Comprehensive evaluation of the immune risk phenotype in successfully treated HIV-infected individuals

Abstract

Background: Despite 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.

Methods: 18 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.

Results: Compared 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).

Conclusions: Our 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.

Fig 1. Relationship between the Immune Risk Phenotype (IRP) and Staphylococcus Enterotoxin B induced pro-inflammatory cytokine secretion.

Percentages of IL-2, IFN-γ or TNF-α producing T-cells in response to Staphylococcus Enterotoxin B (SEB) stimulation within the CD4+ (A) and CD8+ (B) T-cell compartments. Comparisons were made between uninfected controls (blue), HIV+IRP+ (purple) and HIV+IRP+ (red) subjects. Statistical significance was determined using the Mann–Whitney U test. *p<0.05, **p<0.01, ***p <0.001.

Fig 2. Relationship between the IRP and proliferative capacity.

The percentage of carboxyfluorescein succinimidyl ester (CFSE) positive cells was used to assess the proliferative capacity of lymphocytes after stimulation with phytohemagglutinin, (PHA), anti-CD3 and-CD28, Pokeweed mitogen and Tetanus toxoid. Comparisons were made between uninfected controls (blue), HIV+IRP- (purple) and HIV+IRP+ (red). Statistical significance was determined using the Mann–Whitney U test. *p<0.05, **p<0.01, ***p <0.001.

Fig 3. Relationship between the IRP and T-cell subset distribution.

Percentages of naive (CD45RA+ CD28+CCR7+), central memory (CD45RA-CD28+CCR7+), effector memory (CD45RA- CD28-CCR7-) and terminally differentiated effector memory (TEMRA) (CD45RA+CD28- CCR7-) T-cells were assessed within the CD4+ (A) and CD8+ (B) T-cell compartments. Comparisons were made between the frequency of these subsets in uninfected controls (blue), HIV+IRP- (purple) and HIV+IRP+ (red). Statistical significance was determined using the Mann–Whitney U test. *p<0.05, **p<0.01, ***p <0.001.

Fig 4. Relationship between the IRP and markers of replicative senescence.

The frequency of CD4+ (A) and CD8+ (B) T-cells expressing a both CD57 and KLRG1 was measured and compared in uninfected controls (blue), HIV+IRP- (purple) and HIV+IRP+ (red) sunjects. Statistical significance was determined using the Mann–Whitney U test. *p<0.05, **p<0.01, ***p <0.001.

Fig 5. Relationship between the IRP and lymphocyte telomere length.

Average telomere length (aTL) was measured in kilobases (kb) per diploid genome. Lymphocyte aTL comparisons were made between uninfected controls (blue), HIV+IRP- (purple) and HIV+IRP+ (red). Statistical significance was determined using the Mann–Whitney U test. *p<0.05, **p<0.01, ***p <0.001.