Denise S. Rodrigues

CD8+ T Cells from SIV Elite Controller Macaques Recognize Mamu-B*08-Bound Epitopes and Select for Widespread Viral Variation

Background It is generally accepted that CD8+ T cell responses play an important role in control of immunodeficiency virus replication. The association of HLA-B27 and -B57 with control of viremia supports this conclusion. However, specific correlates of viral control in individuals expressing these alleles have been difficult to define. We recently reported that transient in vivo CD8+ cell depletion in simian immunodeficiency virus (SIV)-infected elite controller (EC) macaques resulted in a brief period of viral recrudescence. SIV replication was rapidly controlled with the reappearance of CD8+ cells, implicating that these cells actively suppress viral replication in ECs. Methods and Findings Here we show that three ECs in that study made at least seven robust CD8+ T cell responses directed against novel epitopes in Vif, Rev, and Nef restricted by the MHC class I molecule Mamu-B*08. Two of these Mamu-B*08-positive animals subsequently lost control of SIV replication. Their breakthrough virus harbored substitutions in multiple Mamu-B*08-restricted epitopes. Indeed, we found evidence for selection pressure mediated by Mamu-B*08-restricted CD8+ T cells in all of the newly identified epitopes in a cohort of chronically infected macaques. Conclusions Together, our data suggest that Mamu-B*08-restricted CD8+ T cell responses effectively control replication of pathogenic SIVmac239. All seven regions encoding Mamu-B*08-restricted CD8+ T cell epitopes also exhibit amino acid replacements typically seen only in the presence of Mamu-B*08, suggesting that the variation we observe is indeed selected by CD8+ T cell responses. SIVmac239 infection of Indian rhesus macaques expressing Mamu-B*08 may therefore provide an animal model for understanding CD8+ T cell-mediated control of HIV replication in humans.

The Antiviral Efficacy of Simian Immunodeficiency Virus-Specific CD8+ T Cells Is Unrelated to Epitope Specificity and Is Abrogated by Viral Escape

ABSTRACT CD8+ T lymphocytes appear to play a role in controlling human immunodeficiency virus (HIV) replication, yet routine immunological assays do not measure the antiviral efficacy of these cells. Furthermore, it has been suggested that CD8+ T cells that recognize epitopes derived from proteins expressed early in the viral replication cycle can be highly efficient. We used a functional in vitro assay to assess the abilities of different epitope-specific CD8+ T-cell lines to control simian immunodeficiency virus (SIV) replication. We compared the antiviral efficacies of 26 epitope-specific CD8+ T-cell lines directed against seven SIV epitopes in Tat, Nef, Gag, Env, and Vif that were restricted by either Mamu-A*01 or Mamu-A*02. Suppression of SIV replication varied depending on the epitope specificities of the CD8+ T cells and was unrelated to whether the targeted epitope was derived from an early or late viral protein. Tat28-35SL8- and Gag181-189CM9-specific CD8+ T-cell lines were consistently superior at suppressing viral replication compared to the other five SIV-specific CD8+ T-cell lines. We also investigated the impact of viral escape on antiviral efficacy by determining if Tat28-35SL8- and Gag181-189CM9-specific CD8+ T-cell lines could suppress the replication of an escaped virus. Viral escape abrogated the abilities of Tat28-35SL8- and Gag181-189CM9-specific CD8+ T cells to control viral replication. However, gamma interferon (IFN-γ) enzyme-linked immunospot and IFN-γ/tumor necrosis factor alpha intracellular-cytokine-staining assays detected cross-reactive immune responses against the Gag escape variant. Understanding antiviral efficacy and epitope variability, therefore, will be important in selecting candidate epitopes for an HIV vaccine.

Immunophenotypic characterization of peripheral T lymphocytes in Mycobacterium tuberculosis infection and disease

The cellular immune response probably plays a pivotal role in determining the clinical outcome after exposure to Mycobacterium tuberculosis. We used multi‐parameter flow‐cytometry to evaluate the distribution of T‐lymphocyte subsets during infection and disease caused by M. tuberculosis. Samples were obtained from 71 volunteers to identify the T CD4+ and CD8+ lymphocyte numbers, and the activation plus memory/naïve phenotypes, as defined by CD38, HLA‐DR, CD45RA and CD27 markers. Subjects were divided into 18 healthy volunteers without detectable reaction to purified protein derivative (PPD−), 18 health care workers with a recent conversion to PPD, 20 patients with active pulmonary tuberculosis (TBC) and 15 patients with treated TBC at 6 months of therapy. By multiple‐comparison analyses, the T CD4+ lymphocyte number of the TBC group was lower than the PPD– group (P < 0·05). This difference was apparently lost after treatment. The higher and the lower number of naïve T CD4+ cells was observed in the PPD– and TBC group, respectively. CD8+ T lymphocytes were also statistically different among the four groups (P = 0·0002), lower in the TBC group (P < 0·05). CD8+ T lymphocyte activation was evaluated by the CD38 and HLA‐DR surface expression. The percentage distribution of these markers was statistically different between the four groups (P = 0·0055). TBC patients had a higher percentage of CD38+ cells and mean fluorescence index, suggesting an overall increase of cell activation. These results suggest that peripheral T lymphocytes reflect cellular activation during TBC, along with possible redistribution of naïve, memory/effector and late differentiated memory/effector phenotypes in the peripheral blood after infection and disease caused by M. tuberculosis.

Neuroinvasion of Fluorescein-Positive Monocytes in Acute Simian Immunodeficiency Virus Infection

ABSTRACT Monocytes and macrophages play a central role in the pathogenesis of human immunodeficiency virus (HIV)-associated dementia. They represent prominent targets for HIV infection and are thought to facilitate viral neuroinvasion and neuroinflammatory processes. However, many aspects regarding monocyte brain recruitment in HIV infection remain undefined. The nonhuman primate model of AIDS is uniquely suited for examination of the role of monocytes in the pathogenesis of AIDS-associated encephalitis. Nevertheless, an approach to monitor cell migration from peripheral blood into the central nervous system (CNS) in primates had been lacking. Here, upon autologous transfer of fluorescein dye-labeled leukocytes, we demonstrate the trafficking of dye-positive monocytes into the choroid plexus stromata and perivascular spaces in the cerebra of rhesus macaques acutely infected with simian immunodeficiency virus between days 12 and 14 postinfection (p.i.). Dye-positive cells that had migrated expressed the monocyte activation marker CD16 and the macrophage marker CD68. Monocyte neuroinvasion coincided with the presence of the virus in brain tissue and cerebrospinal fluid and with the induction of the proinflammatory mediators CXCL9/MIG and CCL2/MCP-1 in the CNS. Prior to neuroinfiltration, plasma viral load levels peaked on day 11 p.i. Furthermore, the numbers of peripheral blood monocytes rapidly increased between days 4 and 8 p.i., and circulating monocytes exhibited increased functional capacity to produce CCL2/MCP-1. Our findings demonstrate acute monocyte brain infiltration in an animal model of AIDS. Such studies facilitate future examinations of the migratory profile of CNS-homing monocytes, the role of monocytes in virus import into the brain, and the disruption of blood-cerebrospinal fluid and blood-brain barrier functions in primates.

Individuals with Pulmonary Tuberculosis Have Lower Levels of Circulating CD1d-Restricted NKT Cells

Mycobacterium tuberculosis (MTB) is a leading cause of mortality worldwide from an infectious agent. Natural killer T (NKT) cells recognize mycobacterial antigens and contribute to anti-MTB immunity in mouse models. NKT cells were measured in subjects with pulmonary tuberculosis, MTB-exposed individuals, and healthy controls. NKT cell levels are selectively lower in peripheral blood mononuclear cells from individuals with pulmonary tuberculosis than in both MTB-exposed subjects and healthy control subjects. This apparent loss of NKT cells from the peripheral blood is sustained during the 6 months after the initiation of MTB treatment. These findings indicate that NKT cells may be an important component of antituberculosis immunity.

Distribution of naive and memory/effector CD4+ T lymphocytes and expression of CD38 on CD8+ T lymphocytes in AIDS patients with tuberculosis

CD(4)(+) and CD(8)(+) T lymphocyte counts, naive and memory/effector CD(4)(+) T subpopulations, and the expression of CD(38) on CD(8)(+) T lymphocytes were evaluated in four groups: AIDS patients with tuberculosis (HIV/TB, n=14), HIV-1 infected patients (HIV, n=10), HIV-1 negative patients with tuberculosis (TB, n=20) and healthy controls (CTL, n=17). TB and HIV had fewer +CD(4)(+) T cells than CTL, with the lowest values observed in TB/HIV (p<0.001). No difference between groups was observed in the percentage of naive and memory/effector subpopulations in +CD(4)(+) T lymphocytes. TB (355 cells/ micro L) and HIV (517 cells/ micro L) had diverging effects on +CD(8)(+) T cell counts, with a marked depletion observed in HIV/TB (196 cells/ micro L). TB and HIV up-regulated CD(38) expression on CD(8)(+) T cells, a finding also present in TB/HIV. While the decrease of +CD(4)(+) T cell counts in HIV/TB may be attributed to HIV and tuberculosis, the decrease of +CD(8)(+) T cell counts is likely to be due to tuberculosis.

Distinct Chemokine Triggers and In Vivo Migratory Paths of Fluorescein Dye-Labeled T Lymphocytes in Acutely Simian Immunodeficiency Virus SIVmac251-Infected and Uninfected Macaques

ABSTRACT To define the possible impact of T-lymphocyte trafficking parameters on simian immunodeficiency virus (SIV) pathogenesis, we examined migratory profiles of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T lymphocytes in acutely SIVmac251-infected and uninfected macaques within 48 h after autologous transfer. Despite significant upregulation of homeostatic chemokine CCL19/macrophage inflammatory protein 3β and proinflammatory chemokine CXCL9/monokine induced by gamma interferon in secondary lymphoid tissue in SIV infection, no differences in CFSE+ T-lymphocyte frequencies or cell compartmentalization in lymph nodes were identified between animal groups. By contrast, a higher frequency of CFSE+ T lymphocytes in the small intestine was detected in acute SIV infection. This result correlated with increased numbers of gut CD4 T lymphocytes expressing chemokine receptors CCR9, CCR7, and CXCR3 and high levels of their respective chemokine ligands in the small intestine. The changes in trafficking parameters in SIV-infected macaques occurred concomitantly with acute gut CD4 T-lymphocyte depletion. Here, we present the first in vivo T-lymphocyte trafficking study in SIV infection and a novel approach to delineate T-lymphocyte recruitment into tissues in the nonhuman primate animal model for AIDS. Such studies are likely to provide unique insights into T-lymphocyte sequestration in distinct tissue compartments and possible mechanisms of CD4 T-lymphocyte depletion and immune dysfunction in simian AIDS.

Interferon-γ and tumour necrosis factor-α production by CD4+ T and CD8+ T lymphocytes in AIDS patients with tuberculosis

Tuberculosis (TB) is usually more severe in HIV‐infected patients, and the immune derangement found in co‐infected patients may differ from that in each isolated disease. Following mitogen stimulation of peripheral blood mononuclear cells (PBMC), interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α production was evaluated in T cells by flow cytometry, and in culture supernatants by enzyme‐linked immunosorbent assay (ELISA) in 33 individuals: 11 AIDS patients with tuberculosis, six asymptomatic HIV‐1‐infected patients, eight patients with tuberculosis and eight healthy controls. The proportion of CD4+ T lymphocytes expressing IFN‐γ did not differ between the groups, whereas a trend towards increased proportions of TNF‐α‐expression in CD4+ T cells was observed in the TB compared to the HIV group, while intermediate values were observed in co‐infected patients. Detection of IFN‐γ and TNF‐α in CD8+ T lymphocytes was higher in TB than in HIV individuals. Co‐infected patients presented intermediate values for IFN‐γ, while TNF‐α detection was similar to that in HIV mono‐infection. In conclusion, the proportion of T cells expressing IFN‐γ was relatively preserved in co‐infected patients compared to TB patients, while the percentage of T cells expressing TNF‐α was decreased, mainly in CD8+ T lymphocytes. However, the marked reduction in T lymphocyte numbers in co‐infected patients led to a striking reduction of both cytokines in PBMC supernatants, a finding that is consistent with the impaired response to Mycobacterium tuberculosis.

Osteíte por BCG Moreau em uma menina vacinada ao nascer

A female infant (age, 2 years and 4 months) from the city of Sao Paulo, Brazil, had been vaccinated with BCG in the maternity ward and presented with a 15-day history of pain in the right leg and impaired ambulation. The patient was treated with a nonsteroidal anti-inflammatory drug and showed improvement. However, two weeks after the drug had been discontinued, she showed worsening. An X-ray of the knee showed an osteolytic lesion in the right distal femoral epiphysis. Magnetic resonance imaging (Figure 1) revealed a centromedial lesion in the right distal femoral epiphysis, with multiple areas of cortical erosion; there was significant cortical discontinuity, and there was no effusion in the posteroinferior portion of the medial femoral condyle. The patient was treated with ceftriaxone for 14 days, with no improvement. A punch biopsy of the right knee showed a granuloma with no AFB. The patient was started on isoniazid, rifampin, and pyrazinamide. Investigation of her parents, siblings, and nannies showed no exposure to pulmonary or extrapulmonary tuberculosis. At admission to our facility, a few days after treatment initiation, the patient was in good general health. The only abnormality on physical examination was right knee edema (distal and proximal to the tibia). The edema was cold and painful on palpation, being accompanied by functional disability.The following tests were performed: tuberculin skin testing with PPD, the induration being 14 mm; X-ray and magnetic resonance imaging of the right knee; enzyme-linked immunospot (ELISPOT) assay, the results being negative; knee biopsy, revealing very little bone tissue with two epithelioid granulomas (one of which had caseous necrosis) and chronic lymphoplasmacytic inflammatory infiltrate; AFB testing, the results being negative; mycobacterial culture, the results being positive; PCR testing, revealing the presence of insertion sequence 6110 (which is characteristic of mycobacteria) and duplication of spacer 33 in the DR region (172-bp amplicon), present only in the Mycobacterium bovis BCG strain and absent in M. tuberculosis; PCR testing, the results being negative for M. tuberculosis; routine tests (complete blood count, ESR determination, HIV testing, and evaluation of liver and kidney function), the results being normal; humoral and cellular immune response testing, the results being normal (Table 1); and chest CT, the findings being normal.The patient achieved a satisfactory clinical improvement and was discharged after 18 months of treatment, at which point an X-ray of the right femur showed normal findings.The BCG vaccine is used in many countries, and there are no strict rules regarding the age of vaccination, the groups that should be vaccinated, the type of vaccine, the concentration of bacilli, the ratio of live to dead bacilli, or the mode of administration. Many strains are used; however, there is uncertainty regarding the total number of viable and nonviable bacilli (which could potentiate the immunity induced by the former) and the ability of PPD to induce allergy, both of which are factors that can have an impact on the potency and complications of the vaccine.

Profound peripheral T-lymphocyte depletion and activation in disseminated tuberculosis

Three HIV-1-seronegative patients with disseminated tuberculosis presented significant depletion of T-cell counts, in CD4+ and/or CD8+ cells, associated with increased expression of activation marker CD38 on CD8+ T-lymphocytes. This finding raises the question of potential mechanisms involved in the activation or loss of T-cells in disseminated tuberculosis.