Andrew Lim

Proportions of circulating T cells with a regulatory cell phenotype increase with HIV-associated immune activation and remain high on antiretroviral therapy

Objective:To examine the relationships between blood CD4 natural regulatory T (Treg) cells, plasma HIV RNA level, CD4 T-cell count and immune activation in untreated HIV-infected patients and immunodeficient patients beginning antiretroviral therapy (ART), using a novel phenotype to define Treg cells (CD25CD127loCD4). Data were compared with established Treg cell markers (FoxP3, CTLA-4 and GITR). Methods:Twenty-nine untreated HIV-infected patients with CD4 T-cell counts of < 300 or > 400/μl were compared in a cross-sectional study and 12 patients beginning combination ART with < 100 CD4 T cells/μl were followed for 1 year on therapy. Three- and four-colour flow cytometry was used to quantitate proportions of Treg cells. Results:In control donors and patients with high CD4 T-cell counts, 28–89% (median 60%) of CD25CD127loCD4 cells were FoxP3, but < 10% expressed GITR or CTLA-4. Immunodeficient patients also had CD4-negative lymphocytes with the phenotype FoxP3CD127lo. Proportions of CD25CD127lo cells and activated (HLA-DRhi) cells in the CD4 T-cell population were increased in patients with low CD4 T cell counts. The proportion of CD25CD127loCD4 T cells correlated positively with plasma HIV RNA level and CD4 T-cell activation, but inversely with CD4 T-cell count. Longitudinal studies of 12 patients receiving ART in two distinct cohorts (Western Australia and Malaysia) showed that the proportion of CD25CD127loCD4 cells decreased slightly over time, but remained above levels seen in non-HIV controls. Conclusions:Proportions of circulating T cells with a regulatory cell phenotype increase with HIV-associated immune activation and remain high after 1 year on ART.

Chemokine Levels and Chemokine Receptor Expression in the Blood and the Cerebrospinal Fluid of HIV-Infected Patients With Cryptococcal Meningitis and Cryptococcosis-Associated Immune Reconstitution Inflammatory Syndrome

BACKGROUND Human immunodeficiency virus-infected patients with treated cryptococcal meningitis who start combination antiretroviral therapy (cART) are at risk of further neurological deterioration, in part caused by paradoxical cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS). We hypothesized that C-IRIS is associated with alterations of chemokine receptor expression on T cells and chemokine concentrations in cerebrospinal fluid (CSF) that enhance recruitment of T-helper 1 cells and/or myeloid cells to the central nervous system. METHODS In a prospective study of 128 human immunodeficiency virus-infected patients with cryptococcal meningitis who received antifungal therapy followed by cART, we examined the proportions of CD4(+) and CD8(+) T cells expressing CCR5 and/or CXCR3, in CSF and whole blood and the concentrations of CXCL10, CCL2, and CCL3 in stored CSF and plasma. RESULTS The proportion of CD4(+) and CD8(+) T cells expressing CXCR3(+)CCR5(+) and the concentrations of CXCL10, CCL2 and CCL3 were increased in CSF compared with blood at cART initiation (P < .0001). Patients with C-IRIS (n = 26), compared with those with no neurological deterioration (n = 63), had higher CSF ratios of CCL2/CXCL10 and CCL3/CXCL10 and higher proportions of CXCR3(+)CCR5(+)CD8(+)T cells in CSF compared with blood at cART initiation (P = .03, .0053, and .02, respectively). CONCLUSION CD8(+) T-cell and myeloid cell trafficking to the central nervous system may predispose patients to C-IRIS.

TLR2-induced cytokine responses may characterize HIV-infected patients experiencing mycobacterial immune restoration disease.

Objectives:Most HIV patients who experience Mycobacterium tuberculosis-associated immune restoration disease (TB IRD) display elevated interferon-gamma (IFN&ggr;) responses against mycobacterial antigens, but these can occur without an IRD. Recognition of mycobacteria-associated molecular patterns through toll-like receptors (TLRs) on dendritic cells and monocytes induces cytokine production. Here, we investigate TLR-induced responses in IRD. Design:Peripheral blood mononuclear cells (PBMCs) were collected at approximately weeks 0, 6, 12, 24 and 48 after antiretroviral therapy from five patients experiencing TB IRD, nine matched non-IRD patients and 15 healthy controls. Methods:IFN&ggr; production by PBMC stimulated with protein purified derivative (PPD) was assessed by ELISpot. TLR2 expression on myeloid dendritic cells (mDCs) and monocytes was assessed by flow cytometry. TNF&agr;, IL-12p40 and IL-10 were measured by ELISA in 24-h cultures of PBMC with lipomannan (mycobacteria-derived TLR2 agonist). Results:TLR2 expression on mDC and monocytes was higher in patients than controls at baseline (P < 0.005). TLR2 expression decreased to normal levels on mDC by week 12, but remained higher on monocytes at week 24 (P = 0.02). At week 24, IRD patients showed higher IFN&ggr; responses to PPD (P = 0.02), TLR2 expression on monocytes (P = 0.006) and lipomannan-induced TNF&agr; production (P = 0.016) than non-IRD patients. Lipomannan-induced TNF&agr; and IL-12p40 responses paralleled TB IRD in the patients with high TLR2 expression. IL-10 levels did not associate with IRD. Conclusion:TLR2-induced pro-inflammatory cytokines by dendritic cells or monocytes may contribute to the pathogenesis of mycobacterial IRD.

Vaccine-induced IgG2 anti-HIV p24 is associated with control of HIV in patients with a 'high-affinity' FcγRIIa genotype

Objectives:We have previously shown that vaccination with a recombinant fowlpox virus carrying the genes for HIV Gag-Pol and interferon-gamma (IFN-γ) was associated with partial control of HIV replication after antiretroviral therapy (ART) was ceased but not with increased anti-HIV T-cell responses. Because IFN-γ enhances IgG2 production, and IgG2 antibodies to HIV antigens and the ‘high-affinity’ polymorphism of FcγRIIa (the major Fc receptor for IgG2) have been associated with a favourable outcome of HIV infection, we examined the association of IgG2 antibodies to HIV p24 and ‘high-affinity’ polymorphisms of FcγRIIa with control of HIV replication in these patients. Methods:Plasma from weeks 0 (cessation of ART 1 week after the last vaccination), 9 and 20 was available from patients who had received the full construct vaccine, a partial construct (without IFN-γ) or placebo. IgG2 and IgG1 anti-p24 and anti-gp41 were assayed and all patients were genotyped for the FcγRIIa 131 R/H polymorphism that affects IgG2 binding. Results:At week 0, IgG2 anti-p24 was present in five of nine full construct patients but none of 14 partial construct or placebo patients and was associated with a smaller increase in plasma HIV RNA over 20 weeks. Patients with IgG2 anti-p24 and the ‘high-affinity’ polymorphism of FcγRIIa exhibited lower HIV replication than other patients at week 20. Conclusion:The role of IgG2 anti-HIV antibodies and FcγRIIa in the control of HIV replication should be investigated further. Inclusion of an IFN-γ gene in DNA vaccine constructs might be a means of enhancing IgG2 antibody production.

Cryptococcosis-IRIS is associated with lower cryptococcus-specific IFN-γ responses before antiretroviral therapy but not higher T-cell responses during therapy.

BACKGROUND Cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS) may be driven by aberrant T-cell responses against cryptococci. We investigated this in human immunodeficiency virus (HIV)-infected patients with treated cryptococcal meningitis (CM) commencing combination antiretroviral therapy (cART). METHODS Mitogen- and cryptococcal mannoprotein (CMP)-activated (CD25+CD134+) CD4+ T cells and -induced production of interferon-gamma (IFN-γ), IL-10, and CXCL10 were assessed in whole blood cultures in a prospective study of 106 HIV-CM coinfected patients. RESULTS Patients with paradoxical C-IRIS (n = 27), compared with patients with no neurological deterioration (no ND; n = 63), had lower CMP-induced IFN-γ production in 24-hour cultures pre-cART and 4 weeks post-cART (P = .0437 and .0257, respectively) and lower CMP-activated CD4+ T-cell counts pre-cART (P = .0178). Patients surviving to 24 weeks had higher proportions of mitogen-activated CD4+ T cells and higher CMP-induced CXCL10 and IL-10 production in 24-hour cultures pre-cART than patients not surviving (P = .0053, .0436 and .0319, respectively). C-IRIS was not associated with higher CMP-specific T-cell responses before or during cART. CONCLUSION Greater preservation of T-cell function and higher CMP-induced IL-10 and CXCL10 production before cART are associated with improved survival while on cART. Lower CMP-induced IFN-γ production pre-cART, but not higher CMP-specific T-cell responses after cART, were risk factors for C-IRIS.

Susceptibility to pulmonary disease due to Mycobacterium avium-intracellulare complex may reflect low IL-17 and high IL-10 responses rather than Th1 deficiency

It remains unclear why some individuals and not others are susceptible to non-tuberculous mycobacterial lung disease (NTMLD). To determine whether NTMLD is associated with defects or biases in Th1/Th2/Th17 immunity, blood leukocytes from NTM patients with nodular bronchiectasis, their adult offspring, and healthy population controls were stimulated with staphylococcal enterotoxin B (SEB), tuberculin and sensitin to measure cytokine production. In response to SEB, NTM patients exhibited higher frequencies of IFNγ-producing CD4(+) T cells than population controls (P<0.001). In supernatant, levels of IL-17 were lower in patients than adult offspring. Sensitin elicited higher IFNγ responses from patients than controls (P<0.05). Patients also produced more IL-10 in supernatant than controls after culture with tuberculin (P<0.01) or sensitin (P<0.05), but IL-10-producing CD4(+) T cells were undetectable. NTMLD is not associated with deficient IFNγ production, but may be associated with reduced Th17 immunity and/or a predisposition towards IL-10 production from non-CD4(+) T cells.

CD4+ and CD8+ T cells expressing FoxP3 in HIV-infected patients are phenotypically distinct and influenced by disease severity and antiretroviral therapy

Objectives:Forkhead box P3 (FoxP3) is critical for the development of CD4+ regulatory T (Treg) cells and is a useful marker to identify this population. Recently, expression of FoxP3 was reported in human CD8+ T cells from the blood of untreated HIV-infected individuals. We assessed whether FoxP3 expression in CD8+ T cells is associated with suppressive potential and/or with HIV-associated immune activation. Methods:FoxP3+CD8+ T cells in non-HIV donors and in untreated and treated HIV-infected patients were identified by flow cytometry, then examined for coexpression of other Treg cell-associated markers [cytotoxic T lymphocyte-associated antigen (CTLA)-4, GITR, and CD45RO], markers of activation [HLA-DRHI, Ki-67, and programmed death (PD)-1], and markers of senescence (CD57 without CD28). Results:Similar proportions of FoxP3-expressing CD4+ and CD8+ T cells coexpressed HLA-DRHI and Ki-67. However, compared with FoxP3+CD4+ cells, FoxP3+CD8+ cells expressed less CTLA-4, CD28, and CD45RO but more PD-1 and CD57. FoxP3-expressing CD4+ and CD8+ cells from untreated patients exhibited higher expression of HLA-DRHI, Ki-67, and PD-1 compared with non-HIV donors and treated patients. Conclusions:FoxP3+CD8+ T cells are phenotypically distinct from FoxP3+CD4+ and FoxP3−CD8+ T cells. Expression of FoxP3 is associated with cellular activation in both CD4+ and CD8+ T cells.

TB-IRIS after initiation of antiretroviral therapy is associated with expansion of preexistent th1 responses against mycobacterium tuberculosis antigens

Background:The role of T-cell responses against Mycobacterium tuberculosis antigens in tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is unclear. Methods:Peripheral blood mononuclear cells from 45 HIV patients with treated TB, of whom 12 developed TB-IRIS, were collected at weeks 0, 2, and 6 of antiretroviral therapy (ART). Production of interferon-gamma (IFN-&ggr;) and interleukin-2 by T cells after stimulation with purified protein derivative (PPD) or early secretory antigenic target-6 (ESAT-6) and T-cell expressions of CCR5 and CXCR3 were assessed by flow cytometry. IFN-&ggr; and CXCL10 were assayed by enzyme-linked immunosorbent assay. Results:TB-IRIS patients had higher proportions of PPD- and ESAT-6–reactive IFN-&ggr;+CD4+ and CD3+CD4− T cells at weeks 0, 2, and 6. IFN-&ggr; levels were also higher in peripheral blood mononuclear cell culture supernatants at all times with PPD but only at weeks 2 and 6 with ESAT-6. There were few differences for interleukin-2. CXCL10 levels in supernatants after PPD and ESAT-6 stimulation were only higher at week 6. CXCR3+/CCR5+CD4+ T cells were higher at week 2, and CCR5+CD4+ T cells were higher at week 6. Conclusions:TB-IRIS is associated with Th1 responses against M. tuberculosis antigens by CD4+ and CD3+CD4− T cells that are present before ART and amplified afterward. It is unclear if these cause immunopathology or reflect a high pathogen load.

Antibody and B cell responses may control circulating lipopolysaccharide in patients with HIV infection.

Objectives:To examine the relationship between plasma markers of microbial translocation and antibodies to lipopolysaccharide (LPS) and circulating memory B cells in patients with HIV infection. Design:Cross-sectional study in antiretroviral therapy (ART)-naive (n = 23) and ART-treated (n = 27) HIV patients. Methods:Antibodies to LPS and immunoglobulins, assayed in stored serum, and matched memory B-cell counts were correlated with levels of LPS and bacterial 16S ribosome DNA (16S rDNA), assayed in stored plasma. Results:In ART-naive patients, plasma LPS levels correlated inversely with serum levels of IgG and IgA antibodies to LPS (P = 0.03 and 0.006, respectively), serum levels of IgA anti-LPS correlated with total IgA (P < 0.0001) and levels of IgG anti-LPS correlated with IgM+ memory B-cell counts (P = 0.025). In ART-treated patients, plasma LPS levels were not related to levels of LPS antibodies, but were related to CD4+ T-cell and switched memory B-cell counts. There were no correlations with plasma levels of 16S rDNA. Conclusion:Plasma LPS levels were associated with antibody and possibly B-cell responses to LPS in ART-naive HIV patients, whereas they were associated with the degree of immune reconstitution in ART-treated patients.

Compartmentalization of innate immune responses in the central nervous system during cryptococcal meningitis/HIV coinfection

Objective:The role of innate immunity in the pathogenesis of cryptococcal meningitis is unclear. We hypothesized that natural killer (NK) cell and monocyte responses show central nervous system (CNS) compartment-specific profiles, and are altered by antifungal therapy and combination antiretroviral therapy (cART) during cryptococcal meningitis/HIV coinfection. Design:Substudy of a prospective cohort study of adults with cryptococcal meningitis/HIV coinfection in Durban, South Africa. Methods:We used multiparametric flow cytometry to study compartmentalization of subsets, CD69 (a marker of activation), CXCR3 and CX3CR1 expression, and cytokine secretion of NK cells and monocytes in freshly collected blood and cerebrospinal fluid (CSF) at diagnosis (n = 23), completion of antifungal therapy induction (n = 19), and after a further 4 weeks of cART (n = 9). Results:Relative to blood, CSF was enriched with CD56bright (immunoregulatory) NK cells (P = 0.0004). At enrolment, CXCR3 expression was more frequent among blood CD56bright than either blood CD56dim (P <0.0001) or CSF CD56bright (P = 0.0002) NK cells. Antifungal therapy diminished blood (P <0.05), but not CSF CXCR3pos NK-cell proportions nor CX3CR1pos NK-cell proportions. CD56bright and CD56dim NK cells were more activated in CSF than blood (P <0.0001). Antifungal therapy induction reduced CD56dim NK-cell activation in CSF (P = 0.02). Activation of blood CD56bright and CD56dim NK cells was diminished following cART commencement (P <0.0001, P = 0.03). Immunoregulatory NK cells in CSF tended to secrete higher levels of CXCL10 (P = 0.06) and lower levels of tumor necrosis factor &agr; (P = 0.06) than blood immunoregulatory NK cells. CSF was enriched with nonclassical monocytes (P = 0.001), but antifungal therapy restored proportions of classical monocytes (P = 0.007). Conclusion:These results highlight CNS activation, trafficking, and function of NK cells and monocytes in cryptococcal meningitis/HIV and implicate immunoregulatory NK cells and proinflammatory monocytes as potential modulators of cryptococcal meningitis pathogenesis during HIV coinfection.