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• W3095406654 abstract "•Lysosomal acid lipase (LAL) is required for donor T cells to induce GVHD•LAL regulates T cell immunity in GVHD target organs•Pharmacological blockade of LAL effectively ameliorates GVHD•Pharmacological blockade of LAL preserves GVL activity Graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT). Lysosomal acid lipase (LAL) mediates the intrinsic lipolysis of cells to generate free fatty acids (FFAs), which play an essential role in the development, proliferation, and function of T cells. Here, we find that LAL is essential for donor T cells to induce GVHD in murine models of allo-HCT. Specifically, LAL is required for donor T cell survival, differentiation, and alloreactivity in GVHD target organs, but not in lymphoid organs. LAL induces the differentiation of donor T cells toward GVHD pathogenic Th1/Tc1 and Th17 while suppressing regulatory T cell generation. LAL−/− T cells succumb to oxidative stress and become anergic in target organs. Pharmacologically targeting LAL effectively prevents GVHD development while preserving the GVL activity. Thus, the present study reveals the role of LAL in T cell alloresponse and pathogenicity and validates LAL as a target for controlling GVHD and tumor relapse after allo-HCT. Graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT). Lysosomal acid lipase (LAL) mediates the intrinsic lipolysis of cells to generate free fatty acids (FFAs), which play an essential role in the development, proliferation, and function of T cells. Here, we find that LAL is essential for donor T cells to induce GVHD in murine models of allo-HCT. Specifically, LAL is required for donor T cell survival, differentiation, and alloreactivity in GVHD target organs, but not in lymphoid organs. LAL induces the differentiation of donor T cells toward GVHD pathogenic Th1/Tc1 and Th17 while suppressing regulatory T cell generation. LAL−/− T cells succumb to oxidative stress and become anergic in target organs. Pharmacologically targeting LAL effectively prevents GVHD development while preserving the GVL activity. Thus, the present study reveals the role of LAL in T cell alloresponse and pathogenicity and validates LAL as a target for controlling GVHD and tumor relapse after allo-HCT. Graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT) (Ferrara et al., 2009Ferrara J.L. Levine J.E. Reddy P. Holler E. Graft-versus-host disease.Lancet. 2009; 373: 1550-1561Abstract Full Text Full Text PDF PubMed Scopus (1756) Google Scholar). Cell metabolism determines T cell fate and function by regulating nutrition intake and transcription factor expression (Buck et al., 2015Buck M.D. O’Sullivan D. Pearce E.L. T cell metabolism drives immunity.J. Exp. Med. 2015; 212: 1345-1360Crossref PubMed Scopus (716) Google Scholar). The metabolic characteristics of pathogenic T cells are different in various immunological diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and colitis (Biniecka et al., 2011Biniecka M. Fox E. Gao W. Ng C.T. Veale D.J. Fearon U. O’Sullivan J. Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.Arthritis Rheum. 2011; 63: 2172-2182Crossref PubMed Scopus (79) Google Scholar; Gerriets et al., 2014Gerriets V.A. Kishton R.J. Nichols A.G. Macintyre A.N. Inoue M. Ilkayeva O. Winter P.S. Liu X. Priyadharshini B. Slawinska M.E. et al.Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation.J. Clin. Invest. 2014; 125: 194-207Crossref PubMed Scopus (442) Google Scholar; Wahl et al., 2010Wahl D.R. Petersen B. Warner R. Richardson B.C. Glick G.D. Opipari A.W. Characterization of the metabolic phenotype of chronically activated lymphocytes.Lupus. 2010; 19: 1492-1501Crossref PubMed Scopus (66) Google Scholar; Yang et al., 2013Yang Z. Fujii H. Mohan S.V. Goronzy J.J. Weyand C.M. Phosphofructokinase deficiency impairs ATP generation, autophagy, and redox balance in rheumatoid arthritis T cells.J. Exp. Med. 2013; 210: 2119-2134Crossref PubMed Scopus (224) Google Scholar). Among these diseases, colitis shares many immunological similarities with gut GVHD, which is the most common GVHD target organ, potentially leading to life-threatening complications (Naymagon et al., 2017Naymagon S. Naymagon L. Wong S.Y. Ko H.M. Renteria A. Levine J. Colombel J.F. Ferrara J. Acute graft-versus-host disease of the gut: considerations for the gastroenterologist.Nat. Rev. Gastroenterol. Hepatol. 2017; 14: 711-726Crossref PubMed Scopus (76) Google Scholar). Fatty acid (FA) metabolism has been implicated in GVHD development after allo-HCT. A study by Gatza et al., 2011Gatza E. Wahl D.R. Opipari A.W. Sundberg T.B. Reddy P. Liu C. Glick G.D. Ferrara J.L. Manipulating the bioenergetics of alloreactive T cells causes their selective apoptosis and arrests graft-versus-host disease.Sci. Transl. Med. 2011; 3: 67ra8Crossref PubMed Scopus (134) Google Scholar demonstrated that the oxidation of FAs (FAO) in mitochondria is responsible for the generation of alloreactive T cells, which are the driving force in GVHD. Therefore, blocking FAO via targeting mitochondrial F(1)F(0) adenosine triphosphate synthase (F(1)F(0)-ATPase) or Cpt1a (the enzyme responsible for FA uptake into mitochondria) (Byersdorfer et al., 2013Byersdorfer C.A. Tkachev V. Opipari A.W. Goodell S. Swanson J. Sandquist S. Glick G.D. Ferrara J.L. Effector T cells require fatty acid metabolism during murine graft-versus-host disease.Blood. 2013; 122: 3230-3237Crossref PubMed Scopus (106) Google Scholar) induces the apoptosis of alloreactive T cells. However, no attempt has been made to block the resources of cytosolic FAs for tricarboxylic acid (TCA)-dependent FAO in mitochondria to control GVHD. Lipolysis of stored lipids generates FAs that can be used as energy substrates through FAO in the TCA cycle (Zechner et al., 2012Zechner R. Zimmermann R. Eichmann T.O. Kohlwein S.D. Haemmerle G. Lass A. Madeo F. FAT SIGNALS--lipases and lipolysis in lipid metabolism and signaling.Cell Metab. 2012; 15: 279-291Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar). Several enzymes regulate the release of FAs from lipid droplets under changing nutrition state. Lysosomal acid lipase (LAL) is an intracellular lipase that catalyzes the hydrolysis of cholesteryl esters and triglycerides in lysosomes at acidic pH (Qu et al., 2009Qu P. Du H. Wilkes D.S. Yan C. Critical roles of lysosomal acid lipase in T cell development and function.Am. J. Pathol. 2009; 174: 944-956Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). LAL plays a central role in lipid metabolism in lymphocytes and is required for the normal development, maturation, and functionality of this type of cell (Qu et al., 2009Qu P. Du H. Wilkes D.S. Yan C. Critical roles of lysosomal acid lipase in T cell development and function.Am. J. Pathol. 2009; 174: 944-956Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In addition, in the absence of LAL, T cell receptor (TCR) activation, T cell proliferation, and cytokine secretion are tremendously impaired (Schlager et al., 2017Schlager S. Vujic N. Korbelius M. Duta-Mare M. Dorow J. Leopold C. Rainer S. Wegscheider M. Reicher H. Ceglarek U. et al.Lysosomal lipid hydrolysis provides substrates for lipid mediator synthesis in murine macrophages.Oncotarget. 2017; 8: 40037-40051Crossref PubMed Scopus (33) Google Scholar). LAL supports the metabolic reprogramming necessary for CD8 memory (CD8mem) development (O’Sullivan et al., 2014O’Sullivan D. van der Windt G.J. Huang S.C. Curtis J.D. Chang C.H. Buck M.D. Qiu J. Smith A.M. Lam W.Y. DiPlato L.M. et al.Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development.Immunity. 2014; 41: 75-88Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). However, how LAL regulates alloreactive T cell metabolism, survival, activation, and GVHD pathogenesis has not been studied. Recently, LAL has been shown to affect T cell differentiation, as CD4 T cells deficient for LAL have a reduced ability to differentiate into T helper 1 and 2 (Th1/Th2) cells while increasing the generation of regulatory T cells (Tregs) (Qu et al., 2009Qu P. Du H. Wilkes D.S. Yan C. Critical roles of lysosomal acid lipase in T cell development and function.Am. J. Pathol. 2009; 174: 944-956Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Because Th1 cells are pathogenic and Tregs are suppressive in GVHD (Nguyen et al., 2018bNguyen H.D. Kuril S. Bastian D. Yu X.Z. T-Cell Metabolism in Hematopoietic Cell Transplantation.Front. Immunol. 2018; 9: 176Crossref PubMed Scopus (20) Google Scholar), LAL targeting may be beneficial for controlling GVHD. In the present study, we found that LAL was required for donor T cells to induce GVHD after allo-HCT. LAL-deficient T cells retained sufficient anti-tumor activity to prevent tumor relapse. The pharmacological blockade of LAL effectively prevented or treated GVHD while maintaining the graft versus leukemia (GVL) effect. Our study therefore validated LAL in T cells as a potential target for controlling GVHD and tumor relapse after allo-HCT. Given that LAL-specific inhibitors have been traditionally used for the prevention or treatment of obesity in clinics, the outcome of this study is of high translational potential. FAs serve not only as fuel for cells but also as components of cell membrane phospholipids and glycolipids. In our previously published work, we found that donor T cells accumulated long-chain FAs in allogeneic recipients, which likely resulted from a decline in FAO and an increase in lipid hydrolysis (Nguyen et al., 2016Nguyen H.D. Chatterjee S. Haarberg K.M. Wu Y. Bastian D. Heinrichs J. Fu J. Daenthanasanmak A. Schutt S. Shrestha S. et al.Metabolic reprogramming of alloantigen-activated T cells after hematopoietic cell transplantation.J. Clin. Invest. 2016; 126: 1337-1352Crossref PubMed Scopus (76) Google Scholar). Among other enzymes, lysosomal acid lipase (LAL) is an important lipase responsible for hydrolyzing lipids in the droplets to free FAs and lysolipids during stress conditions (Gomaraschi et al., 2019Gomaraschi M. Bonacina F. Norata G.D. Lysosomal Acid Lipase: From Cellular Lipid Handler to Immunometabolic Target.Trends Pharmacol. Sci. 2019; 40: 104-115Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar; Rader, 2015Rader D.J. Lysosomal Acid Lipase Deficiency--A New Therapy for a Genetic Lipid Disease.N. Engl. J. Med. 2015; 373: 1071-1073Crossref PubMed Scopus (28) Google Scholar). Unlike regulatory or memory T cells, effector T cells are known to require appreciable amounts of extracellular-free FA (Nguyen et al., 2018bNguyen H.D. Kuril S. Bastian D. Yu X.Z. T-Cell Metabolism in Hematopoietic Cell Transplantation.Front. Immunol. 2018; 9: 176Crossref PubMed Scopus (20) Google Scholar; Tijaro-Ovalle et al., 2019Tijaro-Ovalle N.M. Karantanos T. Wang H.T. Boussiotis V.A. Metabolic Targets for Improvement of Allogeneic Hematopoietic Stem Cell Transplantation and Graft-vs.-Host Disease.Front. Immunol. 2019; 10: 295Crossref PubMed Scopus (16) Google Scholar). Furthermore, LAL was found to play a critical role in T cell development and function, while being dispensable for Tregs (Qu et al., 2009Qu P. Du H. Wilkes D.S. Yan C. Critical roles of lysosomal acid lipase in T cell development and function.Am. J. Pathol. 2009; 174: 944-956Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We therefore hypothesized that LAL is essential for T cell pathogenicity in the induction of GVHD. To test this hypothesis, we used LAL-deficient mice (LAL−/−) as donors in allogeneic bone marrow transplantation (allo-BMT). Although T cell development in the thymus was partially impaired in the absence of LAL (Qu et al., 2009Qu P. Du H. Wilkes D.S. Yan C. Critical roles of lysosomal acid lipase in T cell development and function.Am. J. Pathol. 2009; 174: 944-956Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), we verified that immune phenotypes, including percentages of T cells, B cells, monocytes, and multiple T cell subsets (memory and regulatory), were comparable in peripheral lymphoid organs of young mice, regardless of LAL expression (data not shown). We next examined the effect of LAL on T cell survival, proliferation, and activation under polyclonal activation by CD3/CD28 in vitro. Upon stimulation, LAL-deficient T cells (both CD4 and CD8) exhibited increased necrosis (higher percentage of 7AAD+annexin V+) (Figures 1A and 1C ), decreased proliferation (lower percentage of carboxyfluorescein diacetate succinimidyl ester [CFSE] diluted) (Figures 1B and 1D), lower levels of pro-inflammatory cytokine production (interferon γ [IFN-γ] and tumor necrosis factor α [TNF-α]) (Figures 1E and 1G), and decreased FA oxidation (lower percentage of peroxisome proliferator-activated receptor gamma, PPRγ+) (Figures 1F and 1H), as compared to wild-type (WT) counterparts. To understand the role of LAL in alloantigen-induced T cell response, we performed an in vitro mixed lymphocyte reaction (MLR) assay in which CFSE-labeled bulk T cells from LAL+/+ and LAL−/− FVB mice were stimulated with T cell-depleted (TCD) splenocytes from BALB/c mice. Consistent with polyclonal stimulation, LAL−/− CD4+ T cells had less cell survival, reduced proliferation reflected by lower Ki67 expression and CFSE, and reduced activation reflected by low levels of IFN-γ production as compared to WT T cells (Figures S1A and S1B). Under this condition, LAL−/− CD4+ T cells also exhibited lower lipid content, FAO, reactive oxidative species (ROS) formation, and co-inhibitory molecules, which were reflected by BODIPY uptake, Cpt1a expression, 2′,7′-dichlorofluorescin diacetate (DCFDA) intensity, and programmed cell death-1 (PD-1)/Lag3 expression, respectively (Figure S1C). We interpret that these reductions resulted from lower T cell activation in the absence of LAL. Similar trends were observed on LAL−/− CD8+ T cells, although the levels of compromise were less profound (data not shown). The dysfunction of LAL−/− CD4+ T cell responses may be due to reduced survival in culture. To test the hypothesis, we stimulated LAL +/+ and LAL−/− T cells with allogenic antigen-presenting cells (APCs) as in Figure S1, but also supplemented N-acetyl cysteine (NAC), a known antioxidant. We found that additional NAC significantly reduced proliferation, IFN-γ production and PD-1 expression of LAL+/+, but not LAL−/− T cells (Figure S2). These results are consistent with higher ROS generation by LAL+/+ T cells, and suggest that inferior survival may not be the reason for T cell dysfunction in the absence of LAL. The dysfunction of LAL−/− CD4+ T cell responses may be due to impaired lipid metabolism. To test this hypothesis, we used media containing lipid-free serum (LF). Interestingly, while LF media reduced the proliferation and activation of LAL+/+ T cells, it increased the proliferation and activation of LAL−/− T cells (Figures S2A and S2B). These results indicate that external lipids facilitate optional T cell activation, but LAL dictates its contribution to T cell response. Because T cell survival and activation are required for donor T cells to induce GVHD, we hypothesized that targeting LAL in donor T cells would alleviate GVHD severity. Using major histocompatibility complex (MHC)-mismatched B6 (H2b) → BALB/c (H2d) and FVB (H2q)→B6 (H2b) BMT models, we found that LAL-deficient donor T cells induced significantly less severe GVHD compared with WT control T cells, which is reflected by recipient survival and clinical scores (Figures 2A–2D). Because the primary purpose of allo-HCT is to eradicate hematological malignance (Bleakley and Riddell, 2004Bleakley M. Riddell S.R. Molecules and mechanisms of the graft-versus-leukaemia effect.Nat. Rev. Cancer. 2004; 4: 371-380Crossref PubMed Scopus (319) Google Scholar), we asked whether LAL-deficient T cells still retained sufficient activity to mediate the GVL response. Using luciferase-transduced P815 mastocytoma in the haploidentical B6 (H2b) →BDF1 (H2b/d) BMT model, we observed that all of the recipients transplanted with T cell-depleted bone marrow cells (TCD-BMs) plus P815 died of tumor relapse, as indicated by strong luminescent signals (Figures 2E and 2G). The recipients with additional LAL+/+ T cells all died within 20 days after BMT without tumor signal and significant GVHD severity (Figures 2E–2G), indicating that they died from GVHD, not tumor relapse. In contrast, 50% of the recipients with additional LAL−/− T cells survived without GVHD clinical signs until day 60 after transplant, and the majority of them were tumor free (Figures 2E and 2G). These data suggest that LAL is essential in donor T cells to induce GVHD, while it is dispensable for the GVL effect. For translational purposes, we tested the LAL-specific inhibitor of LAL orlistat (Huang et al., 2014Huang S.C. Everts B. Ivanova Y. O’Sullivan D. Nascimento M. Smith A.M. Beatty W. Love-Gregory L. Lam W.Y. O’Neill C.M. et al.Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages.Nat. Immunol. 2014; 15: 846-855Crossref PubMed Scopus (680) Google Scholar) on the T cell alloresponse. Similar to LAL deficiency, we found that orlistat significantly inhibited proliferation, activation, and FA metabolism of CD4 and CD8 T cells, as reflected by Ki67 expression, IFN-γ production, and Cpt1a expression, respectively (Figures S3A–S3D). It is worth noting that orlistat treatment also increased Foxp3 expression on CD4 T cells, although not as profoundly as in the absence of LAL (Figure S3C). These results demonstrate that LAL can be targeted pharmacologically through orlistat. We next assessed the effect of orlistat GVH and GVL responses. By titrating the doses, orlistat was found to be not toxic when administrated to BMT recipients at 8–20 mg/kg/day for 14 days (Figure 3A). Treatment with orlistat effectively prevented GVHD development in recipients, indicated by the improvement of survival and lower clinical scores (Figures 3A and 3B). To investigate the effect of orlistat on the GVL activity against leukemia and lymphoma, we used two GVHD/GVL models. In the B6 to BDF1 model with P815 leukemia, all of the recipients with BM alone died from tumor relapse, reflected by heavy tumor signals before death (Figures 3C–3E). The recipients transplanted with donor T cells and treated with vehicle died from GVHD with little tumor signal (Figure 3E). However, orlistat treatment rescued 40% of the recipients from lethal GVHD and tumor relapse (Figures 3C–3E). To extend the observations to a different model, we used the B6 to BALB/c BMT model in combination with host-derived mixed-lineage leukemia (MLL-AF9). Orlistat treatment improved recipient survival and decreased GVHD clinical signs (Figures S4A and S4B). As expected, all of the recipients with BM alone died from tumor relapse, reflected by MLL in peripheral blood but no GVHD clinical signs before death (Figure S4A–S4D). Orlistat treatment did not alter the outcomes of the recipients with BM alone, indicating that orlistat had little or no direct effect on leukemia growth. However, orlistat treatment maintained the GVL effect, as the recipients under orlistat treatment did not exhibit leukemia (Figures S4C and S4D). These results illustrate that pharmacologically targeting LAL may be an effective approach for the control of GVHD while sparing the GVL effect. To understand how LAL affects T cell immunity in vivo, we performed allo-BMT and analyzed donor T cells in recipient spleens (Figure 4) and guts (Figure 5) 7 days after transplant. Consistent with our observations in vitro (Figure S1), LAL−/− donor CD4 T cells significantly reduced proliferation (Ki67), migration potential (CXCR3), and intercellular lipid content (BODIPY), but increased Treg generation (Foxp3), compared to LAL+/+ CD4 T cells in recipient spleens (Figures 4B, 4E–4G, and 4I). In contrast to CD4 T cells, the deficiency of LAL had little effect on donor CD8 T cells (Figures 4C–4J). The treatment of recipients with orlistat essentially phenocopied LAL deficiency, except for increasing Treg generation (Figure 4G). Unlike in vitro culture (Figure S1), neither LAL deficiency or orlistat treatment had a significant effect on ROS generation (DCFDA and PD-1 expression of donor T cells (Figures 4H and 4J).Figure 5The Effect of LAL on T Cells in Recipient Intestines during GVHDShow full captionAllo-HCT was set up as described in Figure 4. After 7 days, mice were euthanized, and their intestines were collected. Isolated cells from the intestines were stained and analyzed with flow cytometry.(A) Cells were gated on live donor CD4+ or CD8+ cells for downstream analysis.(B and C) The frequency of CXCR3+ or CD4+/Foxp3+, as well as the mean fluorescence intensity of BODIPY, PD-1, and DCFDA were measured for CD4+ (B) and CD8+ (C) cells.(D–G) Summary graphs of the data.The data are presented as means ± SEMs. The data shown are from 1 of 3 replicate experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Allo-HCT was set up as described in Figure 4. After 7 days, mice were euthanized, and their intestines were collected. Isolated cells from the intestines were stained and analyzed with flow cytometry. (A) Cells were gated on live donor CD4+ or CD8+ cells for downstream analysis. (B and C) The frequency of CXCR3+ or CD4+/Foxp3+, as well as the mean fluorescence intensity of BODIPY, PD-1, and DCFDA were measured for CD4+ (B) and CD8+ (C) cells. (D–G) Summary graphs of the data. The data are presented as means ± SEMs. The data shown are from 1 of 3 replicate experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. As the gut is a main target organ in GVHD, we examined donor T cells in recipient intestines (Figure 5). Unlike in recipient spleens, donor LAL−/− T cells (H2Kd−), especially CD4, significantly decreased in recipient guts as compared to WT counterparts (Figures 5A and 5D). Given that Ki67 expression was comparable (data not shown), we interpret that reduced LAL−/− T cells in recipient guts were due to impaired survival and/or migration (Figures 5B, 5C, and 5F). In sharp contrast to T cells in the spleen, both donor CD4 and CD8 T cells deficient for LAL in the gut had significantly increased intracellular lipid content reflected by BODIPY uptake (Figures 5B, 5C, and 5G), suggesting that T cells require LAL to hydrolyze lipid in the gut. Similarly, donor T cells deficient for LAL also had significantly increased ROS generation reflected by DCFDA intensity in recipient guts (Figures 5B, 5C, and 5H). However, an increase in the level of T cell exhaustion was seen, as reflected by PD-1 expression (Figure 5B, 5C, and 5I). Consistent with results observed in the spleen, Treg generation was also increased in LAL−/− CD4 T cells in the recipient gut (Figures 5B and 5E). By and large, the treatment of recipients with orlistat simulated LAL deficiency, with notable exceptions where orlistat treatment significantly decreased CXCR3 expression and failed to increase Foxp3 expression on donor T cells that differed from the effects of LAL deficiency (Figures 5E and 5F). We observed that LAL regulated T cell response differentially in lymphoid (spleen) and in target (gut) organs in the development of GVHD, which likely results from distinct microenvironments. At 7 days after BMT, we also examined the effects of LAL on cytokine production by donor T cells, but we did not find a major impact of LAL deficiency or orlistat treatment on IFNγ or IL-17 production either in recipient spleens or guts (data not shown). Given that GVHD severity was markedly reduced when targeting LAL either genetically or pharmacologically, we reasoned that the impact of LAL would be more profound later after BMT. We examined the effects of LAL deficiency in T cell activation, cytokine production, and metabolism in recipient spleens 13 days after BMT before the recipients of WT T cells. As shown in Figure S5A, the recipients of LAL−/− T cells had significantly lower clinical scores and body weight losses than those receiving LAL+/+ T cells, confirming a reduced pathogenicity of LAL−/− T cells in the induction of GVHD. We measured mammalian target of rapamycin complex 1 (mTORC1) activity as it reflects T cell activation, and found it was comparable between LAL−/− and LAL+/+ T cells, as demonstrated by the significantly increased density of phospho-S6 (pS6) in donor CD4 and CD8 T cells (Figure S5B). Interestingly, the mitochondrial membrane potential (Figure S5C), mitochondrial number (Figure S5F), and respiration (Figure S5G) were increased in LAL−/− T cells. The migration of donor lymphocytes toward GVHD target organs is required for GVHD development. Chemokine receptors expressed on the surface of lymphocytes is responsible for the trafficking of these cells toward target organs (Zeiser and Blazar, 2017Zeiser R. Blazar B.R. Acute Graft-versus-Host Disease - Biologic Process, Prevention, and Therapy.N. Engl. J. Med. 2017; 377: 2167-2179Crossref PubMed Scopus (573) Google Scholar). We found that LAL−/− CD4 T cells expressed lower gut/lung homing chemokine CXCR3 as compared to that of LAL+/+ CD4 T cells in recipient spleens (Figures S5D and S5E). Cytokine production including IFNγ and IL-17 was not different between LAL−/− and LAL+/+ T cells in recipient spleens (data not shown). These results indicate that LAL−/− T cells did not impair T cell activation and metabolism, but did reduce the T cell migration potential. We next focused on examining the impact of LAL in T cells of the gut, a main GVHD target organ. Gut damage caused by conditioning, such as total body irradiation (TBI), triggers systemic GVHD development (Naymagon et al., 2017Naymagon S. Naymagon L. Wong S.Y. Ko H.M. Renteria A. Levine J. Colombel J.F. Ferrara J. Acute graft-versus-host disease of the gut: considerations for the gastroenterologist.Nat. Rev. Gastroenterol. Hepatol. 2017; 14: 711-726Crossref PubMed Scopus (76) Google Scholar). Given that cell death is a valid indicator of gut damage (Jalili-Firoozinezhad et al., 2018Jalili-Firoozinezhad S. Prantil-Baun R. Jiang A. Potla R. Mammoto T. Weaver J.C. Ferrante T.C. Kim H.J. Cabral J.M.S. Levy O. Ingber D.E. Modeling radiation injury-induced cell death and countermeasure drug responses in a human Gut-on-a-Chip.Cell Death Dis. 2018; 9: 223Crossref PubMed Scopus (106) Google Scholar), we isolated intestinal cells and examined their viability. Both percentage and absolute number of necrotic cells (7-AAD+annexin V+) among host non-hematopoietic cells were significantly lower in the recipients of LAL−/− T cells (Figures 6A and 6B ), suggesting that LAL−/− T cells caused less gut damage. T cell differentiation into pathogenic phenotypes is critical for the development of GVHD. As LAL was previously to be involved in T cell differentiation (Qu et al., 2009Qu P. Du H. Wilkes D.S. Yan C. Critical roles of lysosomal acid lipase in T cell development and function.Am. J. Pathol. 2009; 174: 944-956Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), we assessed the impact of LAL in donor T cell differentiation in vivo. In recipient guts, LAL-deficient T cells exhibited reduced percentages of IFN-γ+ and IL-17+ cells while increasing Foxp3+ cells (Figures 6C–6E), suggesting that LAL promotes Th1/Th17 cell differentiation while suppressing Treg generation. These data suggest that LAL regulates GVHD pathogenicity via modulating donor T cell activation, differentiation, and migration in the recipient gut. We next examined how LAL affects donor T cell fate in the recipient gut. We observed that donor T cells deficient for LAL exhibited significantly higher percentages of necrosis, reflected by 7-AAD+/annexin V+ cells (Figure 7A) and lower levels of proliferation, reflected by Ki67 expression (Figure 7B). Oxidative stress has been implicated in T cell death in GVHD target organs. Free FAs (FFAs) that are generated through lipid hydrolysis and subsequently undergo FAO in mitochondria reduce oxidative stress (Galicia-Vázquez and Aloyz, 2018Galicia-Vázquez G. Aloyz R. Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes.Front. Oncol. 2018; 8: 411Crossref PubMed Scopus (25) Google Scholar). We therefore examined oxidative stress levels via DCFDA staining and found that donor CD4 T cells deficient for LAL showed higher levels of oxidative stress, reflected by higher levels of DCFDA uptake (Figure 7C). LAL deficiency likely caused lipid accumulation in donor T cells in recipient gut tissue as lipid content, reflected by BODIPY uptake, was increased in both donor CD4 and CD8 T cells (Figure 7D). In addition, cpt1a expression levels were significantly decreased in LAL−/− CD4 T cells, suggesting that FAO was decreased in the absence of LAL (Figure 7E). We also observed that exhaustion markers such as PD-1 and LAG-3 were significantly increased in LAL−/− T cells (Figures 7F and 7G). These data suggest that donor T cells in target organs are in a state of anergy/exhaustion in the absence of LAL. Autophagy is required for T cells to survive and maintain function in stressful conditions, such as GVHD target organ environment (Le Texier et al., 2016Le Texier L. Lineburg K.E. Cao B. McDonald-Hyman C. Leveque-El Mouttie L. Nicholls J. Melino M. Nalkurthi B.C. Alexander K.A. Teal B. et al.Autophagy-dependent regulatory T cells are critical" @default.
• W3095406654 created "2020-11-09" @default.
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• W3095406654 creator A5013411286 @default.
• W3095406654 creator A5014068004 @default.
• W3095406654 creator A5027881339 @default.
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• W3095406654 date "2020-10-01" @default.
• W3095406654 modified "2023-10-16" @default.
• W3095406654 title "Lysosomal Acid Lipase Is Required for Donor T Cells to Induce Graft-versus-Host Disease" @default.
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