Previously, we showed that Killer Immunoglobulin-like Receptor (KIR)3DS1 homozygotes (hmz) are more frequent in HIV exposed seronegative (HESN) than in recently HIV infected (HIV+) individuals. KIR3DS1 encodes an activating Natural Killer (NK) cell receptor (NKR). The link between KIR genotype and HIV outcomes likely arises from the function that NK cells acquire through expression of particular NKRs. An initial screen of 97 HESN and 123 HIV+ subjects for the frequency of KIR region gene carriage observed between-group differences for several telomeric KIR region loci. In a larger set of up to 106 HESN and 439 HIV+ individuals, more HESN than HIV+ subjects were KIR3DS1 homozygotes, lacked a full length KIR2DS4 gene and carried the telomeric group B KIR haplotype motif, TB01. TB01 is characterized by the presence of KIR3DS1, KIR2DL5A, KIR2DS3/5 and KIR2DS1, in linkage disequilibrium with each other. We assessed which of the TB01 encoded KIR gene products contributed to NK cell responsiveness by stimulating NK cells from 8 HIV seronegative KIR3DS1 and TB01 motif homozygotes with 721.221 HLA null cells and evaluating the frequency of KIR3DS1+/-KIR2DL5+/-, KIR3DS1+/-KIR2DS1+/-, KIR3DS1+/-KIR2DS5+/- NK cells secreting IFN-γ and/or expressing CD107a. A higher frequency of NK cells expressing, versus not, KIR3DS1 responded to 721.221 stimulation. KIR2DL5A+, KIR2DS1+ and KIR2DS5+ NK cells did not contribute to 721.221 responses or modulate those by KIR3DS1+ NK cells. Thus, of the TB01 KIR gene products, only KIR3DS1 conferred responsiveness to HLA-null stimulation, demonstrating its ligation can activate ex vivo NK cells

The engagement of activating NK receptors (aNKR) stimulates NK cell activity, provided that interactions between inhibitory NK receptors (iNKR) with their HLA ligands do not override them. Abs bound to target cells can also activate NK cells by engaging the CD16 aNKR. NK cell education status is an important factor for Ab‐dependent NK cell activation (ADNKA) of some NK cell subsets. However, whether NK cell education also influences Ab‐dependent cellular cytotoxicity (ADCC) levels is not fully known. ADCC‐GranToxiLux (GTL) assays measured ADCC activity as the frequency of granzyme B positive (%GzB+) target cells. Target cells were anti‐HIV Immunoglobulin G (HIVIG)‐opsonized CEM‐NKr.CCR5 (CEM) cells. Lymphocytes and sorted single positive (SP) NKG2A+, KIR2DL1+, KIR2DL3+, and KIR3DL1+ NK cells, to self‐ and nonself HLA, were used as effectors in ADCC‐GTL assays to examine how education status influenced ADCC activity. ADNKA activity was assessed by stimulating lymphocytes with HIVIG‐opsonized CEMs and measuring the frequency of NK cell populations defined by their expression of iNKRs, along with IFN‐γ, CCL4, and CD107a functions. ADCC: the %GzB+ CEM cells generated by self‐ versus nonself HLA‐specific SPiNKR did not differ. ADNKA: More NK cells educated through KIR2DL1 and KIR3DL1, but not KIR2DL3, responded to ADNKA than their uneducated counterparts. CD16 engagement induced ADCC and ADNKA activity. With the proviso that groups’ sizes were small, our results support the notion that NK cell education does not influence ADCC levels but does contribute to ADNKA activity.

BackgroundDespite successful treatment and CD4+ T-cell recovery, HIV-infected individuals often experience a profound immune dysregulation characterized by a persistently low CD4:CD8 T-cell ratio. This residual immune dysregulation is reminiscent of the Immune Risk Phenotype (IRP) previously associated with morbidity and mortality in the uninfected elderly (>85 years). The IRP consists of laboratory markers that include: a low CD4:CD8 T-cell ratio, an expansion of CD8+CD28- T-cells and cytomegalovirus (CMV) seropositivity. Despite the significant overlap in immunological phenotypes between normal aging and HIV infection, the IRP has never been evaluated in HIV-infected individuals. In this pilot study we characterized immune changes associated with the IRP in a sample of successfully treated HIV-infected subjects.Methods18 virologically suppressed HIV-infected subjects were categorized into 2 groups based on their IRP status; HIV+IRP+, (n = 8) and HIV+IRP-, (n = 10) and compared to 15 age-matched HIV uninfected IRP negative controls. All individuals were assessed for functional and phenotypic immune characteristics including: pro-inflammatory cytokine production, antigen-specific proliferation capacity, replicative senescence, T-cell differentiation and lymphocyte telomere length.ResultsCompared to HIV-infected subjects without an IRP, HIV+IRP+ subjects exhibited a higher frequency of TNF-α-producing CD8+ T-cells (p = 0.05) and a reduced proportion of CD8+ naïve T-cells (p = 0.007). The IRP status was also associated with a marked up-regulation of the replicative senescence markers CD57 and KLGR1, on the surface of CD8+T-cells (p = 0.004). Finally, HIV+IRP+ individuals had a significantly shorter mean lymphocyte telomere length than their non-IRP counterparts (p = 0.03).ConclusionsOur findings suggest that, despite similar levels of treatment-mediated viral suppression, the phenotypic and functional immune characteristics of HIV+IRP+ individuals are distinct from those observed in non-IRP individuals. The IRP appears to identify a subset of treated HIV-infected individuals with a higher degree of immune senescence.

NKG2C is an activating NK cell receptor encoded by a gene having an unexpressed deletion variant. Cytomegalovirus (CMV) infection expands a population of NKG2C + NK cells with adaptive-like properties. Previous reports found that carriage of the deleted NKG2C - variant was more frequent in people living with HIV (PLWH) than in HIV - controls unexposed to HIV. The frequency of NKG2C + NK cells positively correlated with HIV viral load (VL) in some studies and negatively correlated with VL in others. Here, we investigated the link between NKG2C genotype and HIV susceptibility and VL set point in PLWH. NKG2C genotyping was performed on 434 PLWH and 157 HIV exposed seronegative (HESN) subjects. Comparing the distribution of the three possible NKG2C genotypes in these populations revealed that the frequency of NKG2C +/+ and NKG2C +/- carriers did not differ significantly between PLWH and HESN subjects, while that of NKG2C -/- carriers was higher in PLWH than in HESNs, in which none were found (p=0.03, χ 2 test). We were unable to replicate that carriage of at least 1 NKG2C - allele was more frequent in PLWH. Information on the pre-treatment VL set point was available for 160 NKG2C +/+ , 83 NKG2C +/- and 6 NKG2C -/- PLWH. HIV VL set point was similar between NKG2C genotypes. The frequency of NKG2C + CD3 - CD14 - CD19 - CD56 dim NK cells and the mean fluorescence intensity (MFI) of NKG2C expression on NK cells was higher on cells from CMV + PLWH who carried 2, versus 1, NKG2C + alleles. We observed no correlations between VL set point and either the frequency or the MFI of NKG2C expression. IMPORTANCE We compared NKG2C allele and genotype distributions in subjects who remained HIV uninfected despite multiple HIV exposures (HESNs) with those in PLWH. This allowed us to determine whether NKG2C genotype influenced susceptibility to HIV infection. The absence of the NKG2C -/- genotype among HESN subjects but not PLWH suggested that carriage of this genotype was associated with HIV susceptibility. We calculated the VL set point in a subset of 252 NKG2C genotyped PLWH. We observed no between-group differences in the VL set point in carriers of the three possible NKG2C genotypes. No significant correlations were seen between the frequency or MFI of NKG2C expression on NK cells with VL set point in cytomegalovirus co-infected PLWH. These findings suggested that adaptive NK cells played no role in establishing the in VL set point, a parameter that is a predictor of the rate of treatment-naïve HIV disease progression.

Objective. During the course of HIV infection, progressive immune deficiency occurs. The aim of this prospective substudy was to evaluate the recovery of functional immunity in a subset of patients from the GRACE (Gender, Race, And Clinical Experience) study treated with a DRV/r-based regimen. Methods. The recovery of functional immunity with a darunavir/ritonavir-based regimen was assessed in a subset of treatment-experienced, HIV-1 infected patients from the GRACE study. Results. 19/32 patients (59%) enrolled in the substudy were virologically suppressed (<50 copies/mL). In these patients, median (range) CD4+ cell count increased from 222 (2, 398) cells/mm3 at baseline to 398 (119, 812) cells/mm3 at Week 48. CD8+% decreased significantly from baseline to Week 48 (P = .03). Proliferation of CD4+ lymphocytes in response to CD3+/CD28+, phytohemagglutinin, and pokeweed was significantly increased (P < .01) by Week 12. Proliferation in response to Candida and tetanus was significantly increased by Week 48 (P < .01 and P = .014, resp.). Staphylococcal enterotoxin B-stimulated tumor necrosis factor-alpha and interleukin-2 in CD4+ cells was significantly increased by Week 12 (P = .046) and Week 48 (P < .01), respectively. Conclusions. Darunavir/ritonavir-based therapy demonstrated improvements in CD4+ cell recovery and association with progressive functional immune recovery over 48 weeks. This trial is registered with NCT00381303.

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Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV+ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV+PLWH and CMV+HIV- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV+PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons.

Background People living with HIV (PLHIV) who have low CD4 counts require advanced clinical care (ACC) to minimise morbidity and mortality risk. These patients include immunological non-responders (INRs) with low CD4 counts despite a suppressed viral load. Objectives To determine the proportion of patients with low CD4 counts after antiretroviral therapy (ART) initiation and to describe INRs within that group. Methods Routine Three Interlinked Electronic Registers.Net (TIER.Net) data from four South African districts were analysed for adult PLHIV on ART > 12 months. Immunological non-responders were defined as patients on ART > 4 years who were virally suppressed (viral load < 1000 copies/mL) with a CD4 count ≤ 350 cell/mm 3 . Results Baseline CD4 was recorded for 80.9% of the 869 571 patients newly initiating ART, with 37.2% of those starting ART since 2017 having baseline counts ≤ 200 cells/mm 3 . Amongst all 1 178 190 patients on ART, only 46.5% had a CD4 test after ART initiation and of these, 14.3% had CD4 ≤ 200 cells/mm 3 . This proportion was highest amongst patients on ART ≤ 2 years (19.7%) ( p < 0.001). Amongst virally suppressed patients, 20.0% were INRs. Immunological non-response was significantly more likely amongst patients on second-line ART (adjusted odds ratio [aOR] 1.79), those aged 35-45 and ≥ 45 years (aOR 1.15 and 1.50, respectively), males (aOR 2.28) and patients with confirmed TB (aOR 2.49), and was significantly less likely in cases with higher baseline CD4 count (aOR 0.35). Conclusion CD4 testing subsequent to ART initiation is poorly implemented and there is a notable proportion of patients with low CD4 counts. Guidelines regarding CD4 testing and ACC need to be more widely implemented to identify patients with low CD4 counts and improve their outcomes.