Immunological Profiles of Immune Restoration Disease Presenting
Immunological Profiles of Immune Restoration Disease Presenting
Objectives: A proportion of HIV patients beginning antiretroviral therapy (ART) develop immune restoration disease (IRD). Immunological characteristics of IRD were investigated in a cohort of HIV patients beginning therapy in Kuala Lumpur, Malaysia.
Methods: Peripheral blood mononuclear cells were collected at weeks 0, 6, 12, 24 and 48 of ART from five patients experiencing IRD [two with cryptococcal and three with Mycobacterium tuberculosis (Mtb) disease], eight non-IRD controls who had begun ART with CD4 T-cell counts of <100 cells/µL and 17 healthy controls. Leukocytes producing interferon-gamma (IFNγ) were quantified by enzyme-linked immunospot assay after stimulation with purified protein derivative (PPD), early secretory antigenic target-6 (ESAT-6), Cryptococcus neoformans or Cytomegalovirus antigens. Plasma immunoglobulin (IgG) antibodies reactive with these antigens were assessed by enzyme-linked immunosorbent assay. Proportions of activated (HLA-DR) and regulatory (CD25 CD127 and CTLA-4) CD4 T-cells were quantified by flow cytometry.
Results: Plasma HIV RNA declined and CD4 T-cell counts rose within 8-27 weeks on ART. Mtb IRD patients displayed elevated IFNγ responses and/or plasma IgG to PPD, but none responded to ESAT-6. Cryptococcal IRD occurred in patients with low baseline CD4 T-cell counts and involved clear IFNγ and antibody responses to cryptococcal antigen. Proportions of activated and regulatory CD4 T-cells declined on ART, but remained higher in patients than in healthy controls. At the time of IRD, proportions of activated CD4 T-cells and regulatory CD4 T-cells were generally elevated relative to other patients.
Conclusions: Cryptococcal and Mtb IRD generally coincide with peaks in the proportion of activated T-cells, pathogen-specific IFNγ responses and reactive plasma IgG. IRD does not reflect a paucity of regulatory CD4 T-cells.
Successful antiretroviral therapy (ART) suppresses the replication of HIV and allows the recovery of CD4 T-cell numbers within a few months. However, recovery of antigen-specific CD4 T-cell responses is variable among HIV patients on ART. Approximately 10-40% of patients beginning ART with advanced immunodeficiency experience immune restoration disease (IRD).
Clinically, IRD patients have atypical presentation of disease associated with common opportunistic pathogens such as Mycobacterium avium complex (MAC), Mycobacterium tuberculosis (Mtb), Cytomegalovirus (CMV), Varicella zoster virus (VZV), hepatitis C virus (HCV) or Cryptococcus neoformans. The timing of these events coincides with increases in CD4 T-cell counts on ART, suggesting that restored immune responses against antigens of viable or non-viable pathogens can be immunopathological rather than protective. However, few longitudinal sample sets have been available to characterize these responses.
Bourgarit et al. described dramatic interferon-γ (IFNγ) responses by cells from seven patients with previously treated tuberculosis (TB) who developed Mtb IRD ('paradoxical' IRD). Purified protein derivative (PPD) induced Th1-related cytokines and chemokines, including IL-2, IL-12, IFNγ and IP-10 (CXCL10). Production of TNFα, IL-6, IL-1ß, IL-10, RANTES and MCP-1 by cultured peripheral blood mononuclear cells (PBMCs) in response to PPD and CMV were also elevated at the time of IRD, suggesting broad-based immune activation. Their results are consistent with our finding that MAC IRD reflects restoration of delayed-type hypersensitivity (DTH) responses to mycobacterial antigens. Bourgarit et al. demonstrated that cells from Mtb IRD patients did not respond to secreted mycobacterial proteins such as the early secretory antigenic target (ESAT)-6 and 85B protein. These antigens are only released by viable bacteria, so 'paradoxical' IRD may be induced by residual antigens from treated infections. This requires confirmation.
Elevated humoral immune responses have been associated with IRD. Patients who developed CMV retinitis as an IRD had a steady increase in plasma IgG reactive to CMV antigens in the first year of ART. Similarly, HIV-HCV co-infected patients who developed hepatotoxicity and clinical hepatitis had increased HCV core-specific IgG. Serum antibodies against several mycobacterial antigens (notably PGL-Tb1) mark symptomatic Mtb, but have not been addressed in an IRD. Levels of pathogen-specific antibodies before and during ART may help to predict and/or diagnose IRD, or shed light on pathogenic mechanisms.
In patients with no previous diagnosis of cryptococcal infection, presentations of cryptococcal disease in the context of increased CD4 T-cell counts and decreased plasma HIV RNA on ART are defined as 'unmasking' IRD. They usually present as central nervous system disease (meningitis or focal enhancing mass lesions) early after ART or as lymphadenitis within 15 months on ART. Histological examination of lymph nodes may show granulomatous inflammation, necrosis or suppuration. Cryptococcal IRD patients may have higher baseline HIV-1 RNA levels and initial cerebrospinal fluid (CSF) cryptococcal antigen titres than patients with no IRD. They may also have higher lumbar puncture opening pressures and leukocyte counts than untreated patients with AIDS-related cryptococcal disease. Cellular and humoral immune responses have not been monitored in cryptococcal IRD.
Patients who experienced IRD provoked by CMV, MAC and/or HCV infections retain elevated levels of plasma and cellular markers of immune activation for several years. This includes CCR3, CCR5, plasma interleukin-(IL)-6 and soluble IL-6 receptor (sIL-6R). Natural regulatory CD25CD4 T-cells (T-regs) can suppress the activation, proliferation and production of cytokines by CD4 and CD8 T-cells. Depletion of T-regs from PBMCs of HIV-infected patients increased HIV- and CMV-specific responses by CD4 and CD8 T-cells. Hence a deficiency in T-regs may promote the excessive T-cell responses characteristic of an IRD. However, proportions of T-regs and activated CD4 T-cells are correlated during the first year of ART, so T-reg populations may increase after an IRD as a consequence of immune activation.
In this study, regulatory T-cells were identified as CD4CD25CD127 and CD4CTLA-4 T-cells. Low expression of IL-7 receptor (CD127) with co-expression of CD25 identifies CD4 T-regs expressing the transcription factor FoxP3. These markers differentiate T-regs from activated T-cells, which may also express CD25. CD4 T-cells expressing the inhibitory receptor cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) were assayed because they influence down-regulation of effector T-cell activation.
We conducted a longitudinal study in a cohort of HIV patients beginning ART with a range of opportunistic infections to investigate the immunological characteristics of IRD; specifically pathogen-specific IFNγ responses, IgG antibody responses and the proportions of activated (HLA-DR) and regulatory (CD25CD127 or CTLA-4) CD4 T-cells. This is the first report of the immunology of cryptococcal IRD.
Abstract and Introduction
Abstract
Objectives: A proportion of HIV patients beginning antiretroviral therapy (ART) develop immune restoration disease (IRD). Immunological characteristics of IRD were investigated in a cohort of HIV patients beginning therapy in Kuala Lumpur, Malaysia.
Methods: Peripheral blood mononuclear cells were collected at weeks 0, 6, 12, 24 and 48 of ART from five patients experiencing IRD [two with cryptococcal and three with Mycobacterium tuberculosis (Mtb) disease], eight non-IRD controls who had begun ART with CD4 T-cell counts of <100 cells/µL and 17 healthy controls. Leukocytes producing interferon-gamma (IFNγ) were quantified by enzyme-linked immunospot assay after stimulation with purified protein derivative (PPD), early secretory antigenic target-6 (ESAT-6), Cryptococcus neoformans or Cytomegalovirus antigens. Plasma immunoglobulin (IgG) antibodies reactive with these antigens were assessed by enzyme-linked immunosorbent assay. Proportions of activated (HLA-DR) and regulatory (CD25 CD127 and CTLA-4) CD4 T-cells were quantified by flow cytometry.
Results: Plasma HIV RNA declined and CD4 T-cell counts rose within 8-27 weeks on ART. Mtb IRD patients displayed elevated IFNγ responses and/or plasma IgG to PPD, but none responded to ESAT-6. Cryptococcal IRD occurred in patients with low baseline CD4 T-cell counts and involved clear IFNγ and antibody responses to cryptococcal antigen. Proportions of activated and regulatory CD4 T-cells declined on ART, but remained higher in patients than in healthy controls. At the time of IRD, proportions of activated CD4 T-cells and regulatory CD4 T-cells were generally elevated relative to other patients.
Conclusions: Cryptococcal and Mtb IRD generally coincide with peaks in the proportion of activated T-cells, pathogen-specific IFNγ responses and reactive plasma IgG. IRD does not reflect a paucity of regulatory CD4 T-cells.
Introduction
Successful antiretroviral therapy (ART) suppresses the replication of HIV and allows the recovery of CD4 T-cell numbers within a few months. However, recovery of antigen-specific CD4 T-cell responses is variable among HIV patients on ART. Approximately 10-40% of patients beginning ART with advanced immunodeficiency experience immune restoration disease (IRD).
Clinically, IRD patients have atypical presentation of disease associated with common opportunistic pathogens such as Mycobacterium avium complex (MAC), Mycobacterium tuberculosis (Mtb), Cytomegalovirus (CMV), Varicella zoster virus (VZV), hepatitis C virus (HCV) or Cryptococcus neoformans. The timing of these events coincides with increases in CD4 T-cell counts on ART, suggesting that restored immune responses against antigens of viable or non-viable pathogens can be immunopathological rather than protective. However, few longitudinal sample sets have been available to characterize these responses.
Bourgarit et al. described dramatic interferon-γ (IFNγ) responses by cells from seven patients with previously treated tuberculosis (TB) who developed Mtb IRD ('paradoxical' IRD). Purified protein derivative (PPD) induced Th1-related cytokines and chemokines, including IL-2, IL-12, IFNγ and IP-10 (CXCL10). Production of TNFα, IL-6, IL-1ß, IL-10, RANTES and MCP-1 by cultured peripheral blood mononuclear cells (PBMCs) in response to PPD and CMV were also elevated at the time of IRD, suggesting broad-based immune activation. Their results are consistent with our finding that MAC IRD reflects restoration of delayed-type hypersensitivity (DTH) responses to mycobacterial antigens. Bourgarit et al. demonstrated that cells from Mtb IRD patients did not respond to secreted mycobacterial proteins such as the early secretory antigenic target (ESAT)-6 and 85B protein. These antigens are only released by viable bacteria, so 'paradoxical' IRD may be induced by residual antigens from treated infections. This requires confirmation.
Elevated humoral immune responses have been associated with IRD. Patients who developed CMV retinitis as an IRD had a steady increase in plasma IgG reactive to CMV antigens in the first year of ART. Similarly, HIV-HCV co-infected patients who developed hepatotoxicity and clinical hepatitis had increased HCV core-specific IgG. Serum antibodies against several mycobacterial antigens (notably PGL-Tb1) mark symptomatic Mtb, but have not been addressed in an IRD. Levels of pathogen-specific antibodies before and during ART may help to predict and/or diagnose IRD, or shed light on pathogenic mechanisms.
In patients with no previous diagnosis of cryptococcal infection, presentations of cryptococcal disease in the context of increased CD4 T-cell counts and decreased plasma HIV RNA on ART are defined as 'unmasking' IRD. They usually present as central nervous system disease (meningitis or focal enhancing mass lesions) early after ART or as lymphadenitis within 15 months on ART. Histological examination of lymph nodes may show granulomatous inflammation, necrosis or suppuration. Cryptococcal IRD patients may have higher baseline HIV-1 RNA levels and initial cerebrospinal fluid (CSF) cryptococcal antigen titres than patients with no IRD. They may also have higher lumbar puncture opening pressures and leukocyte counts than untreated patients with AIDS-related cryptococcal disease. Cellular and humoral immune responses have not been monitored in cryptococcal IRD.
Patients who experienced IRD provoked by CMV, MAC and/or HCV infections retain elevated levels of plasma and cellular markers of immune activation for several years. This includes CCR3, CCR5, plasma interleukin-(IL)-6 and soluble IL-6 receptor (sIL-6R). Natural regulatory CD25CD4 T-cells (T-regs) can suppress the activation, proliferation and production of cytokines by CD4 and CD8 T-cells. Depletion of T-regs from PBMCs of HIV-infected patients increased HIV- and CMV-specific responses by CD4 and CD8 T-cells. Hence a deficiency in T-regs may promote the excessive T-cell responses characteristic of an IRD. However, proportions of T-regs and activated CD4 T-cells are correlated during the first year of ART, so T-reg populations may increase after an IRD as a consequence of immune activation.
In this study, regulatory T-cells were identified as CD4CD25CD127 and CD4CTLA-4 T-cells. Low expression of IL-7 receptor (CD127) with co-expression of CD25 identifies CD4 T-regs expressing the transcription factor FoxP3. These markers differentiate T-regs from activated T-cells, which may also express CD25. CD4 T-cells expressing the inhibitory receptor cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) were assayed because they influence down-regulation of effector T-cell activation.
We conducted a longitudinal study in a cohort of HIV patients beginning ART with a range of opportunistic infections to investigate the immunological characteristics of IRD; specifically pathogen-specific IFNγ responses, IgG antibody responses and the proportions of activated (HLA-DR) and regulatory (CD25CD127 or CTLA-4) CD4 T-cells. This is the first report of the immunology of cryptococcal IRD.
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