Dynamics of T-Lymphocyte Activation Related to Paradoxical Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome in Persons With Advanced HIV

Most persons living with HIV (PLWH) experience a significant restoration of their immunity associated with successful inhibition of viral replication after antiretroviral therapy (ART) initiation. Nevertheless, with the robust quantitative and qualitative restoration of CD4(+) T-lymphocytes, a fraction of patients co-infected with tuberculosis develop immune reconstitution inflammatory syndrome (TB-IRIS), a dysregulated inflammatory response that can be associated with significant tissue damage. Several studies underscored the role of adaptive immune cells in IRIS pathogenesis, but to what degree T lymphocyte activation contributes to TB-IRIS development remains largely elusive. Here, we sought to dissect the phenotypic landscape of T lymphocyte activation in PLWH coinfected with TB inititating ART, focusing on characterization of the profiles linked to development of TB-IRIS. We confirmed previous observations demonstrating that TB-IRIS individuals display pronounced CD4(+) lymphopenia prior to ART initiation. Additionally, we found an ART-induced increase in T lymphocyte activation, proliferation and cytotoxicity among TB-IRIS patients. Importantly, we demonstrate that TB-IRIS subjects display higher frequencies of cytotoxic CD8(+) T lymphocytes which is not affected by ART. Moreover, These patients exhibit higher levels of activated (HLA-DR(+)) and profilerative (Ki-67(+)) CD4(+) T cells after ART commencenment than their Non-IRIS counterparts. Our network analysis reveal significant negative correlations between Total CD4(+) T cells counts and the frequencies of Cytotoxic CD8(+) T cells in our study population which could suggest the existance of compensatory mechanisms for Mtb-infected cells elimination in the face of severe CD4(+) T cell lymphopenia. We also investigated the correlation between T lymphocyte activation profiles and the abundance of several inflammatory molecules in plasma. We applied unsupervised machine learning techniques to predict and diagnose TB-IRIS before and during ART. Our analyses suggest that CD4(+) T cell activation markers are good TB-IRIS predictors, whereas the combination of CD4(+) and CD8(+) T cells markers are better at diagnosing TB-IRIS patients during IRIS events Overall, our findings contribute to a more refined understanding of immunological mechanisms in TB-IRIS pathogenesis that may assist in new diagnostic tools and more targeted patient management

Disparate Immunoregulatory Potentials for Double-Negative (CD4(−) CD8(−)) αβ and γδ T Cells from Human Patients with Cutaneous Leishmaniasis

Although most T lymphocytes express the αβ T-cell receptor and either CD4 or CD8 molecules, a small population of cells lacking these coreceptors, CD4(−) CD8(−) (double negative [DN]) T cells, has been identified in the peripheral immune system of mice and humans. To better understand the role that this population may have in the human immune response against Leishmania spp., a detailed study defining the activation state, cytokine profile, and the heterogeneity of DN T cells bearing αβ or γδ T-cell receptors was performed with a group of well-defined cutaneous leishmaniasis patients. Strikingly, on average 75% of DN T cells from cutaneous leishmaniasis patients expressed the αβ T-cell receptor, with the remainder expressing the γδ receptor, while healthy donors displayed the opposite distribution with ∼75% of the DN T cells expressing the γδ T-cell receptor. Additionally, αβ DN T cells from cutaneous leishmaniasis patients are compatible with previous antigen exposure and recent activation. Moreover, while αβ DN T cells from Leishmania-infected individuals present a proinflammatory cytokine profile, γδ DN T cells express a regulatory profile exemplified by interleukin-10 production. The balance between these subpopulations could allow for the formation of an effective cellular response while limiting its pathogenic potential

Evaluation of the interference of platelets in reticulocyte counting by flow cytometry using thiazole orange

ABSTRACT Introduction: The reticulocyte count by flow cytometry (FC) - an automated counting method - can present errors due to the presence of interfering factors, contributing to a slight increase in results. However, automated methods have large advantages over the manual method, taken as reference, what justifies efforts to improve their quality. Objective: Evaluate platelet interference with the reticulocyte count by FC, using thiazole orange (TO) (FC/TO). Materials and methods: The method of reticulocyte count by FC/TO and a modified automated equivalent method, which excluded CD61-positive cells (platelets) from analysis (FC/TO/MOD), were compared to the manual method. Conclusion: Results were analyzed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) to assess interchangeability between the methods, by linear regression analysis and paired t-test. The exclusion of interfering fragments from result analysis by the modified method produced results in closer proximity to those of the reference method

Characterization of monocyte subsets in malaria patients.

<p>(A) Representative dot plots showing the gate strategy for the identification of CD14⁺CD16⁻ (green gate), CD14⁺CD16⁺ (red gate), CD14^loCD16⁺ (blue gate) monocyte subsets. CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes are represented by green, red and blue symbols. (B) Frequencies of CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^lo monocytes within PBMC from <i>P. vivax</i>-infected patients before (BT, filled symbols) and 30–45 days after treatment (AT, open symbols) (n = 28). (C) Mean fluorescence intensity (MFI) of CCR2 (BT, n = 28 and AT, n = 18), CX3CR1 (BT, n = 26 and AT, n = 19), CCR7 (BT, n = 28 and AT, n = 19) and LFA-1 (BT, n = 15 and AT, n = 11) (from the top to the bottom) was evaluated on monocyte subsets (CD14⁺CD16⁻ (left panel), CD14⁺CD16⁺ (middle panel), CD14^loCD16⁺ (right panel)) from <i>P. vivax</i>-infected subjects, before and 30–45 days after treatment. Dotted lines represent medians of a given measurements from healthy donors. (D) Scattered dot plots (left panels) and representative histograms (right panels) showing MFI of the molecules described above on CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes from <i>P. vivax</i>-infected patients before treatment (open histograms). Levels of the molecules above were measured by flow cytometry. Circles indicate individual patients and lines represent median values and interquartile ranges. (E) Levels of molecules expressed by the monocyte subsets analyzed according to D. * <i>p</i><0.05, **0.05><i>p</i>>0.01, ***<i>p</i><0.01.</p

<i>P. vivax</i>-infected patients display higher levels of cytokines accompanied by increased frequencies of circulating monocytes.

<p>(A) IL-6, IL-8 and IL-10 were measured in plasma of <i>P. vivax</i>-infected individuals before (BT, black circles) and 30–45 days after (AT, grey circles) treatment (n = 20). Levels of cytokine were measured by Cytometric Bead Array. (B) Leukocyte counts and frequencies from <i>P. vivax</i>-infected individuals before (BT, black circles) and after (AT, grey circles) treatment were assessed at a clinical laboratory (n = 33). C) Representative density plots of CD14⁺ monocytes (left panel) and frequencies of CD14⁺ monocytes (right panel) within PBMCs from <i>P. vivax</i>-infected individuals (BT, n = 28 and AT, n = 20). Circles indicate individual patients and lines represent median values and interquartile ranges. Dotted lines represent medians of a given measurements from healthy donors. *<i>p</i><0.05, ***<i>p</i><0.01.</p

Monocyte subsets from <i>P. vivax</i>-infected patients display a highly activated phenotype.

<p>CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes are represented by green, red and blue symbols. (A) Mean fluorescence intensity (MFI) of HLA-DR (BT, n = 11 and AT, n = 11), CD106 (BT, n = 26 and AT, n = 19), CD54 (BT, n = 28 and AT, n = 19) and CD31 (BT, n = 25 and AT, n = 20) (from the top to the bottom) was evaluated on monocyte subsets (CD14⁺CD16⁻ (left panel), CD14⁺CD16⁺ (middle panel), CD14^loCD16⁺ (right panel)) from <i>P. vivax</i>-infected subjects, before (filled symbols) and 30–45 days after treatment (open symbols). Dotted lines represent medians of given measurements from healthy donors. (B) Scattered dot plots (left panels) and representative histograms (right panels) showing MFI of the molecules described above on CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes from <i>P. vivax</i>-infected patients before treatment. Levels of the molecules above were measured by flow cytometry. Circles indicate individual patients and lines represent median values and interquartile ranges. (C) Levels of molecules expressed by the monocyte subsets analyzed according to B. *<i>p</i><0.05, **0.05><i>p</i>>0.01, ***<i>p</i><0.01.</p

CD16⁺CD14⁺ monocytes display pronounced phagocytic ability than other subsets.

<p>CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes are represented by green, red, and blue symbols, respectively. <i>P. vivax</i>-infected reticulocytes (Pv-Ret) were purified, CFSE labeled (A, B, C) or not (D, E) and cultured with PBMC from acutely <i>P. vivax</i>-infected subjects. (A) Mean fluorescence intensity (MFI) of CFSE within CD14⁺CD16⁻, CD14⁺CD16⁺, CD14^loCD16⁺ monocytes exposed to <i>P. vivax</i> was measured by flow cytometry after 4 hours of culture. Each circle represents a single patient (n = 12) and lines represent median values and interquartile ranges (B) Correlation between <i>P. vivax</i> phagocytosis (CFSE MFI) and CD54, CD11a or CD31 expression by CD14⁺CD16⁻, CD14⁺CD16⁺, CD14^loCD16⁺ monocytes. Circles indicate individual patients (n = 13). (C) Phagocytosis of Pv-Ret by CD14+ cells was measured in the absence of serum, presence of inactivated serum and presence of serum, and in the presence of anti-CD54, CD11a and CD31 blocking antibodies (lower graph). Bars represent median values and interquartile ranges (n = 6). (D) MFI of CFSE within CD14⁺CD16⁻, CD14⁺CD16⁺, CD14^loCD16⁺ monocytes exposed to <i>P. vivax</i> was measured by flow cytometry after 4 and 12 hours of culture. Connecting circles represent values of CFSE MFI of a single patient after 4 and 12 hours of culture, (n = 12) (E) Frequencies of TNF-α producing monocyte subsets were measured after culture with medium alone, Pv-Ret or LPS. Bars represent median values and interquartile ranges (n = 6). (F) ROS production was detected by measuring MFI (top panel) and the proportions (middle panel) of carboxy-H₂DCDA⁺ CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^loCD16⁺ monocytes after 3 hours incubation with <i>P. vivax</i>-infected reticulocytes. Bars represent median values and interquartile ranges (n = 9). Representative histograms showing MFI of carboxy-H₂DCFDA expressing CD14⁺CD16⁻, CD14⁺CD16⁺, CD14^loCD16⁺ monocytes (bottom panel). Grey histogram represents CD14⁺ cells cultured in the absence of Pv-Ret. (G) Mitochondrial ROS was assessed in monocyte subsets from <i>P. vivax</i>-infected patients measuring MitoSox by flow cytomery after culture with <i>P. vivax</i>-infected reticulocytes (n = 4). Connecting circles represent frequencies of different monocyte subsets producing mitochondrial ROS. Symbols represent individual subject. Representative histograms showing MFI of MitoSox expressing CD14⁺CD16⁻, CD14⁺CD16⁺, CD14^loCD16⁺ monocytes (bottom panel). Grey histogram represents MitoSox- cells. *<i>p</i><0.05, **0.05><i>p</i>>0.01, ***<i>p</i><0.01.</p