Pain is prevalent in sickle cell disease (SCD) patients who display great heterogeneity in pain severity and frequency. Hypothesizing that inflammatory factors are involved in the pathogenesis of SCD pain, we focused on the IL1A C/T polymorphism rs1800587 that is an SNP located in a cis-transcriptional regulatory region.We genotyped IL1A rs1800587 and performed association studies with phenotype data obtained by a multidimensional pain assessment tool using the PAINReportIt® Questionnaire.Each T allele was associated with a 3.9 increase in composite pain index score (p = 0.04) as determined by multiple linear regression.IL1A rs1800587 may influence chronic pain in SCD.
T-cell acute lymphoblastic leukemias (T-ALLs) are clonal lymphoid malignancies with a poor prognosis, and still a lack of effective treatment. Here we examined the interactions between the mammalian target of rapamycin (mTOR) inhibitor rapamycin and idarubicin (IDA) in a series of human T-ALL cell lines Molt-4, Jurkat, CCRF-CEM and CEM/C1. Co-exposure of cells to rapamycin and IDA synergistically induced T-ALL cell growth inhibition and apoptosis mediated by caspase activation via the intrinsic mitochondrial pathway and extrinsic pathway. Combined treatment with rapamycin and IDA down-regulated Bcl-2 and Mcl-1, and inhibited the activation of phosphoinositide 3-kinase (PI3K)/mTOR and extracellular signal-related kinase (ERK). They also played synergistic pro-apoptotic roles in the drug-resistant microenvironment simulated by mesenchymal stem cells (MSCs) as a feeder layer. In addition, MSCs protected T-ALL cells from IDA cytotoxicity by up-regulating ERK phosphorylation, while rapamycin efficiently reversed this protective effect. Taken together, we confirm the synergistic antitumor effects of rapamycin and IDA, and provide an insight into the potential future clinical applications of combined rapamycin-IDA regimens for treating T-cell malignancies.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Group 1 CD1 molecules, CD1a, CD1b and CD1c, present lipid antigens from Mycobacterium tuberculosis (Mtb) to T cells. Mtb lipid-specific group 1 CD1-restricted T cells have been detected in Mtb-infected individuals. However, their role in protective immunity against Mtb remains unclear due to the absence of group 1 CD1 expression in mice. To overcome the challenge, we generated mice that expressed human group 1 CD1 molecules (hCD1Tg) and a CD1b-restricted, mycolic-acid specific TCR (DN1Tg). Using DN1Tg/hCD1Tg mice, we found that activation of DN1 T cells was initiated in the mediastinal lymph nodes and showed faster kinetics compared to Mtb Ag85B-specific CD4+ T cells after aerosol infection with Mtb. Additionally, activated DN1 T cells exhibited polyfunctional characteristics, accumulated in lung granulomas, and protected against Mtb infection. Therefore, our findings highlight the vaccination potential of targeting group 1 CD1-restricted lipid-specific T cells against Mtb infection. https://doi.org/10.7554/eLife.08525.001 eLife digest Most cases of tuberculosis are caused by a bacterium called Mycobacterium tuberculosis, which is believed to have infected one third of the world’s population. Most of these infections are dormant and don’t cause any symptoms. However, active infections can be deadly if left untreated and often require six months of treatment with multiple antibiotics. One reason why these infections are so difficult to treat is because the M. tuberculosis cell walls contain fatty molecules known as mycolic acids, which make the bacteria less susceptible to antibiotics. These molecules also help the bacteria to subvert and then hide from the immune system. The prevalence of the disease and the increasing problem of antibiotic resistance have spurred the search for an effective vaccine against tuberculosis. While most efforts have focused on using protein fragments in tuberculosis vaccines, some evidence suggests that human immune cells can recognize fatty molecules such as mycolic acids and that these cells could help manage and control M. tuberculosis infections. However, it has been difficult to determine whether these immune cells genuinely play a protective role against the disease because most vaccine research uses mouse models and mice do not have an equivalent of these immune cells. Now, Zhao et al. have engineered a “humanized” mouse model that produces the fatty molecule-specific immune cells, and show that these mice do respond to the presence of mycolic acids. Infecting the genetically engineered mice with M. tuberculosis revealed that the fatty molecule-specific immune cells were quickly activated within lymph nodes at the center of the chest. These cells later accumulated at sites in the lung where the bacteria reside, and ultimately protected against M. tuberculosis infection. The results show that these specific immune cells can counteract M. tuberculosis, and highlight the potential of using mycolic acids to generate an effective vaccine that provides protection against tuberculosis. https://doi.org/10.7554/eLife.08525.002 Introduction The CD1 family of antigen presenting molecules presents self and microbial lipids to T cells (Van Rhijn et al., 2013; De Libero and Mori, 2014; Adams, 2014). Two major groups of CD1 isoforms have been identified in humans: group 1 CD1 (CD1a, CD1b, and CD1c) and group 2 CD1 (CD1d) (Adams, 2014). While CD1d is broadly expressed, group 1 CD1 expression is limited to cortical thymocytes and professional antigen presenting cells (Barral and Brenner, 2007). Humans express all CD1 isoforms while mice only express CD1d. Due to the lack of a suitable small animal model to study group 1 CD1-restricted T cells, CD1d-restricted NKT cells have been better studied. A large proportion of CD1d-restricted NKT cells express an invariant TCR α chain and are known as iNKT cells (Bendelac et al., 2007). Unlike conventional T cells, which are positively selected by thymic epithelial cells (TECs), hematopoietic cells (HCs) select iNKT cells (Bendelac et al., 2007). The unique developmental selection program is thought to drive their pre-activated phenotype, which allows for rapid effector function manifestations upon TCR stimulation (Bendelac et al., 2007). Unlike iNKT cells, group 1 CD1-restricted T cells are known to have diverse TCR usage (Grant et al., 1999; Vincent et al., 2005; Felio et al., 2009). Group 1 CD1-restricted T cell responses have mostly been characterized in the context of mycobacterial antigens, although recent studies have shown that humans have a significant proportion of autoreactive group 1 CD1-restricted T cells (de Jong et al., 2010; de Lalla et al., 2011). The Mtb cell wall is lipid rich (60% of its cell wall is composed of lipids) and contains a plethora of lipid antigens that are presented by group 1 CD1 molecules (Van Rhijn et al., 2013; De Libero and Mori, 2014). Among group 1 CD1 molecules, CD1b presents the largest pool of Mtb-derived lipids like mycolic acid (MA), glucose monomycolate, glycerol monomycolate, diacylated sulfoglycolipids, lipoarabinomannan and phosphatidylinositol mannoside to cognate T cells (Van Rhijn et al., 2013; De Libero and Mori, 2014). Of the Mtb lipids mentioned above, MAs are the major lipid constituents of the Mtb cell envelope and considered a potent Mtb virulence factor (Barry et al., 1998; Karakousis et al., 2004). Interestingly, MA-specific CD1b-restricted T cells have been detected in the blood as well as disease sites of Mtb-infected individuals (Montamat-Sicotte et al., 2011). Tuberculosis (TB), the disease caused by Mtb, is a global health burden, especially in developing countries and amongst HIV/AIDS patients. Additionally, due to the emergence of multidrug-resistant Mtb and the lack of an effective vaccine to prevent pulmonary TB in adults, it is important to decipher the role of various T cell subsets in Mtb infection for the development of better preventive or therapeutic vaccines (Ottenhoff et al., 2012). The subunit vaccines currently under development for Mtb utilize peptide or protein antigens which target MHC-restricted conventional T cells (Dorhoi and Kaufmann, 2014), but the utility in targeting lipid antigens has not been explored. Since CD1 molecules are non-polymorphic, CD1-restricted Mtb lipid antigens are likely to be recognized by most individuals, making them attractive vaccine targets (Barral and Brenner, 2007). Several lines of evidence suggest that Mtb lipid-specific group 1 CD1-restricted T cells contribute to anti-mycobacterial immunity. Investigation of group 1 CD1-restricted T cell lines derived from healthy or mycobacteria-infected individuals has revealed that these T cells are cytotoxic and produce IFN-γ and TNF-α, cytokines critical for protective immunity to TB (Van Rhijn et al., 2013). Moreover, group 1 CD1-restricted Mtb lipid-specific T cells are found in higher frequencies in individuals exposed to Mtb compared with control populations, suggesting that they are activated following infection with Mtb (Moody, 2000; Ulrichs et al., 2003; Gilleron, 2004; Layre et al., 2009; Moody et al., 2000). Additionally, a robust Mtb lipid-specific group 1 CD1-restricted T cell response has been detected in Mtb-infected human group 1 CD1 transgenic mice (Felio et al., 2009). However, it remains unclear whether this unique T cell subset plays a protective role during the course of infection. In this study, we generated transgenic mice expressing mycolic acid-specific CD1b-restricted TCR (DN1Tg) and human group 1 CD1 molecules (hCD1Tg). Using this mouse model, we found that DN1 T cells were selected most efficiently by group 1 CD1-expressing HCs in the thymus. Upon adoptive transfer of DN1 T cells to Mtb-infected hCD1Tg mice, DN1 T cells were first activated in the mediastinal lymph nodes, exhibiting faster kinetics than Ag85B-specific CD4+ T cells. DN1 T cells were cytotoxic, polyfunctional and contributed to anti-mycobacterial immunity by reducing bacterial burdens in the lung, spleen and liver. Thus, this study provides the first direct demonstration that group 1 CD1-restricted Mtb lipid-specific T cells play a protective role during Mtb infection. Results Generation of a mycolic acid-specific CD1b-restricted TCR transgenic mouse model We developed human CD1 transgenic mice, which expressed group 1 CD1 molecules in a similar pattern to that observed in humans. Using this model, we demonstrated the feasibility to study group 1 CD1-restricted T cell responses in aerosol infection with Mtb (Felio et al., 2009). To facilitate the direct analysis of Mtb lipid-specific group 1 CD1-restricted T cells, we generated a novel transgenic mouse strain that expressed a human/mouse chimeric TCR, composed of variable region from human T cell clone DN1 (Grant et al., 1999), specific for CD1b/mycolic acid (MA), and mouse TCR constant region (Figure 1A). DN1Tg founders and their progeny were screened for the presence of TRAV13-2-TRAJ57 gene fragment by PCR and for the surface expression of human Vβ5.1 (TRBV5-1) by flow cytometry (Figure 1B,C). Subsequently, DN1Tg mice were bred onto hCD1Tg/Rag-/- background to eliminate the expression of endogenous TCR. All DN1Tg mice used in this study were on a Rag-/- background. To examine whether the development of DN1 T cells was dependent on group 1 CD1 molecules, we compared DN1 T cells in WT and hCD1Tg backgrounds. We found that both frequency and absolute number of DN1 T cells were greatly reduced in DN1Tg mice compared with DN1Tg/hCD1Tg mice in all tested organs (Figure 1D–F). This suggested that group 1 CD1 supported the development of DN1 T cells. Notably, unlike CD1d-restricted iNKT cells, DN1 T cells from the spleen and lymph nodes of DN1Tg/hCD1Tg mice exhibited a naïve phenotype (characterized by low expression levels of T cell activation markers such as CD69 and CD44) similar to conventional CD8+ T cells and were either CD8αβ+ or CD4-CD8- (DN). In addition, DN1 thymocytes from DN1Tg/hCD1Tg mice did not express PLZF, the master transcription factor for innate T cell lineages (Figure 1G) (Kovalovsky et al., 2008; Savage et al., 2008). Figure 1 Download asset Open asset Development of DN1 T cells is dependent on the presence of group 1 CD1 molecules. (A) Schematic diagram of DN1 TCR construct used to generate DN1Tg mice. (B) The presence of DN1 TCR in the genomic DNA of transgenic mice was examined by PCR using primers specific for TRAV13-2 and TRAJ57. DN1 plasmid was used as a positive control (Ctrl). (C) DN1 T cells in the spleen of DN1Tg+ and DN1Tg- mice (in a B6 background) were detected by FACS using anti-mouse TCRβ and anti-human Vβ5.1 mAbs. (D) Lymphocytes from the thymus, spleen and liver of DN1Tg/hCD1Tg and DN1Tg mice (in the Rag-deficient background) were analyzed for the presence of DN1 T cells (TCRβ+hVβ5.1+). (E, F) Bar graphs depict the mean and SEM of the percentages (in the lymphocyte gate) and absolute numbers of DN1 T cells from DN1Tg/hCD1Tg and DN1Tg mice (n=3–8 per group). ***p<0.001; **p<0.01; *p<0.05. (G) Expression of indicated markers (black line) on DN1 T cells (TCRβ+hVβ5.1+) from DN1Tg/hCD1Tg/Rag-/- mice, type I NKT cells (CD1d/αGalCer tetramer+TCRβ+) from WT mice, and conventional CD8+ T cells (TCRβ+CD8+) from WT mice, compared with isotype control (gray filled). The expression of CD4 and CD8 on DN1 T cells and type I NKT cells were shown in the dot plots. Cells isolated from the thymus were used for PLZF staining. Results are representative of 3 experiments. https://doi.org/10.7554/eLife.08525.003 CD1b-expressing hematopoietic cells (HCs) most efficiently select DN1 T cells Unlike conventional T cells, which are positively selected by TECs, iNKT cells are exclusively selected by CD1d-expressing thymocytes (Bendelac, 1995; Coles and Raulet, 2000). Several studies have demonstrated the correlation between positive selection on HCs and a pre-activated T cell phenotype of innate-like T cells (Bendelac et al., 2007; Cho et al., 2011; Bediako et al., 2012). Given that DN1 T cells exhibited a naïve surface phenotype, one would expect DN1 T cells to be positively selected by TECs. To test this hypothesis, we adoptively transferred bone marrow from DN1Tg and DN1Tg/hCD1Tg mice (in the Rag-deficient background) into irradiated CD45.1 congenic WT and hCD1Tg recipients. 5 weeks after transfer, DN1 T cells were identified by CD45.2and hVβ5.1 surface expression in different groups (Figure 2A). The percentage (Figure 2B) and absolute number (Figure 2C) of DN1 T cells were significantly higher in mice with group 1 CD1-expressing HCs compared to mice that only had group 1 CD1-expressing TECs. This suggested that HCs most efficiently mediate the positive selection of DN1 T cells. As a small number of DN1 T cells developed in mice that lack CD1b (Figure 2A), it is possible that mouse CD1d is responsible for their selection. We compared the percentage of DN1 T cells in the spleen and thymus of DN1Tg/hCD1Tg (CD1d+), DN1Tg/hCD1Tg/CD1d-/-, DN1Tg (CD1d+), and DN1Tg/CD1d-/- mice (all in the Rag-deficient background). We found that the percentage of DN1 T cells was comparable in DN1Tg/hCD1Tg and DN1Tg/hCD1Tg/CD1d-/- mice. In addition, DN1 T cells were barely detectable in the thymus and spleen of DN1Tg and DN1Tg/CD1d-/- mice. These data suggest that CD1d does not contribute to the thymic selection of DN1 T cells (Figure 2—figure supplement 1). Figure 2 with 1 supplement see all Download asset Open asset CD1b-expressing hematopoietic cells are the major cell type that medicates the positive selection of DN1 T cells. Bone marrow from DN1Tg and DN1Tg/hCD1Tg mice (in the Rag-deficient background) were adoptively transferred into irradiated CD45.1 congenic WT and hCD1Tg recipients and the development of DN1 T cells were examined 5 weeks later. (A) Dot plots depict the proportion of CD45.2+ hVβ5.1+ cells in the lymphocyte gate. Data are representative of 2 experiments with 4–6 mice in each group. (B, C) Bar graphs depict the mean ± SEM of the percentage and absolute number of CD45.2+ hVβ5.1+ cells in each group. Statistical significance was evaluated by comparing HC, TEC and None group with HC+TEC group. ***p<0.001; **p<0.01; *p<0.05. (D) CD1b expression on TEC (CD45-MHCII+) and DP thymocytes from WT and hCD1Tg mice were shown as MFI values (n=3 per group). (E) Percentage of CD8+ DN1 T cells in the spleen of HC+TEC, HC or TEC groups of mice. (F) Expression of indicated markers on DN1 thymocytes that developed in HC+TEC, HC or TEC groups. ***p<0.001; **p<0.01; *p<0.05. Results are representative of 2 experiments with 3 mice per group. https://doi.org/10.7554/eLife.08525.004 Comparing CD1b expression on TECs and CD4+CD8+ (DP) thymocytes revealed that DP thymocytes express significantly higher levels of CD1b than TECs (Figure 2D). Thus, CD1b-expressing thymocytes may be better suited to mediate the positive selection of DN1 T cells. Since DN1 T cells could also be selected by TECs, albeit with much lower efficiency compared to HCs, we compared the phenotype of DN1 T cells that developed in mice expressing CD1b on both HC and TEC, HC only and TEC only. DN1 T cells in the periphery of these three groups had a comparable proportion of CD8+/DN T cells (Figure 2E). In addition, DN1 T cells in the thymus of these three groups expressed similar levels of PLZF and CD44 (Figure 2F). However, DN1 T cells selected by HCs expressed higher levels of CD5 (Figure 2F), a surrogate marker for the TCR signaling strength in developing thymocytes, suggesting they might receive stronger TCR signals. DN1 T cells exhibit effector functions in response to mycolic acid stimulation and Mtb infection The human DN1 T cell line had been shown to secrete Th1 cytokines when stimulated with MA presented by CD1b+ APCs (Beckman et al., 1994). To test whether DN1 T cells that developed in DN1Tg/hCD1Tg mice retained the same functional properties as the original human T cell line, we stimulated lymph node cells from DN1Tg/hCD1Tg mice with un-pulsed or MA-pulsed bone marrow derived dendritic cells (BMDCs) to detect IFN-γ production and antigen-specific cytotoxicity (Figure 3A,B). DN1 T cells produced IFN-γ in response to MA-pulsed hCD1Tg DCs but not WT DCs or un-pulsed DC, suggesting the activation of DN1 T cells required both antigen and group 1 CD1 molecules. In addition, DN1 T cells showed cytotoxic activity against MA-pulsed hCD1Tg DCs. MA is located within the Mtb cell wall, either covalently attached via arabinogalactan to the cell wall peptidoglycan, or non-covalently associated in the form of trehalose dimycolate (Barry et al., 1998; Karakousis et al., 2004). To determine whether DN1 T cells can be activated by naturally processed MA, we set up co-culture of DN1 T cells with Mtb-infected BMDCs. As shown in Figure 3C, co-culture of DN1 T cells with Mtb-infected hCD1Tg DCs led to up-regulation of activation markers CD69 and CD44 on DN1 T cells. Furthermore, DN1 T cells produced multiple cytokines, of which the production of IFN-γ, TNF-α, IL-13 and IL-6 was dependent on the presence of group 1 CD1 molecules (Figure 3D), with the exception of IL-17. It is possible that co-stimulatory molecules and/or cytokines induced by Mtb-infected DCs can stimulate DN1 T cells to secrete this cytokine independent of TCR-CD1b interaction. Collectively, our data demonstrated that DN1 T cells became activated and exhibited effector functions in response to MA-pulsed or Mtb-infected DC in a group 1 CD1-dependent manner. Figure 3 Download asset Open asset DN1 T cells acquire effector functions in response to MA-pulsed DC and Mtb-infected DC. (A) DN1 T cells isolated from lymph nodes of DN1Tg/hCD1Tg/Rag-/- mice were co-cultured with un-pulsed or MA-pulsed WT or hCD1Tg DC and IFN-γ producing cells were determined by ELISPOT assays. (B) DN1 T cells were stimulated by hCD1Tg BMDCs pulsed with MA for 7 days and then tested for cytotoxic activity against hCD1Tg BMDCs with or without MA. Data are representative of 3 experiments (mean ± SEM of triplicate cultures). (C, D) DN1 T cells isolated from lymph nodes of DN1Tg/hCD1Tg/Rag-/- mice were co-cultured with Mtb-infected BMDC for 48 hr. Activation markers on DN1 T cells were detected by flow cytometry and cytokines in the supernatant were detected by CBA flex set. ***p<0.001; **p<0.01; *p<0.05. Results are representative of 2 experiments with 3 mice per experiment. https://doi.org/10.7554/eLife.08525.006 DN1 T cell-mediated control of Mtb is dependent on antigen-presentation by group 1 CD1-expressing DCs and IFN-γ production Macrophages are known as primary host cells for Mtb. Whereas BMDCs and a subset of myeloid DCs from hCD1Tg mice expressed high levels of CD1b, the expression of CD1b was almost undetectable on bone marrow derived macrophages (BMDMs) (Figure 4A), similar to the observation in human monocyte derived macrophages. Accordingly, we detected only minimal DN1 T cell activation when stimulated with Mtb-infected BMDMs (Figure 4B). While our data showed that Mtb-infected macrophages do not directly present antigen to DN1 T cells, apoptotic vesicles released from Mtb-infected macrophages have been shown to transfer mycobacterial antigens to uninfected APCs, such as DCs (Ulrichs et al., 2003; Schaible et al., 2003), which could in turn activate DN1 T cells. To explore whether and how DN1 T cells can control Mtb in infected macrophages, Mtb-infected BMDMs from WT or hCD1Tg mice were cultured together with DN1 T cells in the presence or absence of uninfected WT or hCD1Tg DCs. After 7 days, we determined the number of colony forming units (CFU) to investigate whether DN1 T cells inhibited intracellular bacterial growth within BMDMs. As expected, addition of DN1 T cells alone did not have a significant effect on bacterial burdens in Mtb-infected BMDMs. However, when DN1 T cells were added together with uninfected hCD1Tg DCs to infected BMDMs, the number of CFU decreased significantly compared with controls (Figure 4C). For comparison, we also used Mtb-infected BMDCs from WT or hCD1Tg mice as targets. We found that DN1 T cells efficiently controlled Mtb growth within infected hCD1Tg DCs but not WT DCs. Similarly, if DN1 T cells and uninfected hCD1Tg DCs were added to Mtb-infected WT DCs, Mtb growth was significantly inhibited (Figure 4D). These data indicated that group 1 CD1-expressing DCs mediated activation of DN1 T cells, which in turn controlled bacterial growth not only in the group 1 CD1-expressing DCs but also in macrophage and group 1 CD1-negative DCs. In addition, through a cytokine-blocking assay, we found that IFN-γ but not TNF-α was crucial for mediating anti-mycobacterial function of DN1 T cells (Figure 4E). Figure 4 Download asset Open asset DN1 T cell-mediated control of Mtb is dependent on the antigen presentation by group 1 CD1-expressing DCs and IFN-γ production. (A) CD1b expression on BMDM and BMDC was detected using flow cytometry. (B) BMDMs were in vitro infected with Mtb (MOI=5) and DN1 T cells were added 1 day after infection. After 48 hr co-culture, activation markers on DN1 T cells were detected by flow cytometry. (C, D) WT and hCD1Tg BMDMs and BMDCs were infected with Mtb. 1 day later, DN1 T cells with or without uninfected WT or hCD1Tg DCs were added into the culture for another 6 days. At day 7 post-infection, cells were lysed for CFU assay. Bar represents mean and SEM from replicate cultures (n = 6). (E) DN1 T cells were added into Mtb-infected BMDCs in the presence of control Ig (Ig), anti-IFN-γ or anti-TNFα. At day 7 post-infection, cells were lysed for CFU assay. % reduction was calculated as 100x[(BMDCalone- BMDCwith DN1)/ BMDCalone]. Results are representative of 2–3 experiments. ***p<0.001; **p<0.01; *p<0.05. https://doi.org/10.7554/eLife.08525.007 Activation of DN1 T cells is initiated in mediastinal lymph nodes (MLN) in response to aerosol Mtb infection To study when and where group 1 CD1-restricted Mtb lipid-specific T cells are first presented with antigens after aerosol infection with Mtb, we adoptively transferred naïve DN1 T cells into CD45.1 congenic hCD1Tg mice that had been infected with Mtb 7 days earlier. The expression of CD69 on DN1 T cells from various organs was monitored. The up-regulation of CD69 on DN1 T cells was first observed as early as day 11 post-infection in the lung-draining MLN, but not in other tissues examined. By day 15 after infection, the activation of DN1 T cells was also detected in the lung and spleen (Figure 5A,B). Although bacterial burdens were much higher in the lung than in the MLN (Figure 5C), DN1 T cell activation correlated with the first appearance of bacteria in MLN. Collectively, these data indicate that activation of DN1 T cells is initiated in MLN when Mtb disseminate from the site of primary infection (lung) to MLN. Figure 5 Download asset Open asset Activation of DN1 T cells is initiated in mediastinal lymph nodes after aerosol Mtb infection. (A) 3x106 naïve DN1 T cells were adoptively transferred into Mtb infected CD45.1 congenic hCD1Tg mice at day 7 post-infection. CD69 expression was detected on DN1 T cells (hVβ5+TCRβ+) from the MLN, lung, spleen, and inguinal lymph nodes (ILN) of recipient mice at indicated time points. (B) Bar graphs depict the mean and SEM of the percentages of CD69hi population among DN1 T cells (n=3 each time point). (C) Bacterial CFU in MLN and lung at indicated time points. Each symbol represents the bacteria burden in the MLN or lung of an individual mouse at the indicated time point. Horizontal bars represent the mean CFU counts ± SEM for each group. Results are representative of 2 experiments with 3 mice per time point. ***p<0.001; **p<0.01; *p<0.05. https://doi.org/10.7554/eLife.08525.008 DN1 T cells are activated earlier compared with Ag85B-specific CD4+ T cells after Mtb infection Several studies have shown that activation of Mtb-specific CD4+ conventional T cells is also initiated in the MLN (Wolf et al., 2008; Reiley et al., 2008; Gallegos et al., 2008). To compare the kinetics of DN1 T cell priming with conventional T cells after aerosol Mtb infection, we co-transferred naïve DN1 T cells with CD4+ TCR transgenic T cells specific to a peptide from Mtb Ag85B (P25 T cells) to Mtb-infected mice to monitor their activation and proliferation. CellTrace Violet dye labeled P25 T cells and CFSE labeled DN1 T cells were co-transferred in equal numbers to CD45.1 congenic hCD1Tg mice that were infected 7 days earlier. Similar to the observation in Figure 5A, up-regulation of CD69 on DN1 T cells started at day 11 post infection while CD69 was up-regulated on a small percentage of P25 T cells in MLN 13 days after infection (Figure 6A,C). Additionally, cell division was detected on DN1 T cells by day 13 and on P25 T cells by day 15 in the MLN (Figure 6B,D). Also, compared to P25 T cells, a greater proportion of DN1 T cells in the lung and spleen expressed CD69 and underwent cell division at day 15 post-infection (Figure 6E,F). Taken together, our data suggests that activation of MA-specific CD1b-restricted T cells occurs earlier than Ag85B-specific MHC II-restricted CD4+ T cells during Mtb infection. Figure 6 Download asset Open asset DN1 T cells are activated earlier than Ag85 specific CD4+ T cells after Mtb infection. (A, B) 3x106 CFSE-labeled DN1 T cells and 3x106 CellTrace Violet-labeled P25 T cells were co-transferred into Mtb infected CD45.1 congenic hCD1Tg mice. CD69 expression, CFSE and CellTrace Violet were detected on DN1 T cells and P25 T cells from MLN at indicated time points. (C, D) Bar graphs depict the mean and SEM of the percentages of CD69hi and CFSE/Violetlow populations among DN1 and P25 T cells. (E, F) CD69 expression, CFSE and CellTrace Violet were detected on DN1 and P25 T cells from lungs (E) and spleens (F) at day 13 and day 15 post-infection. Results are representative of 2 experiments with 3–4 mice per experiments. ***p<0.001; **p<0.01; *p<0.05. https://doi.org/10.7554/eLife.08525.009 Adoptive transfer of DN1 T cells confers protection against Mtb Group 1 CD1-restricted human T cell clones show effector functions including cytotoxic activity and cytokine production in response to Mtb-specific lipid antigens. However, whether group 1 CD1-restricted T cells confer protection against Mtb infection remains unknown. To address this question, DN1 effector T cells were adoptively transferred to hCD1Tg/Rag-/- mice and recipient mice were subsequently challenged with virulent Mtb via aerosol route. 4 weeks after infection, the number of bacteria in the lung, spleen and liver was determined. As shown in Figure 7A, DN1 T cells decreased the number of viable bacteria in hCD1Tg/Rag-/- recipient mice in all tested organs as compared to mice that received no DN1 T cell transfer. Moreover, adoptive transfer of DN1 T cells to Rag-/- recipient mice did not significantly reduce bacterial burdens suggesting that DN1 T cells confer protection in an hCD1Tg-dependent manner (Figure 7A). We also compared the protective capacity of DN1 T cells with non-relevant Listeria LemA-specific H2-M3-restricted D7 T cells (Figure 7—figure supplement 1). D7 T cells did not significantly reduce bacterial burdens in the lung of hCD1Tg/Rag-/- recipient mice compared to mice that received no T cell transfer (Figure 7—figure supplement 1). Figure 7 with 1 supplement see all Download asset Open asset DN1 T cells contribute to protective immunity against Mtb infection. Effector DN1 T cells (5-7x106 cells) were transferred into hCD1Tg/Rag-/- or Rag-/- mice 1 day before infection. Mice were euthanized at 4 week post-infection. (A) The number of bacteria in the lung, spleen and liver of individual mouse in each group. Horizontal bars represent the mean CFU counts ± SEM for each group. (B, C) Cells harvested from lungs of hCD1Tg/Rag-/- or Rag-/- mice were stimulated with un-pulsed or MA-pulsed DC and intracellular stained for the indicated cytokines (B) and CD107a expression (C). (D) Immunohistochemistry staining of anti-CD3 (brown cells) of the lung section from indicated groups. Pictures show granuloma area in infected lung tissues. (E) Bar graphs depict the mean and SEM of number of CD3+ cells per mm2 within granuloma areas (n=3–6 mice each group). ***p<0.001; **p<0.01; *p<0.05. https://doi.org/10.7554/eLife.08525.010 DN1 T cells isolated from the lung of infected hCD1Tg/Rag-/- mice produced multiple cytokines (e.g. TNF-α, IFN-γ, and IL-2, Figure 7B) and expressed CD107a, a surrogate marker of cytotoxic activity (Cho et al., 2011), after ex vivo MA stimulation (Figure 7C). To further visualize the location and distribution of DN1 T cells, DN1 T cells in the lung were stained by immunohistochemistry using anti-CD3 antibody. A significantly higher number of DN1 T cells were seen within pulmonary granulomas of hCD1Tg/Rag-/- mice that received DN1 T cells compared to control groups (Figure 7D,E). In summary, these data demonstrate that DN1 T cells accumulate in granulomas and contribute to protective immunity against Mtb by producing multiple Th1-related cytokines and exerting cytotoxicity. Discussion In this stud
Abstract Psoriasis, a chronic inflammatory skin disease, is associated with hyperlipidemia. While conventional T cells usually exacerbate psoriasis, the role of self-lipid reactive CD1-restricted T cells requires further study. CD1 molecules present lipid antigens to T cells and are divided in to two groups. Group 1 includes CD1a, CD1b, CD1c while CD1d belongs to group 2. Humans express both group 1 and group 2 CD1 molecules whereas mice only have CD1d. Thus, due to the lack of a suitable animal model, the role of autoreactive group 1 CD1-restricted T cells in hyperlipidemia-associated inflammatory diseases is unknown. To overcome this challenge, our lab generated mice that expressed human CD1b and a CD1b-autoreactive T cell receptor (hCD1Tg/HJ1Tg). hCD1Tg/HJ1Tg mice were crossed to the ApoE-/- background to examine the role of CD1b-restricted T cells in hyperlipidemia (hCD1Tg/HJ1Tg/ApoE-/-). Interestingly, hCD1Tg/HJ1Tg/ApoE-/- mice developed severe psoriasis-like skin inflammation characterized by T cell and neutrophil infiltration along with a Th17-biased cytokine milieu. They also showed significantly increased polar lipid accumulation in the skin as compared to hCD1Tg/HJ1Tg/ApoE+ mice. This suggested that the presence of excessive lipids in the skin resulted in activation of CD1b-autoreacitve T cells, leading to the onset of skin pathology. This work demonstrates that autoreactive group 1 CD1-reactive T cells might serve as a link between inflammatory processes and hyperlipidemia.
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