Stim1 Deletion Synthetically Rescues Ezh2-Null Effector T Cells and Alloimmunity

Abstract Graft-versus-host disease (GVHD) remains a major barrier for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have identified the central role of the histone methyltransferase Ezh2 in regulating allogeneic T-cell expansion, differentiation and function. Conditional loss of Ezh2 in donor T cells inhibits GVHD in mice due to the inability of alloreactive T cells to persist. However, the molecular mechanism by which Ezh2 deficiency causes alloreactive T cell death remains unknown. Here we demonstrate that genetic deletion of Stromal Interaction Molecule (Stim) 1, a dynamic endoplasmic reticulum Ca2+ sensor and regulator of Ca2+ signaling, rescues antigen-activated Ezh2-null (Ezh2-/-) T cells, leading to restored persistence of alloreactive effector T cells in mice and severe GVHD. Using RNA-sequencing analysis, we found Ezh2-deficiency led to the upregulation of multiple genes (e.g., Ifng, Prf1, Ccl5, Ccl4, Upp1 and Spp1) known to be regulated by Ca2+ signals through calcineurin (CN), the primary target of the immunosuppressant cyclosporine A (CsA). This reverse correlation between Ezh2 inhibition and CsA-treatment for gene expression suggests that Ezh2 may antagonize Ca2+ signaling in activated T cells. Calcium signaling assays revealed higher cytosolic Ca2+ uptake and more frequent Ca2+ oscillations in Ezh2-/- T cells. Moreover, Ezh2-/- T cells exhibited significantly increased polarization of Stim1 and Orai1 in the cellular membrane. These data reveal an unexpected role of Ezh2 as a negative regulator of Ca2+ entry, thereby serving as a 'brake' for Ca2+ signaling. Using the C57BL/6 (B6) into Balb/c mouse GVHD model, we found significantly fewer Ezh2-/- or Stim1-/- IFN-g-secreting effector T cells compared to the WT counterparts on day 8 or 14 post-transplantation. In contrast, deleting Stim1 from Ezh2-/- donor T cells rescued the cells in the spleen and liver, producing even more donor T cells and IFN-g-secreting effector T cells compared to WT T cells and inducing severe GVHD. We further examined the cell autonomous effect of Stim1 deletion on the rescue of Ezh2-/- T cells by mixing WT T cells (B6/SJL, CD45.1) with Ezh2- and/or Stim1- conditional knockout T cells (i.e., Ezh2-/-, Stim1-/- or Ezh2-/- x Stim1-/- B6 T cells (CD45.2)) at a ratio of 1:1 before transferring into the Balb/c mice. While loss of either Ezh2 or Stim1 led to lower frequency of IFN-g+IL-2+ effector T cells, combined deletion of both genes restored the frequency and number of IFN-g+IL-2+ effector T cells to that of WT T cells. Thus, Stim1-mediated Ca2+ signals are crucial for mediating cell death in alloantigen-driven Ezh2-/- effector T cells. To further determine whether the inhibition of CN-NFAT contributes to the rescue, we treated T cell receptor (TCR)-activated Ezh2-/- T cells with CsA or the calcium release-activated channel specific inhibitor BTP2, respectively, in vitro. While BTP2 dramatically improved the survival of IFN-g-producing effector T cells, CsA did not, suggesting the involvement of CN-NFAT-independent pathways. Ca2+ overload is known to impair mitochondrial function and cause massive cell death. As compared to TCR-activated WT T cells, activated Ezh2-/- T cells displayed significantly less ATP, lower mitochondrial membrane potential, enlarged mitochondrial mass, and decreased capacity to upregulate oxidative phosphorylation. Stim1 deletion largely reversed the metabolic defect in Ezh2-/- T cells, indicating the critical role of mitochondrial metabolism in rescuing these T cells. Considered together, our findings identify the remarkable coordination between Ezh2- and Stim1-regulated effector T cell persistence. As such, these investigations may lead to new approaches to inhibit GVHD, with broad implications to defining fundamental mechanisms of T cell differentiation for control of adaptive immunity, such as tumor immunity and autoimmunity. Disclosures Reshef: Incyte: Consultancy; Takeda Pharmaceuticals: Consultancy; Pfizer: Consultancy; Kite Pharma: Consultancy; Atara Biotherapeutics: Consultancy; Bristol-Myers Squibb: Consultancy.

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TIM-3 drives temporal differences in restimulation-induced cell death sensitivity in effector CD8+ T cells in conjunction with CEACAM1

Cell Death and Disease ◽

10.1038/s41419-021-03689-6 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Camille M. Lake ◽

Kelsey Voss ◽

Bradly M. Bauman ◽

Katherine Pohida ◽

Timothy Jiang ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Late Stage ◽

Surface Expression ◽

Effector T Cells ◽

T Cell Response ◽

Tcr Signaling ◽

Activated T Cells

AbstractImmune homeostasis depends upon effective clearance of pathogens while simultaneously preventing autoimmunity and immunopathology in the host. Restimulation-induced cell death (RICD) is one such mechanism where by activated T cells receive subsequent antigenic stimulation, reach a critical signal threshold through the T cell receptor (TCR), and commit to apoptosis. Many details of this process remain unclear, including the role of co-stimulatory and co-inhibitory proteins that influence the TCR signaling cascade. Here we characterize the role of T cell immunoglobulin and mucin domain containing 3 (TIM-3) in RICD regulation. TIM-3 protected newly activated CD8+ effector T cells from premature RICD during clonal expansion. Surprisingly, however, we found that TIM-3 potentiated RICD in late-stage effector T cells. The presence of TIM-3 increased proximal TCR signaling and proapoptotic protein expression in late-stage effector T cells, with no consistent signaling effects noted in newly activated cells with or without TIM-3. To better explain these differences in TIM-3 function as T cells aged, we characterized the temporal pattern of TIM-3 expression in effector T cells. We found that TIM-3 was expressed on the surface of newly activated effector T cells, but remained largely intracellular in late-stage effector cells. Consistent with this, TIM-3 required a ligand to prevent early RICD, whereas ligand manipulation had no effects at later stages. Of the known TIM-3 ligands, carcinoembryonic antigen‐related cell adhesion molecule (CEACAM1) showed the greatest difference in surface expression over time and also protected newly activated cells from premature RICD, with no measurable effects in late-stage effectors. Indeed, CEACAM1 enabled TIM-3 surface expression on T cells, implying a co-dependency for these proteins in protecting expanding T cells from premature RICD. Our findings suggest that co-signaling proteins like TIM-3 and CEACAM1 can alter RICD sensitivity at different stages of the effector T cell response, with important implications for checkpoint blockade therapy.

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Graded RhoA GTPase Expression in Treg Cells Distinguishes Tumor Immunity From Autoimmunity

Frontiers in Immunology ◽

10.3389/fimmu.2021.726393 ◽

2021 ◽

Vol 12 ◽

Author(s):

Khalid W. Kalim ◽

Jun-Qi Yang ◽

Vishnu Modur ◽

Phuong Nguyen ◽

Yuan Li ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Treg Cell ◽

Tumor Immunity ◽

Th17 Cell ◽

Treg Cells ◽

Effector T Cells ◽

Conditional Knockout ◽

Cell Plasticity ◽

Cell Homeostasis

RhoA of the Rho GTPase family is prenylated at its C-terminus. Prenylation of RhoA has been shown to control T helper 17 (Th17) cell-mediated colitis. By characterizing T cell-specific RhoA conditional knockout mice, we have recently shown that RhoA is required for Th2 and Th17 cell differentiation and Th2/Th17 cell-mediated allergic airway inflammation. It remains unclear whether RhoA plays a cell-intrinsic role in regulatory T (Treg) cells that suppress effector T cells such as Th2/Th17 cells to maintain immune tolerance and to promote tumor immune evasion. Here we have generated Treg cell-specific RhoA-deficient mice. We found that homozygous RhoA deletion in Treg cells led to early, fatal systemic inflammatory disorders. The autoimmune responses came from an increase in activated CD4+ and CD8+ T cells and in effector T cells including Th17, Th1 and Th2 cells. The immune activation was due to impaired Treg cell homeostasis and increased Treg cell plasticity. Interestingly, heterozygous RhoA deletion in Treg cells did not affect Treg cell homeostasis nor cause systemic autoimmunity but induced Treg cell plasticity and an increase in effector T cells. Importantly, heterozygous RhoA deletion significantly inhibited tumor growth, which was associated with tumor-infiltrating Treg cell plasticity and increased tumor-infiltrating effector T cells. Collectively, our findings suggest that graded RhoA expression in Treg cells distinguishes tumor immunity from autoimmunity and that rational targeting of RhoA in Treg cells may trigger anti-tumor T cell immunity without causing autoimmune responses.

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Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype

Blood ◽

10.1182/blood.v116.21.588.588 ◽

2010 ◽

Vol 116 (21) ◽

pp. 588-588

Author(s):

Karrune Woan ◽

Fengdong Cheng ◽

Hongwei Wang ◽

Jennifer Rock-Klotz ◽

Zi Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cell Function ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Egfp Expression ◽

In Vivo Models

Abstract Abstract 588 We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models. 1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100. 2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25. Disclosures: No relevant conflicts of interest to declare.

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An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes

Blood ◽

10.1182/blood-2009-06-225987 ◽

2010 ◽

Vol 115 (2) ◽

pp. 265-273 ◽

Cited By ~ 212

Author(s):

Graziella Curtale ◽

Franca Citarella ◽

Claudia Carissimi ◽

Marina Goldoni ◽

Nicoletta Carucci ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Cell Death ◽

T Cell ◽

T Lymphocytes ◽

T Cell Activation ◽

Cell Activation ◽

Interleukin 2 ◽

Activation Induced Cell Death

Abstract Activation of the T cell–mediated immune response has been associated with changes in the expression of specific microRNAs (miRNAs). However, the role of miRNAs in the development of an effective immune response is just beginning to be explored. This study focuses on the functional role of miR-146a in T lymphocyte–mediated immune response and provides interesting clues on the transcriptional regulation of miR-146a during T-cell activation. We show that miR-146a is low in human naive T cells and is abundantly expressed in human memory T cells; consistently, miR-146a is induced in human primary T lymphocytes upon T-cell receptor (TCR) stimulation. Moreover, we identified NF-kB and c-ETS binding sites as required for the induction of miR-146a transcription upon TCR engagement. Our results demonstrate that several signaling pathways, other than inflammation, are influenced by miR-146a. In particular, we provide experimental evidence that miR-146a modulates activation-induced cell death (AICD), acting as an antiapoptotic factor, and that Fas-associated death domain (FADD) is a target of miR-146a. Furthermore, miR-146a enforced expression impairs both activator protein 1 (AP-1) activity and interleukin-2 (IL-2) production induced by TCR engagement, thus suggesting a role of this miRNA in the modulation of adaptive immunity.

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β2- Adrenergic Signaling Regulates Graft Versus Host Disease after Allogenic Transplantation While Preserving Graft Versus Leukemia Effect

Blood ◽

10.1182/blood-2019-123139 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1915-1915 ◽

Cited By ~ 1

Author(s):

Hemn Mohammadpour ◽

Joseph L. Sarow ◽

George L. Chen ◽

Cameron R. MacDonald ◽

Umesh Sharma ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Adrenergic Receptor ◽

Graft Versus Host ◽

Graft Versus Leukemia ◽

Donor T Cells ◽

Β2 Adrenergic Receptor ◽

Ifn Γ

β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P< 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P<0.001). We showed that treatment increased the expression of IL-10 and decreased the expression of IFN-γ and IL-17 in CD4+ T cells. Interestingly, we found that β2-AR agonist treatment significantly increased the generation of myeloid derived suppressor cells (MDSCs) from WT BM without any effect on β2-AR-/- BM both in vitro and in vivo, suggesting an important role of β2-AR signaling in the generation of MDSCs. To investigate the effect of Bambuterol on GvL, the A20 lymphoma cell line was injected 4 hours before allo-HCT. Using two different doses of T cells (0.5 × 106 and 0.2 × 106) in B6 → BALB/c model, we found that Bambuterol preserved GvL by inducing CD44+ CD62L- NKG2D+ effector cells and CD44+ CD62L+ central memory cells. Since β2-AR agonists can affect cardiac function, we measured heart rate (HR) and blood pressure (BP) using a tail-cuff. There was no difference in BP and HR at day 21 and 28 after allo-HCT between mice receiving Bambuterol compared to mice receiving vehicle. In conclusion, these data reveal how β-AR signaling can influence donor T cell differentiation and function in murine GvHD models without decreasing GvL effect pointing to the feasibility of manipulation of β2-AR signaling to ameliorate clinical GvHD. Disclosures No relevant conflicts of interest to declare.

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The actin-capping protein alpha-adducin is required for T-cell costimulation

10.1101/580191 ◽

2019 ◽

Cited By ~ 1

Author(s):

Timothy J. Thauland ◽

Manish J. Butte

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Conditional Knockout ◽

Capping Protein ◽

Actin Capping Protein ◽

Actin Capping ◽

Spectrin Network

AbstractAlpha-adducin (Add1) is a critical component of the actin-spectrin network in erythrocytes, acting to cap the fast-growing, barbed ends of actin filaments, and recruiting spectrin to these junctions. Add1 is highly expressed in T cells, but its role in T-cell activation has not been examined. Using a conditional knockout model, we show that Add1 is necessary for complete activation of CD4+ T cells in response to low levels of antigen but is dispensable for CD8+ T cell activation and response to infection. Surprisingly, costimulatory signals through CD28 were completely abrogated in the absence of Add1. This study is the first to examine the role of actin-capping in T cells, and it reveals a previously unappreciated role for the actin cytoskeleton in regulating costimulation.

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Leveraging the Treg-intrinsic CTLA4–PKCη signaling pathway for cancer immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-002792 ◽

2021 ◽

Vol 9 (9) ◽

pp. e002792

Author(s):

Hsin-Yu Liu ◽

Christophe Pedros ◽

Kok-Fai Kong ◽

Ann J Canonigo-Balancio ◽

Wen Xue ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Antitumor Immunity ◽

Effector T Cells ◽

Clinical Approach ◽

Antitumor Effects ◽

Tumor Implantation ◽

Therapeutic Model

BackgroundOur previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCη) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These studies have employed adoptive transfer of germline PKCη-deficient (Prkch−/−) Tregs into Prkch+/+ mice prior to tumor implantation. Here, we extended these findings into a biologically and clinically more relevant context.MethodsWe have analyzed the role of PKCη in antitumor immunity and the tumor microenvironment (TME) in intact tumor-bearing mice with Treg-specific or CD8+ T cell-specific Prkch deletion, including in a therapeutic model of combinatorial treatment. In addition to measuring tumor growth, we analyzed the phenotype and functional attributes of tumor-infiltrating immune cells, particularly Tregs and dendritic cells (DCs).ResultsUsing two models of mouse transplantable cancer and a genetically engineered autochthonous hepatocellular carcinoma (HCC) model, we found, first, that mice with Treg-specific Prkch deletion displayed a significantly reduced growth of B16–F10 melanoma and TRAMP-C1 adenocarcinoma tumors. Tumor growth reduction was associated with a less immunosuppressive TME, indicated by increased numbers and function of tumor-infiltrating CD8+ effector T cells and elevated expression of the costimulatory ligand CD86 on intratumoral DCs. In contrast, CD8+ T cell-specific Prkch deletion had no effect on tumor growth or the abundance and functionality of CD8+ effector T cells, consistent with findings that Prkch−/− CD8+ T cells proliferated normally in response to in vitro polyclonal or specific antigen stimulation. Similar beneficial antitumor effects were found in mice with germline or Treg-specific Prkch deletion that were induced to develop an autochthonous HCC. Lastly, using a therapeutic model, we found that monotherapies consisting of Treg-specific Prkch deletion or vaccination with irradiated Fms-like tyrosine kinase 3 ligand (Flt3L)-expressing B16–F10 tumor cells post-tumor implantation significantly delayed tumor growth. This effect was more pronounced in mice receiving a combination of the two immunotherapies.ConclusionThese findings demonstrate the potential utility of PKCη inhibition as a viable clinical approach to treat patients with cancer, especially when combined with adjuvant therapies.

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Genomic Loss of the Mismatched HLA Locus in Leukemia Is a Major Mechanism of in Vivo Escape from T Cell Immunosurveillance Following Haploidentical HSCT

Blood ◽

10.1182/blood.v112.11.828.828 ◽

2008 ◽

Vol 112 (11) ◽

pp. 828-828 ◽

Cited By ~ 2

Author(s):

Luca Vago ◽

Serena Kimi Perna ◽

Monica Zanussi ◽

Benedetta Mazzi ◽

Maria Teresa Lupo Stanghellini ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Direct Evidence ◽

De Novo ◽

Patient Specific ◽

Post Transplant ◽

Donor T Cells ◽

Leukemia Relapse

Abstract Hematopoietic Stem Cell Transplantation (HSCT) from haploidentical family donors is a promising therapeutic option for nearly all patients suffering from high-risk leukemia. Until now, its application has been limited by the prolonged immunodeficiency that patients suffer as a consequence of graft T cell depletion, used to prevent severe Graft versus Host Disease (GvHD). When efficient strategies to control GvHD are applied, adoptive immunotherapy with donor T cells grants a significant advantage for immune reconstitution. However, direct evidence for the role of haploidentical donor T cells in controlling leukemia relapse is still missing. Here we report on the in vivo selection of de novo mutant variants of acute myeloid leukemia (AML), accounting for relapse after haploidentical HSCT and adoptive transfer of donor T cells. These novel variants of AML were observed in 5 out of 17 (29%) patients suffering from disease relapse in a series of 43 patients transplanted at the San Raffaele Hospital in Milan from 2002 to 2008. All patients received a myeloablative conditioning regimen and high doses of haploidentical donor stem cells (median 10.2×106 CD34+ cells/kg, range 4.6–15.5). Donor T lymphocytes were infused as part of the graft (n=21, median 438×106 CD3+ cell/kg, range 179–796) or as post-transplant add-backs (n=22, median 111×105 CD3+ cell/kg, range 1–900). Human Leukocyte Antigen (HLA) genomic typing was routinely used for post-transplant donor-recipient chimerism assessment. The five patients with de novo mutant variants of the original leukemia came to our attention because patient-specific HLA alleles could not be detected in bone marrow samples harvested at disease relapse, nor in subsequently sorted AML blasts. A Loss of Heterozygosity (LOH) study was performed on purified blasts from these patients, and demonstrated that patient-specific HLA alleles were lost due to extensive events of homologous recombination, encompassing a region of chromosome 6 comprising the entire HLA locus. We show that donor T cells capable of recognizing the original, HLA-heterozygous, leukemia were efficiently transferred from the haploidentical donor to the patient, granting an in vivo cytotoxic, cytokine and proliferative anti-tumor response by specific recognition of the mismatched HLA molecules. However, consistent with genomic loss of the patientspecific HLA locus in disease recurrence, the same alloreactive T cells were unable to recognize the mutant variant of the leukemia, harvested at the time of relapse. This observation strongly suggests that the genomic rearrangements we identified granted the disease an in vivo selective advantage in escaping from an established donor T cell response. Taken together, our data show that adoptive transfer of alloreactive donor T cells in haploidentical HSCT is efficient in providing a patient-specific antileukemic effect, and that the loss of this effect is an important mechanism underlying the outgrowth of relapsing disease. The frequency we documented for this phenomenon calls for routine assessment of the leukemia HLA genotype in the post-transplant follow-up and for careful consideration in the choice of a putative second haploidentical donor in case of leukemia relapse. Ultimately, our data provide the first direct evidence for the role of donor T cell alloreactivity in controlling minimal residual disease after haploidentical HSCT, favoring the use of donor T cell-based immunotherapeutic strategies to exploit alloreactivity for the cure of high-risk leukemia.

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The T Cell Cytolytic Molecules Fas Ligand and TRAIL, the Trafficking Molecules CCR9, β7 Integrin and PSGL-1, and the Immune Modulating Molecules OX40, CEACAM1, and CTLA4 Are Required for Thymic Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v112.11.65.65 ◽

2008 ◽

Vol 112 (11) ◽

pp. 65-65 ◽

Cited By ~ 5

Author(s):

Il-Kang Na ◽

Sydney X. Lu ◽

Gabrielle L. Goldberg ◽

Daniel Daniel Hirschhorn-Cymerman ◽

Christopher G. King ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cell Trafficking ◽

T Cell Trafficking ◽

Cytolytic Function ◽

Alloreactive T Cells ◽

Donor T Cells ◽

Trafficking Molecules

Abstract Although thymic graft-versus-host-disease (tGVHD) has been recognized as an important contributor to impaired T cell reconstitution, limited T cell repertoire and increased infection risk in patients with GVHD, the molecular basis of interactions between donor alloreactive T cells, donor bone marrow (BM)-derived thymocytes, and host hematopoietic and non-hematopoietic thymic stromal cells in GVHD has not been well-defined. Here we analyzed the role of molecules relevant for T cell trafficking, cytolytic function, and co-stimulation and co-inhibition of alloreactive T cells in tGVHD. We first demonstrated that thymic output (as measured by RAG2+ splenic recent thymic emigrants) as well as the thymic cellularity (especially of CD4+CD8+ thymocytes) were inversely proportional to numbers of mature donor T cells infused with the allograft, suggesting that tGVHD severity was inversely associated with thymic function. We then studied the migration of alloreactive donor T cells in vivo with bioluminescence imaging (BLI) and found that luciferase-expressing donor T cells infiltrated the thymus within one week after allogeneic bone marrow transplantation (BMT) (Fig. 1). Upon adoptive transfer of CFSE-labeled donor T cells we noted that thymus-infiltrating alloreactive donor T cells were largely fast-proliferating (CFSElo) and highly activated (CD25+ CD44+). We analyzed the importance of T cell trafficking molecules for tGVHD using mice deficient for certain trafficking molecules, and assessed tGVHD by loss of BM-derived CD4+CD8+ thymocytes. We found that CCR9, b7 integrin subunit, and PSGL-1 were all partially required for tGVHD, while L-selectin and aE integrin subunit may be dispensable (Fig. 2A). Similarly, we examined the role of T cell cytolytic pathways for tGVHD, and found that FasL and TRAIL were required for tGVHD, but that perforin and TNF were dispensable (Fig. 2B). Finally, we assessed the role of various T cell co-stimulatory and co-inhibitory molecules for tGVHD, and found that CEACAM1, OX40 and CTLA4 were required, while GITR was partially required and ICOS was dispensable (Fig. 2C). Upon further analysis of donor BM-derived thymocytes, we observed that Bcl-2 expression in donor BM-derived thymocytes was decreased in recipients with GVHD vs. those without GVHD, which suggests that survival of thymocytes is decreased during tGVHD. Hollander and others have previously demonstrated in non-irradiated GVH reaction models that host non-hematopoietic thymic stroma may be an important target for donor alloreactive T cells. We assessed the expression of the death receptors Fas and DR5 in thymic stroma from normal and irradiated (850 cGy) BALB/c mice. We observed that in particular, MHC class II-negative stroma (endothelial cells and fibroblasts), as well as a population of MHC class II-intermediate stroma (epithelial cells) upregulated the expression of both Fas and DR5 after irradiation. Our study defines the specific pathways for cytolysis, trafficking and immune modulation involved in tGVHD and suggests selective therapeutic targets to attenuate tGVHD and improve post-transplant T-cell reconstitution in patients with GVHD. Fig 1. BLI demonstrate a distinct distribution pattern for alloreactive donor T cells in allogeneic BMT recipients, Allogeneic Balb/c recipients show a strong signal on day 4 post-transparent after transfer of 10×108 luc+ splenocytes as measured by total body photon emission. Ex vivo imaging confirms the infiltration of luc+ splenocytes to the thymus. Fig 1. BLI demonstrate a distinct distribution pattern for alloreactive donor T cells in allogeneic BMT recipients, Allogeneic Balb/c recipients show a strong signal on day 4 post-transparent after transfer of 10×108 luc+ splenocytes as measured by total body photon emission. Ex vivo imaging confirms the infiltration of luc+ splenocytes to the thymus. Fig 2. We assessed the role of molecules relevant for T cell trafficking (A), cytolytic function (B), and co-stimulation, co-inhibition (C). Irradiated BALB/c mice received 5×106 T cell depleted C57BL/6 bone marrow + 0.25×106 purified splenic T cells. Absolute numbers of donor-BM-derived CD4+CD8+ thymocytes are shown. Black bars indicate means. p-values were calculated vs. recipients of WT T cells(*p<0.05, **p<0.01) Fig 2. We assessed the role of molecules relevant for T cell trafficking (A), cytolytic function (B), and co-stimulation, co-inhibition (C). Irradiated BALB/c mice received 5×106 T cell depleted C57BL/6 bone marrow + 0.25×106 purified splenic T cells. Absolute numbers of donor-BM-derived CD4+CD8+ thymocytes are shown. Black bars indicate means. . / p-values were calculated vs. recipients of WT T cells(*p<0.05, **p<0.01)

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The Inhibitory Receptor LAG-3 Is Not Essential for Regulatory T Cells Function but Influences Donor T Cell Potency in Acute Graft-Versus-Host-Disease

Blood ◽

10.1182/blood.v118.21.1900.1900 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1900-1900

Author(s):

Emanuela I Sega ◽

Dennis B Leveson-Gower ◽

Mareike Florek ◽

Robert S Negrin

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Treg Function ◽

Donor T Cells ◽

Alloreactive T Cell

Abstract Abstract 1900 GVHD is a major complication of bone marrow transplantation (BMT) and results from donor T cells becoming activated and reacting to host antigens. Recently, lymphocyte activation gene-3 (LAG-3) has emerged as an important molecule, negatively regulating T cell activation and has been proposed to play an important role in CD4+CD25+Foxp3+ regulatory T cell (Treg) function. We investigated the functional in vivo role of LAG-3 in Treg and conventional T cells in murine GVHD with the hypothesis that LAG-3 engagement diminishes alloreactive T cell responses after BMT. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown previously that donor Treg are able to suppress GVHD induced by donor allogeneic conventional T cells (Tcon). The role of LAG-3 in Treg function was evaluated both in vitro and in vivo by directly comparing Treg isolated from LAG-3−/− donor mice to Treg isolated from wild type donors (WT Treg). In vitro, in a mixed lymphocyte reaction assay, LAG-3−/− Treg efficiently suppressed the proliferation of alloreactive T cells in a manner similar to WT Treg. In vivo, a bioluminescent imaging assay (BLI) was utilized that allows for quantitative assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight loss, survival and GVHD score. Both LAG-3−/− Treg and WT Treg were equally potent at suppressing Tcon proliferation as illustrated by BLI of luc+ T cells and demonstrated a significant increase in median survival time (MST) as compared to mice receiving Tcon only (35 days for Tcon vs. 58 and 68 days for WT and LAG-3−/− Treg, respectively, P=0.03), but there was no significant difference in MST between the groups receiving WT and LAG-3−/− Treg. Interestingly, when LAG-3−/− Tcon were used to induce GVHD in the absence of Treg, GVHD lethality was accelerated. Thus, all mice receiving LAG-3−/− Tcon showed decreased survival and significantly lower body weights than mice receiving WT Tcon (P=0.017). GVHD scores of LAG-3−/− Tcon recipients were also significantly higher than WT Tcon recipients at Day 20 post BMT (6.0 vs. 2.2, P=<0.0001). The addition of WT Treg induced only a modest yet statistically significant increase in median survival in mice receiving both LAG-3−/− Tcon and WT Treg as compared to mice receiving LAG-3−/− Tcon alone (45 days vs. 14.5 days, P=0.0075). In contrast, WT Treg more efficiently suppressed the proliferation of WT Tcon, increasing the MST to 70 days versus a MST of 26 days for mice receiving WT Tcon (P=0.0002). Re-isolation experiments using CFSE-labeled Tcon did not show differences in proliferation between WT and LAG-3−/− Tcon at five days following BMT. Since LAG-3 is upregulated as early as 2 days after T cell activation and gradually decreases over the next few days, is it possible that a difference in proliferation could be detected at an earlier timepoint thus explaining the difference in potency between the WT and LAG-3−/− Tcon. Together our results indicate, contrary to previous published results, that the absence of the LAG-3 molecule on Treg does not impair Treg function in our mouse model of acute GVHD. However, the absence of LAG-3 on Tcon induces a more severe GVHD suggesting that LAG-3 engagement on donor T cells diminishes alloreactive T cell response after BMT. Disclosures: No relevant conflicts of interest to declare.

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