but only before cART initiation (during TB treatment). KIR expression by NK cells (CD158a and CD158i) was similar in both groups. CD69 expression by NK cells decreased in all groups. Expression of the NCR repertoire (NKp30, NKp44, NKp46) has similar kinetics in TB-IRIS subjects compared to non-IRIS subjects regardless of the arm analysed. NK cell reconstitution appeared to be better in TB-IRIS subjects. Although NK cell reconstitution is impaired in HIV infection after cART, as previously reported, it does not appear to be affected by the development of IRIS in HIV and TB-infected individuals.

T cell migration and effector function differences in familial adenomatous polyposis patients with APC gene mutations.

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology.

Inflammasome genetic variants are associated with tuberculosis, HIV-1 infection, and TB/HIV-immune reconstitution inflammatory syndrome outcomes.

Background: Tuberculosis (TB) and AIDS are the leading causes of infectious diseases death worldwide. Here, we investigated the relationship between from single nucleotide polymorphisms (SNPs) of the NLRP3, CARD8, AIM2, CASP-1, IFI16, and IL-1ß inflammasome genes, as well as the profiles of secreted proinflammatory cytokines (e.g., IL-1ß, IL-18, IL-33, and IL-6) with the TB clinical profiles, TB-HIV coinfection, and IRIS onset. Methods: The individuals were divided into four groups: TB-HIV group (n=88; 11 of them with IRIS), HIV-1 group (n=20), TB group (n=24) and healthy volunteers (HC) group (n=10), and were followed up at INI/FIOCRUZ and HGNI (Rio de Janeiro/Brazil) from 2006 to 2016. Real-time PCR was used to determine the genotypes of the Single Nucleotide Polymorphism (SNPs), and ELISA was used to measure the plasma cytokine levels. Unconditional logistic regression models were used to perform risk estimations. Results: A higher risk for extrapulmonary TB was associated with the TT genotype (aOR=6.76; P=0.026) in the NLRP3 rs4612666 Single Nucleotide Polymorphism (SNP) and the C-C-T-G-C haplotype (aOR=4.99; P= 0.017) in the NLRP3 variants. This same Single Nucleotide Polymorphism (SNP) was associated with lower risk against extrapulmonary TB when the carrier allele C (aOR=0.15; P=0.021) was present. Among those with HIV-1 infections, a higher risk for TB onset was associated with the GA genotype (aOR=5.5; P=0.044) in the IL1-ß rs1143634 Single Nucleotide Polymorphism (SNP). In contrast, lower risk against TB onset was associated with the A-G haplotype (aOR=0.17; P= 0.026) in the CARD8 variants. Higher IL-6 and IL-33 levels were observed in individuals with TB. A higher risk for IRIS onset was associated with CD8 counts ≤ 500 cells/mm3 (aOR=12.32; P=0.010), the presence of extrapulmonary TB (aOR=6.6; P=0.038), and the CT genotype (aOR=61.06; P=0.026) or carrier allele T (aOR=61.06; P=0.026) in the AIM2 rs2276405 Single Nucleotide Polymorphism (SNP), whereas lower risk against IRIS onset was associated with the AT genotype (aOR=0.02; P=0.033) or carrier allele T (aOR=0.02; P=0.029) in the CARD8 rs2043211 Single Nucleotide Polymorphism (SNP) and the T-G haplotype (aOR=0.07; P= 0.033) in the CARD8 variants. No other significant associations were observed. Conclusions: Our results depict the involvement of genetic polymorphisms of crucial innate immunity genes and proinflammatory cytokines in the clinical outcomes related to TB-HIV coinfection.

Changes in the natural killer cell repertoire and function induced by the cancer immune adjuvant candidate IMMUNEPOTENT-CRP.

Natural killer (NK) cells are CD3-, CD16+, CD56+ that play a crucial role in immune response by recognizing and eliminating a variety of virus-infected, malignant, and antibody-coated target cells. We examined activation; repertoire changes and effector functions of human NK cells normal donors treated with IMMUNEPOTENT-CRP (I-CRP), a bovine dialyzable leukocyte extract (DLE) containing a mixture of low molecular weight molecules. I-CRP induces human NK cells activation and increase CD56Dim CD16- subset, without inducing proliferation. Human NK cells showed an increase on NKp30, NKp44, NKp46, NKG2D, NKG2C and KIR receptors, whereas no significant differences on CD160, CD85j and CD226 where observed. I-CRP-treated human NK cells exhibited an increased degranulation activity against K562 target cells, as shown by CD107a assay, and this correlates with cytotoxicity against K562 cells observed in calcein release assay. These results indicate that I-CRP can modify human NK cells receptor repertoire leading to an increased cytotoxic activity, supporting evidence for its use to stimulate NK cells.

Bovine Dialyzable Leukocyte Extract IMMUNEPOTENT-CRP Induces Selective ROS-Dependent Apoptosis in T-Acute Lymphoblastic Leukemia Cell Lines.

Immunotherapies strengthen the immune system to fight multiple diseases such as infections, immunodeficiencies, and autoimmune diseases, and recently, they are being used as an adjuvant in cancer treatment. IMMUNEPOTENT-CRP (I-CRP) is an immunotherapy made of bovine dialyzable leukocyte extract (bDLE) that has chemoprotective and immunomodulatory effects in different cellular populations of the immune system and antitumor activity in different cancer cell lines. Our recent results suggest that the antineoplastic effect of I-CRP is due to the characteristics of cancer cells. To confirm, we evaluated whether the selectivity is due to cell lineage or characteristics of cancer cells, testing cytotoxicity in T-acute lymphoblastic leukemia cells and their cell death mechanism. Here, we assessed the effect of I-CRP on cell viability and cell death. To determine the mechanism of cell death, we tested cell cycle, mitochondrial and nuclear alterations, and caspases and reactive oxygen species (ROS) and their role in cell death mechanism. Our results show that I-CRP does not affect cell viability in noncancer cells and induces selective cytotoxicity in a dose-dependent manner in leukemic cell lines. I-CRP also induces mitochondrial damage through proapoptotic and antiapoptotic protein modulation (Bax and Bcl-2) and ROS production, nuclear alterations including DNA damage (γ-H2Ax), overexpression of p53, cell cycle arrest, and DNA degradation. I-CRP induced ROS-dependent apoptosis in leukemic cells. Overall, here, we show that I-CRP cytotoxicity is selective to leukemic cells, inducing ROS-dependent apoptosis. This research opens the door to further exploration of their role in the immune system and the cell death mechanism that could potentially work in conjunction with other therapies including hematological malignances.

Clinical and genetic markers associated with tuberculosis, HIV-1 infection, and TB/HIV-immune reconstitution inflammatory syndrome outcomes.

BACKGROUND: Tuberculosis (TB) and AIDS are the leading causes of infectious disease death worldwide. In some TB-HIV co-infected individuals treated for both diseases simultaneously, a pathological inflammatory reaction termed immune reconstitution inflammatory syndrome (IRIS) may occur. The risk factors for IRIS are not fully defined. We investigated the association of HLA-B, HLA-C, and KIR genotypes with TB, HIV-1 infection, and IRIS onset. METHODS: Patients were divided into four groups: Group 1- TB+/HIV+ (n = 88; 11 of them with IRIS), Group 2- HIV+ (n = 24), Group 3- TB+ (n = 24) and Group 4- healthy volunteers (n = 26). Patients were followed up at INI/FIOCRUZ and HGNI (Rio de Janeiro/Brazil) from 2006 to 2016. The HLA-B and HLA-C loci were typed using SBT, NGS, and KIR genes by PCR-SSP. Unconditional logistic regression models were performed for Protection/risk estimation. RESULTS: Among the individuals with TB as the outcome, KIR2DS2 was associated with increased risk for TB onset (aOR = 2.39, P = 0.04), whereas HLA-B*08 and female gender were associated with protection against TB onset (aOR = 0.23, P = 0.03, and aOR = 0.33, P = 0.01, respectively). Not carrying KIR2DL3 (aOR = 0.18, P = 0.03) and carrying HLA-C*07 (aOR = 0.32, P = 0.04) were associated with protection against TB onset among HIV-infected patients. An increased risk for IRIS onset was associated with having a CD8 count ≤500 cells/mm3 (aOR = 18.23, P = 0.016); carrying the KIR2DS2 gene (aOR = 27.22, P = 0.032), the HLA-B*41 allele (aOR = 68.84, P = 0.033), the KIR2DS1 + HLA-C2 pair (aOR = 28.58, P = 0.024); and not carrying the KIR2DL3 + HLA-C1/C2 pair (aOR = 43.04, P = 0.034), and the KIR2DL1 + HLA-C1/C2 pair (aOR = 43.04, P = 0.034), CONCLUSIONS: These results suggest the participation of these genes in the immunopathogenic mechanisms related to the conditions studied. This is the first study demonstrating an association of HLA-B*41, KIR2DS2, and KIR + HLA-C pairs with IRIS onset among TB-HIV co-infected individuals.

High Activation of γδ T Cells and the γδ2pos T-Cell Subset Is Associated With the Onset of Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome, ANRS 12153 CAPRI NK.

Background: Human Immunodeficiency Virus 1 (HIV-1) and Mycobacterium Tuberculosis (Mtb) co-infected patients are commonly at risk of immune reconstitution inflammatory syndrome (IRIS) when initiating antiretroviral treatment (ART). Evidence indicates that innate immunity plays a role in TB-IRIS. Here, we evaluate the phenotype of Gamma-delta (γδ) T cells and invariant Natural Killer (iNK) T cells in tuberculosis-associated IRIS. Methods: Forty-eight HIV+/TB+ patients (21 IRIS) and three control groups: HIV-/TB- (HD, n = 11), HIV+/TB- (n = 26), and HIV-/TB+ (n = 22) were studied. Samples were taken at ART initiation (week 2 of anti-tuberculosis treatment) and at the diagnosis of IRIS for HIV+/TB+; before ART for HIV+/TB-, and at week 2 of anti-tuberculosis treatment for HIV-/TB+ patients. γδ T cells and Invariant natural killer T (iNKT) cells were analyzed by flow cytometry. Results: Before ART, IRIS, and non-IRIS patients showed a similar proportion of γδpos T and iNKT cells. HLA-DR on γδpos T cells and δ2posγδpos T cells was significantly higher in TB-IRIS vs. non-IRIS patients and controls (p < 0.0001). NKG2D expression on γδpos T cells and the δ2posγδpos T cell subset was lower in HIV+/TB+ patients than controls. CD158a expression on γδpos T cells was higher in TB-IRIS than non-IRIS (p = 0.02), HIV+/TB-, and HIV-/TB- patients. Conclusion: The higher activation of γδposT cells and the γδ2posγδpos T cell subset suggests that γδ T cells may play a role in the pathogenesis of TB-IRIS.

Changes in the NK Cell Repertoire Related to Initiation of TB Treatment and Onset of Immune Reconstitution Inflammatory Syndrome in TB/HIV Co-infected Patients in Rio de Janeiro, Brazil-ANRS 12274.

Tuberculosis (TB) is the most common comorbidity and the leading cause of death among HIV-infected individuals. Although the combined antiretroviral therapy (cART) during TB treatment improves the survival of TB/HIV patients, the occurrence of immune reconstitution inflammatory syndrome (IRIS) in some patients poses clinical and scientific challenges. This work aimed to evaluate blood innate lymphocytes during therapeutic intervention for both diseases and their implications for the onset of IRIS. Natural killer (NK) cells, invariant NKT cells (iNKT), γδ T cell subsets, and in vitro NK functional activity were characterized by multiparametric flow cytometry in the following groups: 33 TB/HIV patients (four with paradoxical IRIS), 27 TB and 25 HIV mono-infected subjects (prior to initiation of TB treatment and/or cART and during clinical follow-up to 24 weeks), and 25 healthy controls (HC). Concerning the NK cell repertoire, several activation and inhibitory receptors were skewed in the TB/HIV patients compared to those in the other groups, especially the HCs. Significantly higher expression of CD158a (p = 0.025), NKp80 (p = 0.033), and NKG2C (p = 0.0076) receptors was detected in the TB/HIV IRIS patients than in the non-IRIS patients. Although more NK degranulation was observed in the TB/HIV patients than in the other groups, the therapeutic intervention did not alter the frequency during follow-up (weeks 2-24). A higher frequency of the γδ T cell population was observed in the TB/HIV patients with inversion of the Vδ2+/Vδ2- ratio, especially for those presenting pulmonary TB, suggesting an expansion of particular γδ T subsets during TB/HIV co-infection. In conclusion, HIV infection impacts the frequency of circulating NK cells and γδ T cell subsets in TB/HIV patients. Important modifications of the NK cell repertoire were observed after anti-TB treatment (week 2) but not during the cART/TB follow-up (weeks 6-24). An increase of CD161+ NK cells was related to an unfavorable outcome. Despite the low number of cases, a more preserved NK cell profile was detected in IRIS patients previous to treatment, suggesting a role for these cells in IRIS onset. Longitudinal evaluation of the NK repertoire showed the impact of TB treatment and implicated these cells in TB pathogenesis in TB/HIV co-infected patients.

γδ T-cell subsets in HIV controllers: potential role of Tγδ17 cells in the regulation of chronic immune activation.

OBJECTIVES: HIV controllers (HICs) are rare HIV-infected individuals able to maintain undetectable viremia in the absence of antiretroviral treatment. Although HIV-specific cytotoxic T cells have been well deciphered in HIC, γδ T lymphocytes remain largely uncharacterized. The aim of this study was to analyse phenotypic and functional characteristics of γδ T cells and their relationship with immune activation, which remains abnormally elevated and associated with comorbidities in HICs. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 16 HICs, 16 patients with untreated chronic HIV infection (UT-CHI) and 20 healthy donors. Surface marker expression and cytokine production by γδ T cells were analysed by flow cytometry. RESULTS: Despite normal frequencies of total γδ T cells, the Vδ2/Vδ2 ratio was significantly reduced in HIC, albeit to a lesser extent than UT-CHI patients. Of note, nine HICs showed elevated Vδ2 γδ T cells, as patients with UT-CHI, which was associated with higher CD8 T-cell activation. Interleukin (IL)-17-production by γδ T cells (Tγδ17) was better preserved in HIC than in UT-CHI patients. Proportion of total γδ T cells positively correlated with CD8 T-cell activation and HIV-DNA, IP-10 and sCD14 levels. Conversely, Tγδ17 cells negatively correlated with CD8 T-cell activation and plasma sCD14 levels. Moreover, transforming growth factor (TGF)-ß producing Vδ2 T cells were as dramatically depleted in HIC as in UT-CHI patients. CONCLUSION: The relative preservation of IL-17-producing γδ T cells in HIC and their negative association with immune activation raise the hypothesis that Tγδ17 cells - potentially through prevention of microbial translocation - may participate in the control of chronic systemic immune activation.

Potential Role of Vδ2+ γδ T Cells in Regulation of Immune Activation in Primary HIV Infection.

Although conventional regulatory T cells (Tregs) are sufficient in controlling low residual T-cell activation in ART-treated patients, they are not efficient in controlling exaggerated immune activation associated with high levels of HIV replication in primary HIV infection (PHI). Our previous data suggested that double negative (DN) T cells including mainly γδ DN T cells play a role in the control of immune activation in PHI. Since γδ T cells are capable of exerting regulatory functions, we investigated their implication as Tregs in PHI as well as chronic HIV infection (CHI). In a cross-sectional study of 58 HIV-infected patients, in the primary and the chronic phase either ART-treated or untreated (UT), we analyzed phenotype and cytokine production of γδ T cells using flow cytometry. Cytokine production was assessed following in vitro stimulation with isopentenyl pyrophosphate or plate-bound anti-CD3/anti-CD28 monoclonal antibodies. We found that the proportion of γδ T cells negatively correlated with CD8 T-cell activation in PHI patients. Furthermore, we found that in these patients, the Vδ2 receptor bearing (Vδ2+) γδ T cells were strongly activated, exhibited low terminal differentiation, and produced the anti-inflammatory cytokine, TGF-ß. In contrast, in UT-CHI, we observed a remarkable expansion of γδ T cells, where the Vδ2+ γδ T cells comprised of an elevated proportion of terminally differentiated cells producing high levels of IFN-γ but very low levels of TGF-ß. We also found that this loss of regulatory feature of γδ T cells in CHI was a lasting impairment as we did not find recovery of TGF-ß production even in ART-CHI patients successfully treated for more than 5 years. Our data therefore suggest that during the primary HIV infection, Vδ2+ γδ T cells may act as Tregs controlling immune activation through production of TGF-ß. However, in CHI, γδ T cells transform from an anti-inflammatory into pro-inflammatory cytokine profile and participate in sustenance of immune activation.

Gag-Specific CD8 T-Cell Proliferation Is Associated With Higher Peripheral Blood Levels of Transforming Growth Factor-ß and Gut-Homing T Cells in Youths Perinatally Infected With Human Immunodeficiency Virus-1: The ANRS-EP38-IMMIP Study.

BACKGROUND: Gag-specific T lymphocytes play a key role in the control of human immunodeficiency virus (HIV) replication. Their restoration will be important for future reservoir targeting strategies. In this study, we aimed to identify immune correlates of Gag-specific CD8 T-cell proliferation in youths with perinatally acquired HIV-1 infection. METHODS: The ANRS-EP38-IMMIP study included youths of 15 to 24 years of age. Fifty-three were taking combination anti-retroviral therapy and aviremic at the time of the study and had undergone valid 5-6-carboxyfluorescein diacetate succimidyl ester-based flow cytometry T-cell proliferation assays. Plasma analytes were quantified by enzyme-linked immunosorbent assay or multiplex assays. Peripheral blood cells were phenotyped by flow cytometry. Logistic regression was used to study the association between Gag-specific T-cell proliferation and immune markers. RESULTS: Patients with Gag-specific CD8 T-cell proliferation had higher levels of plasma transforming growth factor (TGF)-ß1, a lower proportion of naive cells among regulatory T cells (Tregs), and higher percentages of CD4 and CD8 T cells expressing the α4ß7 integrin or CD161 molecule than those without a Gag-specific response. These associations were significant based on analyses including potential confounders. CONCLUSIONS: Preserved Gag-specific CD8 T-cell proliferation was associated with higher TGF-ß1 levels and increased percentages of T cells with a gut-homing phenotype at least 15 years after HIV infection during the perinatal period.

Interleukin-1 receptor antagonist, a biomarker of response to anti-TB treatment in HIV/TB co-infected patients.

OBJECTIVES: Despite the high frequency of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) in human immunodeficiency virus (HIV)/TB co-infected patients, no diagnostic test is available. Here, we investigated whether monocyte/macrophage activation markers can predict TB-IRIS occurrence and if they are modulated by anti-TB treatment. METHODS: Frozen plasma was obtained from 127 HIV/TB co-infected adults naïve for antiretroviral therapy, enrolled in the CAMELIA trial, 36 of whom developed TB-IRIS. Concentrations of IL-1Ra, sCD14, and sCD163 were measured at anti-TB treatment onset (baseline), after 8 weeks of anti-TB treatment and at TB-IRIS time. RESULTS: At baseline, IL-1Ra and sCD14 concentrations were similar in TB-IRIS and non-IRIS patients. sCD163 concentrations, although significantly higher in TB-IRIS patients, did not remain associated with TB-IRIS occurrence in multivariate analysis. At the time of TB-IRIS, patients displayed higher concentrations of IL-1Ra (p = 0.002) and sCD14 (p < 0.001). The most striking result was the significant decrease in IL-1Ra after 8 weeks of anti-TB treatment (median reduction: -63% (p < 0.0001)). CONCLUSIONS: None of the biomarkers tested was associated with TB-IRIS occurrence. However, repeated measurement of IL-1Ra could help for the diagnosis of TB-IRIS. The substantial reduction of IL-1Ra under treatment suggests that IL-1Ra could be a surrogate biomarker of anti-TB treatment response in HIV-infected patients.

Natural amines inhibit activation of human plasmacytoid dendritic cells through CXCR4 engagement.

Plasmacytoid dendritic cells (pDC) are specialized in secretion of type I interferon in response to pathogens. Here we show that natural monoamines and synthetic amines inhibit pDC activation by RNA viruses. Furthermore, a synthetic analogue of histamine reduces type I interferon production in a mouse model of influenza infection. We identify CXC chemokine receptor 4 (CXCR4) as a receptor used by amines to inhibit pDC. Our study establishes a functional link between natural amines and the innate immune system and identifies CXCR4 as a potential 'on-off' switch of pDC activity with therapeutic potential.

Gag-Specific CD4 T Cell Proliferation, Plasmacytoid Dendritic Cells, and Ethnicity in Perinatally HIV-1-Infected Youths: The ANRS-EP38-IMMIP Study.

In perinatally HIV-1-infected youths living in France, we previously reported that Gag-specific CD4 and CD8 T cell proliferation is more frequently detected in patients of black ethnicity than in those of other ethnicities. We observed that black patients had higher levels of dendritic cells (DCs) than other patients. We aimed at studying the association of DC levels with Gag-specific T cell proliferation. The ANRS-EP38-IMMIP study is an observational study of youths aged between 15 and 24 years who were perinatally infected with HIV. A single blood sample was drawn for virological and immunological assays. Data from cART-treated 53 youths with undetectable plasma HIV RNA were analyzed. Gag-specific T cell proliferation was assessed by using a CFSE-based test. Peripheral blood myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) were phenotyped by flow cytometry. Plasma markers were quantified by ELISA or multiplex assays. Logistic regression was used for univariate and multivariate analyses. Patients with Gag-specific CD4 T cell proliferative responses had significantly higher percentages and absolute counts of mDCs and pDCs in the peripheral blood than nonresponding patients. Gag-specific CD4 and CD8 T cell proliferation was associated with lower plasma sCD14 levels. Plasma levels of IFN-α, TRAIL, and chemokines involved in T cell migration to secondary lymphoid organs were not associated with T cell proliferation. Multivariate analysis confirmed the association between Gag-specific CD4 T cell proliferation and pDC levels. In conclusion, DC levels are a robust correlate of the presence of Gag-specific T cell proliferation in successfully treated youths.

CD4 T-Cell Responses in Primary HIV Infection: Interrelationship with Immune Activation and Virus Burden.

Early events during primary HIV infection (PHI) are thought to influence disease outcome. Although a growing body of evidence suggests a beneficial role of HIV-specific CD4 help in HIV infection, it is unclear how early viral replication, systemic immune activation, and antiretroviral therapy (ART) may shape CD4 T-cell responses during PHI, and whether HIV-specific CD4 responses contribute to the high immune activation observed in PHI. Twenty-seven patients with early PHI were included in a prospective longitudinal study and 12 of them received ART after enrollment. Fresh peripheral blood mononuclear cells were used for measurement of ex vivo T-cell activation and of cytokine-producing CD4 T-cells following stimulation with PMA/ionomycin or HIV-1-gag-p24 antigen. Patients were segregated based on CD8 T-cell activation level (i.e., % HLA-DR+CD38+ CD8 T-cells) at baseline (BL). Patients with lower immune activation exhibited higher frequency of bulk CD4 T-cells producing IFN-γ or IL-17 and higher effector-to-regulatory cell ratios. No differences were found in HIV-specific CD4 T-cell frequencies. In contrast, segregation of patients based on plasma viral load (pVL) revealed that patients with higher pVL showed higher cytokine-producing HIV-specific CD4 responses. Of note, the frequency of IFN-γ+ HIV-specific CD4 T cells significantly diminished between BL and month 6 only in ART-treated patients. However, early treatment initiation was associated with better maintenance of HIV-specific IFN-γ+ CD4 T-cells. These data suggest that HIV-specific CD4 responses do not fuel systemic T-cell activation and are driven by viral replication but not able to contribute to its control in the early phase of infection. Moreover, our data also suggest a benefit of early treatment for the maintenance of HIV-specific CD4 T-cell help.

Gag-Specific CD4 and CD8 T-Cell Proliferation in Adolescents and Young Adults with Perinatally Acquired HIV-1 Infection Is Associated with Ethnicity - The ANRS-EP38-IMMIP Study.

The ANRS-EP38-IMMIP study aimed to provide a detailed assessment of the immune status of perinatally infected youths living in France. We studied Gag-specific CD4 and CD8 T-cell proliferation and the association between the proliferation of these cells, demographic factors and HIV disease history. We included 93 youths aged between 15 and 24 years who had been perinatally infected with HIV. Sixty-nine had undergone valid CFSE-based T-cell proliferation assays. Gag-specific proliferation of CD4 and CD8 T cells was detected in 12 (16%) and 30 (38%) patients, respectively. The Gag-specific proliferation of CD4 and CD8 T cells was more frequently observed in black patients than in patients from other ethnic groups (CD4: 32% vs. 4%, P = 0.001; CD8: 55% vs. 26%, P = 0.02). Among aviremic patients, the duration of viral suppression was shorter in CD8 responders than in CD8 nonresponders (medians: 54 vs. 20 months, P = 0.04). Among viremic patients, CD8 responders had significantly lower plasma HIV RNA levels than CD8 nonresponders (2.7 vs. 3.7 log10 HIV-RNA copies/ml, P = 0.02). In multivariate analyses including sex and HIV-1 subtype as covariables, Gag-specific CD4 T-cell proliferation was associated only with ethnicity, whereas Gag-specific CD8 T-cell proliferation was associated with both ethnicity and the duration of viral suppression. Both CD4 and CD8 responders reached their nadir CD4 T-cell percentages at younger ages than their nonresponder counterparts (6 vs. 8 years, P = 0.04 for both CD4 and CD8 T-cell proliferation). However, these associations were not significant in multivariate analysis. In conclusion, after at least 15 years of HIV infection, Gag-specific T-cell proliferation was found to be more frequent in black youths than in patients of other ethnic groups, despite all the patients being born in the same country, with similar access to care.

S100A9 Tetramers, Which are Ligands of CD85j, Increase the Ability of MVAHIV-Primed NK Cells to Control HIV Infection.

Natural killer (NK) cells are the major antiviral effector population of the innate immune system. We previously found that S100A9 is a novel ligand of the receptor CD85j and that S100A9 tetramers enhance the anti-HIV activity of NK cells. Also, we found that dendritic cells (DCs) infected by the HIV vaccine candidate, MVAHIV, prime NK cells to specifically control HIV infection in autologous CD4(+) T cells. In this study, we analyzed whether stimulation of NK cells by S100A9 tetramers prior to the priming by MVAHIV-infected DCs modulates the subsequent anti-HIV activity of NK cells. We found that S100A9 tetramers activate NK cells and that DCs enhance the anti-HIV activity of NK cells. Interestingly, we observed that stimulation of NK cells by S100A9 tetramers, prior to the priming, significantly increased the subsequent anti-HIV activity of NK cells and that the enhanced anti-HIV activity was observed following different conditions of priming, including the MVAHIV-priming. As S100A9 tetramers alone directly increase the anti-HIV activity of NK cells and as this increased anti-HIV activity is also observed following the interaction of NK cells with MVAHIV-infected DCs, we propose S100A9 tetramers as potential adjuvants to stimulate the anti-HIV activity of NK cells.

Phenotype alterations in regulatory T-cell subsets in primary HIV infection and identification of Tr1-like cells as the main interleukin 10-producing CD4+ T cells.

BACKGROUND: Conventional regulatory T cells (Tregs) can suppress human immunodeficiency virus type 1 (HIV-1)-specific immune responses but cannot control immune activation in primary HIV infection. Here, we characterized Treg subsets, using recently defined phenotypic delineation, and analyzed the relative contribution of cell subsets to the production of immunosuppressive cytokines in primary HIV infection. METHODS: In a longitudinal prospective study, ex vivo phenotyping of fresh peripheral blood mononuclear cells from patients with primary HIV infection was performed at baseline and month 6 of follow-up to characterize Treg subsets, immune activation, and cytokine production in isolated CD4(+) T cells. RESULTS: The frequency of CD4(+)CD25(+)CD127(low) Tregs and the distribution between the naive, memory, and activated/memory Treg subsets was similar in patients and healthy donors. However, Tregs from patients with primary HIV infection showed peculiar phenotypic profiles, such as elevated FoxP3, ICOS, and CTLA-4 expression, with CTLA-4 expression strikingly increased in all Treg subsets both at baseline and month 6 of follow-up. The great majority of interleukin 10 (IL-10)-producing CD4(+) T cells were FoxP3(neg) (ie, Tr1-like cells). In contrast to conventional Tregs, Tr1-like cells were inversely correlated with immune activation and not associated with lower effector T-cell responses. CONCLUSION: FoxP3(neg) Tr1-like cells-major contributors to IL-10 production-may have a beneficial role by controlling immune activation in early HIV infection.

Natural killer cell responses to dendritic cells infected by the ANRS HIV-1 vaccine candidate, MVAHIV.

Innate mechanisms are critical for the development of the host immune responses to antigen. Particularly, early interaction between natural killer (NK) cells and dendritic cells (DC) greatly impacts the establishment of both innate and adaptive immune responses. In this study, using an autologous in vitro co-culture system we analyzed the NK cell response against MVAHIV-infected DC as well as the subsequent ability of these MVAHIV-primed NK cells to control HIV-1 infection in autologous DC. We found that NK cells responded early to MVAHIV- or MVAWT-infected DC in terms of degranulation and cytokine production. After a 4-day priming of NK cells by MVAHIV- or MVAWT-infected DC we observed an enhanced proliferation and modulation in the NK cell receptor repertoire expression. Interestingly, we found that MVAHIV-primed NK cells had a significant higher ability to control HIV-1 infection in autologous DC compared to MVAWT-primed NK cells; and this enhanced anti-HIV-1 activity appeared to be HIV-specific as MVAHIV-primed NK cells did not have a better ability to control other viral infections or respond against tumoral cells. Furthermore, we observed that NK cell receptors NKG2D and NKp46 modulate the priming of NK cells. This data provides evidence that in vitro NK cells can be primed by viral vector-infected DC, in the context of a NK/DC culture, to specifically target viral infected cells.