Faculty Bibliography

BACKGROUND:HIV disease progresses more rapidly in children than adults with mortality rates exceeding 50% by 2 years of age without antiretroviral therapy (ART) in sub-Saharan Africa. Recent World Health Organization (WHO) guidelines recommend universal treatment for all living persons with HIV, yet there is limited supporting evidence in pediatric populations. The objective of this study was to determine whether CD4 cell counts reflect immunological markers associated with disease progression in ART naïve perinatally-infected HIV+ children and adolescents and their response to ART. METHODS:PBMC and plasma samples were collected from 71 HIV negative and 132 HIV+ children (65 ART naïve and 67 on ART) between ages 1-19 years from Mombasa, Kenya. Untreated HIV+ subjects were sub-categorized by high or low CD4 T cell counts. Immune activation markers CD38, HLA-DR and Ki67 were analyzed by flow cytometry. Plasma soluble CD14 (sCD14) was quantified by ELISA. RESULTS:HIV-infected children and adolescents with preserved CD4 cell counts had depleted CD4 percentages and CD4:CD8 ratios, and high immune activation levels. ART initiation rapidly and persistently reversed T cell activation, but failed to normalize CD4:CD8 ratios and plasma sCD14 levels. CONCLUSIONS:Diminished CD4 percentages and CD4:CD8 ratios along with profound immune activation occur independent of CD4 cell count thresholds in ART naïve HIV+ children and adolescents. Immediate ART initiation, as recommended in the most recent WHO guidelines may protect them from pathologic sequelae associated with persistent inflammation.

Regulatory T cells (Tregs) are functionally suppressive CD4 T cells, critical for establishing peripheral tolerance and controlling inflammatory responses. Previous reports of Tregs during chronic HIV disease have conflicting results with higher or lower levels compared to controls. Identifying true Tregs with suppressive activity proves challenging during HIV infection, as traditional Treg markers, CD25 and FOXP3, may transiently up-regulate expression as a result of immune activation. Helios is an Ikaros family transcription factor that marks natural Tregs with suppressive activity and does not up-regulate expression after activation. Coexpression of FOXP3 and Helios has been suggested as a highly specific marker of "bona fide" Tregs. We evaluated Treg subsets by FOXP3 co-expressed with either CD25 or Helios and their association with HIV disease progression in perinatally-infected HIV positive children. Identifying Tregs by FOXP3 coexpression with Helios rather than CD25 revealed markedly higher Treg frequencies, particularly in HIV+ children. Regardless of ART, HIV infected children had a selective expansion of memory FOXP3+Helios+ Tregs. The rise in memory Tregs correlated with declining HIV clinical status, indicated by falling CD4 percentages and CD4:CD8 ratios and increasing HIV plasma viremia and immune activation. In addition, untreated HIV+ children exhibited an imbalance between the levels of Tregs and activated T cells. Finally, memory Tregs expressed immune activation markers CD38 and Ki67 and exhaustion marker, PD-1, that tightly correlated with a similar phenotype in memory CD4 T cells. Overall, HIV infected children had significant disruptions of memory Tregs that associated with advancing HIV disease.

Mucosal-associated invariant T cells (MAIT) are innate T cells restricted by major histocompatibility related molecule 1 (MR1) presenting riboflavin metabolite ligands derived from microbes. Specificity to riboflavin metabolites confers MAIT cells a broad array of host-protective activity against gram-negative and -positive bacteria, mycobacteria, and fungal pathogens. MAIT cells are present at low levels in the peripheral blood of neonates and gradually expand to relatively abundant levels during childhood. Despite no anti-viral activity, MAIT cells are depleted early and irreversibly in HIV infected adults. Such loss or impaired expansion of MAIT cells in HIV-positive children may render them more susceptible to common childhood illnesses and opportunistic infections. In this study we evaluated the frequency of MAIT cells in perinatally HIV-infected children, their response to antiretroviral treatment and their associations with HIV clinical status and related innate and adaptive immune cell subsets with potent antibacterial effector functions. We found HIV+ children between ages 3 to 18 years have significantly decreased CD8+ MAIT cell frequencies compared to uninfected healthy children. Remarkably, CD8 MAIT levels gradually increased with antiretroviral therapy, with greater recovery when treatment is initiated at a young age. Moreover, diminished CD8+ MAIT cell frequencies are associated with low CD4:CD8 ratios and elevated sCD14, suggesting a link with HIV disease progression. Last, CD8+ MAIT cell levels tightly correlate with other antibacterial and mucosa-protective immune subsets, namely, neutrophils, innate-like T cells, and Th17 and Th22 cells. Together these findings suggest that low frequencies of MAIT cells in HIV positive children are part of a concerted disruption to the innate and adaptive immune compartments specialized in sensing and responding to pathogenic or commensal bacteria.

Background: A subset of T cells comprised of mucosal associated invariant T (MAIT), natural killer T (NKT), and gammadelta T cells exhibit features of innate cells such as invariant TCR and recognition of non-peptide antigens. These unconventional T cell subsets can secrete pro-inflammatory cytokines or display cytotoxic activity in response to bacterial and fungal derived glycolipids or metabolites. HIV+ adults have lower MAIT and NKT and higher gammadelta T cells in the peripheral blood. Less is known about their changes in HIV-infected children. We sought to determine whether innate-like T cells are disrupted in treated and untreated HIV+ children. Methods: We evaluated peripheral blood samples of 76 perinatally-infected HIV+ and 40 HIV- children between 3-12 years old from Mombasa, Kenya. The HIV+ cohort included 39 ART naive (ART-) and 37 children on ART (ART+). Cryopreserved PBMCs were thawed and stained with surface antibodies CD3, CD4 and CD8 with Valpha7.2 and CD161 (MAIT), gammadelta TCR, and Valpha24Jalpha18 TCR (NKT) then analyzed by flow cytometry. Plasma sCD14 was measured by ELISA. Statistical analysis was performed on GraphPad Prism with Mann-Whitney or Spearman's correlation tests. Results: HIV+ children had decreased levels of MAIT in CD8+ T cells (ART- p=0.0156; ART+ p=0.0137), which correlated positively with CD4:CD8 ratios (r=0.37, p=0.0018). ART+ had lower MAIT levels in CD4+ T cells (p=0.0090); both HIV+ groups maintained CD4-CD8- MAIT cell frequencies and MAIT cell numbers in total T cells. Plasma sCD14 levels were higher in HIV+ (p=0.0068). In HIV+ children CD8+ MAIT cells inversely correlated with sCD14 (r= -0.33, p=0.0049). NKT cells were also lower in ART- (p=0.0001) and ART+ (p=0.0140) and correlated with CD4:CD8 (r=0.25, p=0.0417). Conversely, gammadelta T cells were persistently elevated in HIV+ (ART- p=0.0064; ART+ p<0.0001) and correlated positively with sCD14 (r=0.36, p=0.0025). Interestingly, in HIV+ children gammadelta T cells negatively correlated with CD8+ MAIT (r= -0.36, p=0.0024) and NKT (r= -0.27, p=0.0063) cells and the ratio of gammadeltaT/MAIT cells positively correlated with sCD14 (r=.0.37, p=0.0019). Conclusions: CD8+ MAIT and NKT cells are relatively lower in HIV+ children despite ART compared to HIV- children. This decline in innate-like T cells worsens with diminished immune status. Loss of MAIT cells from the periphery may reflect migration to mucosal tissues in response to microbial translocation. Increases in gammadelta T cells may be a compensatory response to decreased MAIT and NKT cells in HIV infected children

A subset of human regulatory T cells (Tregs) secretes IL-17 and thus resembles Th17 effector cells. How IL-17+ Tregs differentiate from naive precursors remains unclear. In this study, we show that IL-17-producing T cells can differentiate from CCR6+ naive T cell precursors in the presence of IL-2, IL-1beta, TGF-beta, and IL-23. CCR6+ naive T cells are present in adult peripheral and umbilical cord blood and in both conventional T naive and FOXP3+ naive Treg subsets. IL-17+ cells derived from CCR6+ naive Tregs (referred to as IL-17+ Tregs) express FOXP3 but not HELIOS, another Treg-associated transcription factor, and these cells display suppressor capacity and a surface phenotype resembling memory Tregs. Remarkably, the IL-17+ Treg compartment was preferentially reduced relative to the canonical Th17 and Treg compartments in a subset of HIV+ subjects, suggesting a specific perturbation of this subset during the course of disease. Our findings that CCR6+ naive precursors contain a predetermined reservoir to replenish IL-17-secreting cells may have implications in balancing the Th17 and IL-17+ Treg compartments that are perturbed during HIV infection and potentially in other inflammatory diseases.

Background: Regulatory T cells (Tregs) mediate immune tolerance during autoimmune disease and chronic infections. During HIV infection, Tregs may act either beneficially to curb immune activation or pathologically to suppress HIV-specific immune responses. Previous reports of Tregs during chronic HIV have conflicting results with higher or lower levels compared to controls. Identifying true Tregs with suppressive activity proves challenging during HIV infection, as traditional Treg markers, CD25 and FOXP3, may transiently upregulate expression as a result of immune activation. Helios is a recently identified transcription factor that marks natural Tregs with suppressive activity. Moreover FOXP3+Helios+ CD4 T cells do not produce the cytokine IL-17, and have been called "bona fide" Tregs. We sought to identify these bona fide Tregs in vertically infected HIV positive children. Methodology: We evaluated Treg levels by flow cytometry in the peripheral blood of 60 children from Bomu Hospital in Mombasa, Kenya. The cohort included age-matched children between 3 to 12 years old in the following categories: HIV negative (HIV-), HIV positive antiretroviral therapy naive (ART-), and HIV positive on antiretroviral therapy (ART+). Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved from each subject. Thawed PBMCs were stained for surface antibodies CD3, CD4, CD25, CD38, CD45RO, and intracellular transcription factors FOXP3 and Helios. All statistical analysis was performed with GraphPad Prism software using Mann-Whitney or Spearman's correlation tests. Results: HIV+ children (ART- and ART+) expressed higher levels of FOXP3 and Helios in CD4 T cells compared with HIV- controls (ART-: p=0.0012, ART+: p=0.0057). FOXP3+Helios+ expression inversely correlated with the percent of CD4 T cells (p<0.0001, r= -0.4883), despite nearly normal CD4 levels in ART+ children. As previously reported, HIV infected children had higher immune activation as measured by CD38+HLA-DR+ expression on CD8+ T c!

Vitamin D deficiency is common in HIV-infected populations. In resource-limited settings, vitamin D deficiency has been shown to affect HIV disease progression and mortality in pregnant women, and also increases mother-to-child HIV transmission and mortality in their infants. This study sought to investigate vitamin D status in HIV-infected women compared to healthy controls in a high-income country setting and determine variables associated with vitamin D deficiency. We prospectively enrolled 40 women/infant pairs (16 HIV-infected women/HIV-exposed infant pairs and 24 uninfected/unexposed pairs). In serum cord blood, 25-hydroxyvitamin D [25(OH)D] concentrations were suboptimal (<30 ng/ml) in 100% of subjects from both groups. White race, non-Hispanic ethnicity was the only variable associated with higher serum 25(OH)D concentrations. This high prevalence of vitamin D deficiency, especially among HIV-infected women and their infants, deserves further investigation, as it may have a negative impact on maternal and infant health.

Chronic immune activation is a hallmark of HIV infection, yet the underlying triggers of immune activation remain unclear. Persistent antigenic stimulation during HIV infection may also lead to immune exhaustion, a phenomenon in which effector T cells become dysfunctional and lose effector functions and proliferative capacity. Several markers of immune exhaustion, such as PD-1, LAG-3, Tim-3, and CTLA-4, which are also negative regulators of immune activation, are preferentially upregulated on T cells during HIV infection. It is not yet clear whether accumulation of T cells expressing activation inhibitory molecules is a consequence of general immune or chronic HIV-specific immune activation. Importantly, however, in vitro blockade of PD-1 and Tim-3 restores HIV-specific T-cell responses, indicating potential for immunotherapies. In this review we discuss the evolution of our understanding of immune exhaustion during HIV infection, highlighting novel markers and potential therapeutic targets

Background. Identification of the Th17 T cell subset as important mediators of host defense and pathology prompted us to determine their susceptibility to human immunodeficiency virus (HIV) infection. Methods and results. We found that a sizeable portion of Th17 cells express HIV coreceptor CCR5 and produce very low levels of CCR5 ligands macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. Accordingly, CCR5(+) Th17 cells were efficiently infected with CCR5-tropic HIV and were depleted during viral replication in vitro. Remarkably, HIV-infected individuals receiving treatment had significantly reduced Th17 cell counts, compared with HIV-uninfected subjects, regardless of viral load or CD4 cell count, whereas treatment-naive subjects had normal levels. However, there was a preferential reduction in CCR5(+) T cells that were also CCR6 positive, which is expressed on all Th17 cells, compared with CCR6(-)CCR5(+) cells, in both treated and untreated HIV-infected subjects. This observation suggests preferential targeting of CCR6(+)CCR5(+) Th17 cells by CCR5-tropic viruses in vivo. Th17 cell levels also inversely correlated with activated CD4(+) T cells in HIV-infected individuals who are receiving treatment. Conclusions. Our findings suggest a complex perturbation of Th17 subsets during the course of HIV disease potentially through both direct viral infection and virus indirect mechanisms, such as immune activation