A Critical Role for CD8 T Cells in a Nonhuman Primate Model of Tuberculosis

The role of CD8 T cells in anti-tuberculosis immunity in humans remains unknown, and studies of CD8 T cell–mediated protection against tuberculosis in mice have yielded controversial results. Unlike mice, humans and nonhuman primates share a number of important features of the immune system that relate directly to the specificity and functions of CD8 T cells, such as the expression of group 1 CD1 proteins that are capable of presenting Mycobacterium tuberculosis lipids antigens and the cytotoxic/bactericidal protein granulysin. Employing a more relevant nonhuman primate model of human tuberculosis, we examined the contribution of BCG- or M. tuberculosis-elicited CD8 T cells to vaccine-induced immunity against tuberculosis. CD8 depletion compromised BCG vaccine-induced immune control of M. tuberculosis replication in the vaccinated rhesus macaques. Depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a significant decrease in the vaccine-induced immunity against tuberculosis. Consistently, depletion of CD8 T cells in rhesus macaques that had been previously infected with M. tuberculosis and cured by antibiotic therapy also resulted in a loss of anti-tuberculosis immunity upon M. tuberculosis re-infection. The current study demonstrates a major role for CD8 T cells in anti-tuberculosis immunity, and supports the view that CD8 T cells should be included in strategies for development of new tuberculosis vaccines and immunotherapeutics

Global, regional, and national disability-adjusted life-years (DALYs) for 315 diseases and injuries and healthy life expectancy (HALE), 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015.

BACKGROUND: Healthy life expectancy (HALE) and disability-adjusted life-years (DALYs) provide summary measures of health across geographies and time that can inform assessments of epidemiological patterns and health system performance, help to prioritise investments in research and development, and monitor progress toward the Sustainable Development Goals (SDGs). We aimed to provide updated HALE and DALYs for geographies worldwide and evaluate how disease burden changes with development. METHODS: We used results from the Global Burden of Diseases, Injuries, and Risk Factors Study 2015 (GBD 2015) for all-cause mortality, cause-specific mortality, and non-fatal disease burden to derive HALE and DALYs by sex for 195 countries and territories from 1990 to 2015. We calculated DALYs by summing years of life lost (YLLs) and years of life lived with disability (YLDs) for each geography, age group, sex, and year. We estimated HALE using the Sullivan method, which draws from age-specific death rates and YLDs per capita. We then assessed how observed levels of DALYs and HALE differed from expected trends calculated with the Socio-demographic Index (SDI), a composite indicator constructed from measures of income per capita, average years of schooling, and total fertility rate. FINDINGS: Total global DALYs remained largely unchanged from 1990 to 2015, with decreases in communicable, neonatal, maternal, and nutritional (Group 1) disease DALYs offset by increased DALYs due to non-communicable diseases (NCDs). Much of this epidemiological transition was caused by changes in population growth and ageing, but it was accelerated by widespread improvements in SDI that also correlated strongly with the increasing importance of NCDs. Both total DALYs and age-standardised DALY rates due to most Group 1 causes significantly decreased by 2015, and although total burden climbed for the majority of NCDs, age-standardised DALY rates due to NCDs declined. Nonetheless, age-standardised DALY rates due to several high-burden NCDs (including osteoarthritis, drug use disorders, depression, diabetes, congenital birth defects, and skin, oral, and sense organ diseases) either increased or remained unchanged, leading to increases in their relative ranking in many geographies. From 2005 to 2015, HALE at birth increased by an average of 2·9 years (95% uncertainty interval 2·9-3·0) for men and 3·5 years (3·4-3·7) for women, while HALE at age 65 years improved by 0·85 years (0·78-0·92) and 1·2 years (1·1-1·3), respectively. Rising SDI was associated with consistently higher HALE and a somewhat smaller proportion of life spent with functional health loss; however, rising SDI was related to increases in total disability. Many countries and territories in central America and eastern sub-Saharan Africa had increasingly lower rates of disease burden than expected given their SDI. At the same time, a subset of geographies recorded a growing gap between observed and expected levels of DALYs, a trend driven mainly by rising burden due to war, interpersonal violence, and various NCDs. INTERPRETATION: Health is improving globally, but this means more populations are spending more time with functional health loss, an absolute expansion of morbidity. The proportion of life spent in ill health decreases somewhat with increasing SDI, a relative compression of morbidity, which supports continued efforts to elevate personal income, improve education, and limit fertility. Our analysis of DALYs and HALE and their relationship to SDI represents a robust framework on which to benchmark geography-specific health performance and SDG progress. Country-specific drivers of disease burden, particularly for causes with higher-than-expected DALYs, should inform financial and research investments, prevention efforts, health policies, and health system improvement initiatives for all countries along the development continuum. FUNDING: Bill & Melinda Gates Foundation

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015

Forouzanfar MH, Afshin A, Alexander LT, et al. Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. LANCET. 2016;388(10053):1659-1724.Background The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors-the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57.8% (95% CI 56.6-58.8) of global deaths and 41.2% (39.8-42.8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211.8 million [192.7 million to 231.1 million] global DALYs), smoking (148.6 million [134.2 million to 163.1 million]), high fasting plasma glucose (143.1 million [125.1 million to 163.5 million]), high BMI (120.1 million [83.8 million to 158.4 million]), childhood undernutrition (113.3 million [103.9 million to 123.4 million]), ambient particulate matter (103.1 million [90.8 million to 115.1 million]), high total cholesterol (88.7 million [74.6 million to 105.7 million]), household air pollution (85.6 million [66.7 million to 106.1 million]), alcohol use (85.0 million [77.2 million to 93.0 million]), and diets high in sodium (83.0 million [49.3 million to 127.5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Copyright (C) The Author(s). Published by Elsevier Ltd

Immune Distribution and Localization of Phosphoantigen-Specific Vγ2Vδ2 T Cells in Lymphoid and Nonlymphoid Tissues in Mycobacterium tuberculosis Infection▿

Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model of Mycobacterium tuberculosis infection to assess phosphoantigen-specific Vγ2Vδ2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonary M. tuberculosis infection generated diverse distribution patterns of Vγ2Vδ2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vγ2Vδ2 T cells in tissues were associated with M. tuberculosis infection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vγ2Vδ2 T cells were present within TB granulomas. In extrathoracic organs, Vγ2Vδ2 T cells were localized in the interstitial compartment of nonlymphoid tissues, and the interstitial localization was present despite the absence of detectable TB lesions. Finally, Vγ2Vδ2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the γδ T cells present within granulomas. Thus, clonally expanded Vγ2Vδ2 T cells appeared to undergo trans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses to M. tuberculosis infection

Depletion of CD8 lymphocytes in macaques immunized by previous <i>M. tuberculosis</i> infection resulted in a loss of immune control of tuberculosis following re-infection.

<p>(A) Anti-CD8 Ab treatment of the <i>M. tuberculosis</i>-immunized macaques resulted in profound depletion of CD8 lymphocytes in <i>M. tuberculosis</i> re-infection. Shown are absolute levels of blood CD8 cells (/ul) and BAL fluid CD8 cells (×50,000). (B) Depletion of CD8 lymphocytes in the <i>M. tuberculosis</i>-immunized macaques led to higher levels of bacilli in BAL fluids and increased <i>M. tuberculosis</i> RNA in tissues following <i>M. tuberculosis</i> re-infection by aerosol. Numbers of bacilli are shown as CFU counts in 10 ml of BAL fluid; <i>M. tuberculosis</i> RNA was determined as Ag85B RNA copy numbers in 10 mg of tissue <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#ppat.1000392-Shen1" target="_blank">[6]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#ppat.1000392-Huang1" target="_blank">[29]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#ppat.1000392-Shen3" target="_blank">[36]</a>. (C) The CD8 Ab-treated macaques with CD8 depletion developed severe forms of tuberculosis after <i>M. tuberculosis</i> re-infection by aerosol. Macaque ID are indicated for CD8 Ab-treated (right) and isotype control IgG-treated (left) macaques. Shown are the lung surface images of two representative macaques (partially cut in the lung of macaque 2762). Note that the CD8 Ab-treated macaques exhibited pale-colored lungs with dissemination of >0.5 cm coalescing or caseating granulomas or tubercle nodules (the lesion-containing areas are pointed out by large arrows on surface view from 3050) and apparent caseation necrosis. The control macaques displayed no or few small non-caseating granulomas as indicated by small arrows in the lungs. When >0.5 cm coalescing or caseating tubercle nodules both lungs were scored using the scoring system as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#s4" target="_blank">Methods</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000392#ppat.1000392-Barclay1" target="_blank">[41]</a> for the individual macaques, the pathology scores (mean±SD = 72±24) of the CD8-depleted macaques were significantly worse than those (mean±SD = 6±2) of the control animals (p<0.01, by nonparametric <i>t</i> test). (D) Histology of representative granulomas seen in CD8 Ab-treated (right panel) and isotype control IgG-treated (left panel) macaques. Shown are H&E stained lung sections, with macaque ID and magnification indicated in each slide. Note that granulomas in isotype IgG-treated macaques were small, and highly lymphocytic without apparent necrosis. Granulomas in CD8 Ab-treated macaques were large and less lymphocytic, with necrosis seen in the center as pointed out by arrows.</p

Depletion of CD8 lymphocytes in CD8 Ab-treated macaques resulted in a significant decrease in BCG vaccine-induced immunity against tuberculosis after <i>M. tuberculosis</i> infection.

<p>(A) Top-panel photos show that the isotype IgG-treated BCG-vaccinated macaque (7406) exhibited limited numbers of granulomas (small arrows) in the cut-section of the right caudal lobe; the CD8 Ab-treated macaque (7348) showed extensive tuberculosis granulomas (large arrows) in the right caudal lobe, with a unilaterally enlarged hilar lymph node (a large green arrow). A naïve macaque (7419) showed large extensive tuberculosis lesions with caseation (large arrows), and much larger bilateral hilar lymph nodes (large green arrows). Middle- and bottom-panel photos show tuberculosis lesions in cut-sections of lungs of six monkeys in three respective groups. Extent and severity of the lesions could be adjudged based on the examples pointed by arrows at the top panel photos. Overall, CD8 Ab-treated macaques exhibited greater numbers of lung lobes displaying extensive coalescing granulomas than isotype IgG-treated control animals (p<0.05, by nonparametric <i>t</i> test). The CD8 Ab-treated macaques also showed more lobes with extensive caseating and miliary lesions or caseation pneumonia than the isotype IgG-treated controls (p<0.05, by nonparametric <i>t</i> test). Tuberculosis lesions in the CD8 Ab-treated macaques were more likely distributed or disseminated in other lobes or opposite lungs and hilar nodes/pleural. Small vertical/horizontal bars at bottom-left corner of each photo represent the 1-cm scale derived from the fluorescence rulers of each original photo. (B) Gross pathology scores of tuberculosis lesions in CD8 Ab-treated group, Isotype control IgG-treated group and naïve saline-treated group of macaques. Each macaque was given a total gross pathology scores based on the tuberculosis lesions in thoracic and extrathoracic organs. The mean gross pathology score was then calculated for each group of macaques and subjected to statistical analysis. *, P<0.05 (by ANOVA and nonparametric <i>t</i> test) for comparison between CD8 Ab-treated and isotype control IgG-treated groups, and for comparison between CD8 Ab-treated and naïve groups. (C) Histology evaluation of lung tissue sections of CD8 Ab-treated, isotype control IgG-treated and naïve saline-treated macaques. Shown are H&E stained sections taken from four representative macaques for each group, with macaque ID and magnification indicated for each image. Note that the isotype control IgG-treated macaques exhibited well-contained granulomas, which were generally infiltrated by numerous lymphocytes and some neutrophils. The CD8-depleted macaques displayed less-contained granulomas that were more likely to be necrotic (arrows). Naïve macaques showed less lymphocytic and more necrotic tuberculosis lesions than the other two groups.</p

CD8 Ab treatment of BCG-vaccinated macaques resulted in profound depletion of CD8 lymphocytes and mycobacterium-specific CD8 T effector cells during <i>M. tuberculosis</i> infection.

<p>(A) Flow cytometry data showed that anti-CD8 Ab-treated BCG-vaccinated macaques, but not isotype control IgG-treated BCG-vaccinated or naïve saline-treated macaques, exhibited marked decreases in percentages and absolute (data not shown) numbers of CD3+CD8+ and CD3−CD8+ lymphocytes in blood (upper left) and BAL fluid (upper right) during <i>M. tuberculosis</i> infection. Means for six animals are shown for each group, and error bars represent SEM. Arrows indicate the times for the treatment with anti-CD8 Ab or control antibodies. (B) Intracellular cytokine staining data showed that anti-CD8 Ab-treated BCG-vaccinated macaques exhibited an absence of PPD-specific IFNγ-producing CD8 T cells early after <i>M. tuberculosis</i> infection.</p

Depletion of CD8 lymphocytes resulted in a significant decrease in BCG-induced immune control of <i>M. tuberculosis</i>.

<p>(A) CD8 Ab-treated macaques exhibited higher levels of bacilli in BAL fluid than isotype IgG-treated macaques after pulmonary <i>M. tuberculosis</i> infection. Data are means +/− SEM of CFU counts/10 ml BAL fluid derived from 6 macaques for each group; * indicates p<0.05 for a comparison between the CD8 Ab-treated and isotype IgG-treated group. (B) CD8 Ab-treated macaques showed significantly higher levels of bacilli organisms in lung tissues than isotype control macaques after pulmonary <i>M. tuberculosis</i> infection. Data are mean values with SEM error bars of CFU counts/1 ml lung tissue homogenates derived from 6 macaques for each group. Rt, right; Lt, left. Note that right caudal lobe was the <i>M. tuberculosis</i> infection site for each macaque; *, p<0.05 (by both ANOVA and nonparametric <i>t</i> test) for a comparison between the CD8 Ab-treated and isotype control IgG-treated groups.</p