RESEARCH ARTICLE
Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection Edwin Leeansyah1*, Jenny Svärd2, Joana Dias1, Marcus Buggert3, Jessica Nyström3, Máire F. Quigley1, Markus Moll1, Anders Sönnerborg2,3, Piotr Nowak2,3, Johan K. Sandberg1* 1 Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 2 Unit of Infectious Diseases, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 3 Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden * [email protected] (EL); [email protected] (JKS)
OPEN ACCESS Citation: Leeansyah E, Svärd J, Dias J, Buggert M, Nyström J, Quigley MF, et al. (2015) Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection. PLoS Pathog 11(8): e1005072. doi:10.1371/journal.ppat.1005072 Editor: Guido Silvestri, Emory University, UNITED STATES Received: February 25, 2015 Accepted: July 6, 2015 Published: August 21, 2015 Copyright: © 2015 Leeansyah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by grants from the Swedish Research Council, the Swedish Cancer Foundation, the Petrus and Augusta Hedlund Foundation, and the Stockholm County Council (JKS). Further support came from the Lindhés Law Firm, Physicians against AIDS Foundation, the Clas Groschinsky Memorial Fund, and Swedish Society for Medicine (EL). EL was a recipient of a Swedish Institute Postdoctoral Fellowship. JD is supported by Fundação para a Ciência e a Tecnologia (SFRH/BD/ 85290/2012, doctoral fellowship), through program
Abstract Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment in vitro significantly restored MAIT cell effector functions even in the absence of ART. These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.
PLOS Pathogens | DOI:10.1371/journal.ppat.1005072 August 21, 2015
1 / 23
MAIT Cell Arming Is Induced by IL-7 and Defective in HIV-1 Infection
QREN-POPH-typology 4.1. JKS is a Senior Research Fellow of the Swedish Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Author Summary The mucosa-associated invariant T (MAIT) cells recognize antigens that are byproducts of the riboflavin biosynthesis pathway shared by many microbes. These antigens are presented by the MHC class I-like MR1 molecules and trigger rapid activation of MAIT cells in an innate-like fashion with deployment of effector mechanisms including cytokine production and cytolysis. Here, we investigated the MAIT cell response to bacteria in humans infected with HIV-1, and possible means to restore functionality to these cells. MAIT cell dysfunction in HIV-infected patients included an inability to express components of the cytolytic effector machinery. Impairment of the MAIT cell population involved the loss of expression of the transcription factors T-bet and Eomes. Interestingly, IL-7 had strong effects on MAIT cells, including the antigen-independent arming of cytolytic function and enhanced sensitivity for low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively associated with the size of the MAIT cell population, and IL-7 could rescue their function. These findings indicate that MAIT cell impairment in HIV-1 infection is broad-based, includes loss of critical transcription factors, and loss of cytolytic function. Furthermore, the data support the notion that IL-7 is a strong candidate for immunotherapy in diseases associated with MAIT cell loss.
Introduction Mucosa-associated invariant T (MAIT) cells are a recently described subset of unconventional, innate-like T cells that are highly abundant in mucosal tissues, liver and circulation of healthy humans [1–4]. MAIT cells express a semi-invariant T cell receptor (TCR), including Vα7.2 coupled with restricted Jα segments (Jα33, Jα12, or Jα20), and limited Vβ repertoires [5, 6]. Together with their semi-invariant TCR, human MAIT cells are also defined by their high expression of CD161, the IL-18 receptor α subunit (IL-18Rα), and the transcription factor ZBTB16 [7], also known as promyelocytic leukemia zinc finger protein (PLZF) [8, 9]. The vast majority of MAIT cells are either CD8αα or CD8αβ, with some CD4/8 double-negative (DN), and minor CD4+ populations [8–11]. Human MAIT cells acquire innate-like antimicrobial activity in the fetal intestinal mucosa pre-natally, prior to the establishment of the commensal microflora [12]. MAIT cells recognize antigens in complex with the MHC-Ib-related protein (MR1) [2, 4], which displays an extraordinary level of evolutionary conservation among placental and marsupial mammals [4, 13, 14]. MR1 presents microbial vitamin B2 (riboflavin) metabolites from a wide range of microbes [15, 16], including unstable intermediates that are formed from nonenzymatic condensation of the early intermediate of riboflavin biosynthesis 5-amino-6-D-ribitylaminouracil (5-A-RU) with host- or microbe-derived glyoxal or methylglyoxal [15, 17]. MR1 captures these otherwise unstable compounds and presents them to MAIT cells [17]. Once activated by antigens, MAIT cells can rapidly kill infected cells [18, 19], inhibit intracellular microbial growth [20], and produce pro-inflammatory cytokines including IFNγ, TNF, and IL-17 [8, 10–12, 21]. Certain innate cytokines, including IL-12 and IL-18, can stimulate MAIT cells to produce IFNγ independently of the MR1-TCR interaction [22, 23]. These findings are strongly supportive of the notion that MAIT cells perform critical functions in the immune system, also beyond their role as antimicrobial T cells, particularly at mucosal sites. Despite the great advances of antiretroviral therapy (ART) in the management of HIV disease, infected patients are still at an increased risk of microbial co-infections such as Mycobacterium tuberculosis, non-typhoidal Salmonella and Streptococcus pneumoniae [24–27]. Such
PLOS Pathogens | DOI:10.1371/journal.ppat.1005072 August 21, 2015
2 / 23
MAIT Cell Arming Is Induced by IL-7 and Defective in HIV-1 Infection
microbial co-infection burdens are particularly apparent in individuals who are diagnosed at advanced stages, lack access to ART, and those who are non-adherent to therapy and clinical care. Although MAIT cells do not directly recognise viral antigens, indirect involvement of these cells in viral immunopathogenesis can occur, as we originally and others recently described for HIV-1 infection [11, 21, 28–31]. MAIT cell numbers and Th1/17 cytokine production are severely and persistently reduced in chronic HIV-infected patients [11]. The polymicrobial reactivity and breadth of the MAIT cell functional profile most likely contribute to the reported role of MAIT cells in the protection against diverse bacterial and mycobacterial infections in animal models, as well as in severe bacterial infections and pulmonary tuberculosis in humans [8, 20, 32–35]. MAIT cell defects may therefore predispose HIV-infected patients to an increased risk of acquiring microbial co-infections. Furthermore, the loss of MAIT cells is potentially irreversible if the disease is not treated at a very early stage [11, 21, 28–31]. In most settings of HIV-1 infection early diagnosis and treatment is challenging, and this necessitates a strategy where MAIT cells can be rescued through adjunctive immunotherapies. IL-7 is a pleiotropic cytokine that has strong survival and homeostatic effects towards T cells, particularly the memory T cell populations with which MAIT cells show similarities (reviewed in [36]). IL-7 has attracted interest in the context of HIV-1 disease, and was proposed as a potential cytokine intervention therapy in treatment failure as well as in approaches to purge the viral reservoir (reviewed in [37]). Furthermore, IL-7 was recently shown by Tang et al. to enhance MAIT cell Th1/17 cytokine production in response to polyclonal stimulation [38]. In the present study, we investigate the cytolytic function of MAIT cells in the context of HIV-1 infection, the basis for dysfunction of MAIT cells in this disease, and possible approaches to rescue their function. Our findings indicate that arming of cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defects in these cells are associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells in healthy donors, and can partially reverse the functional and transcription factor defects in MAIT cells from HIV-1 infected patients. Thus, inclusion of IL-7 may be considered in immunotherapeutic approaches to restore MAIT cell numbers and function in conditions associated with loss and dysfunction of these cells.
Results MAIT cells express cytolytic proteins at steady state and become rapidly armed into full effector T cells following bacterial stimulation The expression levels of the cytolytic proteins perforin (Prf), granzyme (Grz)A, GrzB, and granulysin (Gnly), as well as the degranulation marker CD107a, were investigated in unstimulated MAIT cells obtained from 20 HIV-uninfected healthy controls (Fig 1A and 1B), and compared with those of non-MAIT, conventional CD4+ and CD8+ T cells (Fig 1B). The majority of MAIT cells expressed Prf and GrzA, whereas Gnly was expressed only by a proportion of MAIT cells and displayed high inter-donor variability (Fig 1B). Interestingly, MAIT cells did not express GrzB at steady state (Fig 1B). This pattern of cytolytic protein expression by MAIT cells was significantly different from non-MAIT, conventional CD4+ and CD8+ T cells (Fig 1B), consistent with recent studies [18, 19]. In addition, GrzA and Gnly in MAIT cells were coexpressed with the pore-forming protein Prf (S1A Fig). We next determined MAIT cell cytolytic protein expression profile following overnight stimulation with mildly PFA-fixed E. coli in 20 healthy individuals, as we have previously shown that such stimulation triggers robust expression of pro-inflammatory cytokines in MAIT cells [11, 12]. Following stimulation, MAIT cells expressed high levels of the
PLOS Pathogens | DOI:10.1371/journal.ppat.1005072 August 21, 2015
3 / 23
MAIT Cell Arming Is Induced by IL-7 and Defective in HIV-1 Infection
Fig 1. MAIT cells are rapidly armed into full effector T cells following bacterial stimulation. (A) The gating strategy to identify Vα7.2+CD161hi MAIT cells through flow cytometry. (B) The comparison of cytolytic proteins (granzyme A (GrzA), granzyme B (GrzB), granulysin (Gnly)), pore-forming protein perforin (Prf), and degranulation marker (CD107a) expression by MAIT cells (red), and CD4+ (blue) and CD8+ (green) conventional T cells at resting state from 20 HIV-uninfected, healthy controls. (C) Expression of cytolytic proteins by MAIT cells following stimulation with PFA-fixed E. coli (orange contour plot) or left unstimulated for 24 h (black dot plot). (D) Activated CD107a+GrzAlo MAIT cells (red contour plot) following bacterial stimulation expressed high levels of GrzB, Prf, and IFNγ, whereas the non-activated CD107a-GrzA+ MAIT cells (black dot plot) were variable for Prf expression and mostly negative for GrzB and IFNγ (n = 20). (E) The kinetics of MAIT cell expression for CD107a (blue line), GrzAlo (red line), and GrzB (black line) after PFA-fixed E. coli stimulation (n = 3). Error bars represent mean and standard deviation. Representative FACS plots from at least 5 healthy donors are shown. Box and whisker plot shows median, IQR and the 10th to the 90th percentile. The Friedman test followed by Dunn’s post-hoc test was used to detect significant differences across multiple, paired samples (B), and the Wilcoxon signed rank test for paired samples (D). NS: not significant (p0.05), *p
Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection Edwin Leeansyah1*, Jenny Svärd2, Joana Dias1, Marcus Buggert3, Jessica Nyström3, Máire F. Quigley1, Markus Moll1, Anders Sönnerborg2,3, Piotr Nowak2,3, Johan K. Sandberg1* 1 Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 2 Unit of Infectious Diseases, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 3 Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden * [email protected] (EL); [email protected] (JKS)
OPEN ACCESS Citation: Leeansyah E, Svärd J, Dias J, Buggert M, Nyström J, Quigley MF, et al. (2015) Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection. PLoS Pathog 11(8): e1005072. doi:10.1371/journal.ppat.1005072 Editor: Guido Silvestri, Emory University, UNITED STATES Received: February 25, 2015 Accepted: July 6, 2015 Published: August 21, 2015 Copyright: © 2015 Leeansyah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by grants from the Swedish Research Council, the Swedish Cancer Foundation, the Petrus and Augusta Hedlund Foundation, and the Stockholm County Council (JKS). Further support came from the Lindhés Law Firm, Physicians against AIDS Foundation, the Clas Groschinsky Memorial Fund, and Swedish Society for Medicine (EL). EL was a recipient of a Swedish Institute Postdoctoral Fellowship. JD is supported by Fundação para a Ciência e a Tecnologia (SFRH/BD/ 85290/2012, doctoral fellowship), through program
Abstract Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment in vitro significantly restored MAIT cell effector functions even in the absence of ART. These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.
PLOS Pathogens | DOI:10.1371/journal.ppat.1005072 August 21, 2015
1 / 23
MAIT Cell Arming Is Induced by IL-7 and Defective in HIV-1 Infection
QREN-POPH-typology 4.1. JKS is a Senior Research Fellow of the Swedish Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Author Summary The mucosa-associated invariant T (MAIT) cells recognize antigens that are byproducts of the riboflavin biosynthesis pathway shared by many microbes. These antigens are presented by the MHC class I-like MR1 molecules and trigger rapid activation of MAIT cells in an innate-like fashion with deployment of effector mechanisms including cytokine production and cytolysis. Here, we investigated the MAIT cell response to bacteria in humans infected with HIV-1, and possible means to restore functionality to these cells. MAIT cell dysfunction in HIV-infected patients included an inability to express components of the cytolytic effector machinery. Impairment of the MAIT cell population involved the loss of expression of the transcription factors T-bet and Eomes. Interestingly, IL-7 had strong effects on MAIT cells, including the antigen-independent arming of cytolytic function and enhanced sensitivity for low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively associated with the size of the MAIT cell population, and IL-7 could rescue their function. These findings indicate that MAIT cell impairment in HIV-1 infection is broad-based, includes loss of critical transcription factors, and loss of cytolytic function. Furthermore, the data support the notion that IL-7 is a strong candidate for immunotherapy in diseases associated with MAIT cell loss.
Introduction Mucosa-associated invariant T (MAIT) cells are a recently described subset of unconventional, innate-like T cells that are highly abundant in mucosal tissues, liver and circulation of healthy humans [1–4]. MAIT cells express a semi-invariant T cell receptor (TCR), including Vα7.2 coupled with restricted Jα segments (Jα33, Jα12, or Jα20), and limited Vβ repertoires [5, 6]. Together with their semi-invariant TCR, human MAIT cells are also defined by their high expression of CD161, the IL-18 receptor α subunit (IL-18Rα), and the transcription factor ZBTB16 [7], also known as promyelocytic leukemia zinc finger protein (PLZF) [8, 9]. The vast majority of MAIT cells are either CD8αα or CD8αβ, with some CD4/8 double-negative (DN), and minor CD4+ populations [8–11]. Human MAIT cells acquire innate-like antimicrobial activity in the fetal intestinal mucosa pre-natally, prior to the establishment of the commensal microflora [12]. MAIT cells recognize antigens in complex with the MHC-Ib-related protein (MR1) [2, 4], which displays an extraordinary level of evolutionary conservation among placental and marsupial mammals [4, 13, 14]. MR1 presents microbial vitamin B2 (riboflavin) metabolites from a wide range of microbes [15, 16], including unstable intermediates that are formed from nonenzymatic condensation of the early intermediate of riboflavin biosynthesis 5-amino-6-D-ribitylaminouracil (5-A-RU) with host- or microbe-derived glyoxal or methylglyoxal [15, 17]. MR1 captures these otherwise unstable compounds and presents them to MAIT cells [17]. Once activated by antigens, MAIT cells can rapidly kill infected cells [18, 19], inhibit intracellular microbial growth [20], and produce pro-inflammatory cytokines including IFNγ, TNF, and IL-17 [8, 10–12, 21]. Certain innate cytokines, including IL-12 and IL-18, can stimulate MAIT cells to produce IFNγ independently of the MR1-TCR interaction [22, 23]. These findings are strongly supportive of the notion that MAIT cells perform critical functions in the immune system, also beyond their role as antimicrobial T cells, particularly at mucosal sites. Despite the great advances of antiretroviral therapy (ART) in the management of HIV disease, infected patients are still at an increased risk of microbial co-infections such as Mycobacterium tuberculosis, non-typhoidal Salmonella and Streptococcus pneumoniae [24–27]. Such
PLOS Pathogens | DOI:10.1371/journal.ppat.1005072 August 21, 2015
2 / 23
MAIT Cell Arming Is Induced by IL-7 and Defective in HIV-1 Infection
microbial co-infection burdens are particularly apparent in individuals who are diagnosed at advanced stages, lack access to ART, and those who are non-adherent to therapy and clinical care. Although MAIT cells do not directly recognise viral antigens, indirect involvement of these cells in viral immunopathogenesis can occur, as we originally and others recently described for HIV-1 infection [11, 21, 28–31]. MAIT cell numbers and Th1/17 cytokine production are severely and persistently reduced in chronic HIV-infected patients [11]. The polymicrobial reactivity and breadth of the MAIT cell functional profile most likely contribute to the reported role of MAIT cells in the protection against diverse bacterial and mycobacterial infections in animal models, as well as in severe bacterial infections and pulmonary tuberculosis in humans [8, 20, 32–35]. MAIT cell defects may therefore predispose HIV-infected patients to an increased risk of acquiring microbial co-infections. Furthermore, the loss of MAIT cells is potentially irreversible if the disease is not treated at a very early stage [11, 21, 28–31]. In most settings of HIV-1 infection early diagnosis and treatment is challenging, and this necessitates a strategy where MAIT cells can be rescued through adjunctive immunotherapies. IL-7 is a pleiotropic cytokine that has strong survival and homeostatic effects towards T cells, particularly the memory T cell populations with which MAIT cells show similarities (reviewed in [36]). IL-7 has attracted interest in the context of HIV-1 disease, and was proposed as a potential cytokine intervention therapy in treatment failure as well as in approaches to purge the viral reservoir (reviewed in [37]). Furthermore, IL-7 was recently shown by Tang et al. to enhance MAIT cell Th1/17 cytokine production in response to polyclonal stimulation [38]. In the present study, we investigate the cytolytic function of MAIT cells in the context of HIV-1 infection, the basis for dysfunction of MAIT cells in this disease, and possible approaches to rescue their function. Our findings indicate that arming of cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defects in these cells are associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells in healthy donors, and can partially reverse the functional and transcription factor defects in MAIT cells from HIV-1 infected patients. Thus, inclusion of IL-7 may be considered in immunotherapeutic approaches to restore MAIT cell numbers and function in conditions associated with loss and dysfunction of these cells.
Results MAIT cells express cytolytic proteins at steady state and become rapidly armed into full effector T cells following bacterial stimulation The expression levels of the cytolytic proteins perforin (Prf), granzyme (Grz)A, GrzB, and granulysin (Gnly), as well as the degranulation marker CD107a, were investigated in unstimulated MAIT cells obtained from 20 HIV-uninfected healthy controls (Fig 1A and 1B), and compared with those of non-MAIT, conventional CD4+ and CD8+ T cells (Fig 1B). The majority of MAIT cells expressed Prf and GrzA, whereas Gnly was expressed only by a proportion of MAIT cells and displayed high inter-donor variability (Fig 1B). Interestingly, MAIT cells did not express GrzB at steady state (Fig 1B). This pattern of cytolytic protein expression by MAIT cells was significantly different from non-MAIT, conventional CD4+ and CD8+ T cells (Fig 1B), consistent with recent studies [18, 19]. In addition, GrzA and Gnly in MAIT cells were coexpressed with the pore-forming protein Prf (S1A Fig). We next determined MAIT cell cytolytic protein expression profile following overnight stimulation with mildly PFA-fixed E. coli in 20 healthy individuals, as we have previously shown that such stimulation triggers robust expression of pro-inflammatory cytokines in MAIT cells [11, 12]. Following stimulation, MAIT cells expressed high levels of the
PLOS Pathogens | DOI:10.1371/journal.ppat.1005072 August 21, 2015
3 / 23
MAIT Cell Arming Is Induced by IL-7 and Defective in HIV-1 Infection
Fig 1. MAIT cells are rapidly armed into full effector T cells following bacterial stimulation. (A) The gating strategy to identify Vα7.2+CD161hi MAIT cells through flow cytometry. (B) The comparison of cytolytic proteins (granzyme A (GrzA), granzyme B (GrzB), granulysin (Gnly)), pore-forming protein perforin (Prf), and degranulation marker (CD107a) expression by MAIT cells (red), and CD4+ (blue) and CD8+ (green) conventional T cells at resting state from 20 HIV-uninfected, healthy controls. (C) Expression of cytolytic proteins by MAIT cells following stimulation with PFA-fixed E. coli (orange contour plot) or left unstimulated for 24 h (black dot plot). (D) Activated CD107a+GrzAlo MAIT cells (red contour plot) following bacterial stimulation expressed high levels of GrzB, Prf, and IFNγ, whereas the non-activated CD107a-GrzA+ MAIT cells (black dot plot) were variable for Prf expression and mostly negative for GrzB and IFNγ (n = 20). (E) The kinetics of MAIT cell expression for CD107a (blue line), GrzAlo (red line), and GrzB (black line) after PFA-fixed E. coli stimulation (n = 3). Error bars represent mean and standard deviation. Representative FACS plots from at least 5 healthy donors are shown. Box and whisker plot shows median, IQR and the 10th to the 90th percentile. The Friedman test followed by Dunn’s post-hoc test was used to detect significant differences across multiple, paired samples (B), and the Wilcoxon signed rank test for paired samples (D). NS: not significant (p0.05), *p
