Generation and Characterization Of a CD30 Positive T-Cell Lymphoma Mouse Model Resembling Human Anaplastic Large Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2450-2450
Author(s):  
Cathrin Klingeberg ◽  
Anna Lena Illert ◽  
Nicolas Schneider ◽  
Christian Peschel ◽  
Cornelius Miething ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Large Cell ◽  
Cre Recombinase ◽  
Double Negative ◽  
Ki 67 ◽  
The Impact

Abstract Anaplastic large cell lymphomas (ALCL) are a subgroup of aggressive Non-Hodgkin-Lymphomas mainly affecting children and young adults. In 60 % of systemic ALCLs, a translocation t(2;5) (p23;q35) resulting in NPM-ALK fusion gene expression is found. The constitutively activation of ALK tyrosine kinase expressed from the NPM-promoter causes increased proliferation and inhibition of apoptosis thereby promoting cell survival and tumorigenesis. Immunphenotypic characterization of human ALCLs revealed highly CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical trials with CD30-coupled antibodies. However, the impact of CD30 on diseases development as well as NPM-ALK signal transduction in course of disease remains unclear and appropriate mouse models to answer these questions are missing. In this regard, we established a retroviral murine bone marrow (BM) transplantation model resembling a human ALCL-like T-cell neoplasia. Therefore we use an inducible Cre/loxP system where NPM-ALK expression is controlled and expressed in a special type of early T-cells. For generation of this vector, we inserted a floxed translational ‘stop-cassette’ between the retroviral promoter MSCV-LTR and the NPM-ALK cDNA, which guaranties specific expression of NPM-ALK only in cells, where the enzyme Cre-recombinase is expressed. Recognition of the loxP-sites by Cre-recombinase leads in our system to deletion of the stop-cassette and consequently NPM-ALK expression. Using different Cre-expressing cell types allowed us to study pathogenesis of ALCL in more detail. In our recent study, we infected bone marrow of transgenic mice expressing Cre-recombinase under the control of the Lck-promotor with our MSCV-Stop-NPM-ALK-IRES-EGFP (MSNAIE) vector and transplanted it into lethally irradiated C57Bl6 recipient mice. With a latency of 4-5 months, these mice developed Thy1.2-positive lymphomas and died from neoplastic infiltration of bone marrow and lymphatic organs with T-cells. Immunphenotypic analyses confirmed T-Cell origin of the lymphomas and showed importantly highly CD30-expression. Staining of the different T-cell-subpopulations demonstrated highest NPM-ALK expression in immature CD4/CD8 double negative T-cells and not fully differentiated CD4/CD8 double positive T-cells. Interestingly, FACS-staining of the proliferation marker Ki-67 revealed highest expression in CD4/CD8 double negative T-cells, in contrast to the other subpopulations where Ki-67 is less detected. Therefore we hypothesized, that the lymphoma initiating cell (LIC) must be within this early T-cell population. Most interestingly we found highest CD30-expression just in the same CD4/CD8 negative T-cell population, pointing to a crucial role of CD30 in lymphoma initiation. To further substantiate our hypothesis we performed secondary and tertiary transplantations with different sorted T-Cell subpopulation and indeed, the immature CD4/CD8 double negative population was able to initiate lymphoma growth in recipient mice. Further transplantations by limited dilution will help to identify the leukemia initiating cell in this model. Taken together, our murine LckCre-NPM-ALK bone marrow transplantation model represents a precise and versatile tool to study disease initiation and development resembling human ALCL. Moreover, the impact of specific proteins (e.g. CD30) in the course of disease can be addressed by combining Knockout (e.g. CD30)/LckCre transgenic mice with our model. To this end we crossed CD30/Lck-Cre mice, and preliminary analysis indicate that CD30 expression seems not to be required for the initial onset of disease. Further characterization of the role of CD30 in ALCL is ongoing. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of the Lymphoma-Initiating Cell Population in a T Cell Lymphoma Mouse Model Resembling Human Anaplastic Large Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2213-2213
Author(s):  
Cathrin Klingeberg ◽  
Stefanie Kreutmair ◽  
Cornelius Miething ◽  
Marie Follo ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Double Negative ◽  
Cell Subpopulations ◽  
T Cell Subpopulations

Abstract In 60% of anaplastic large cell lymphoma (ALCL) patients a translocation t(2;5) (p23;q35) is found, which results in NPM-ALK fusion gene expression and constitutive activation of the ALK tyrosine kinase. Immunophenotypic characterization of human ALCLs revealed highly CD30-positive cells of T- or Null-cell-origin. However, the origin of the lymphoma initiating cell population as well as NPM-ALK signal transduction in course of the disease remains unclear. In this regard, we established a retroviral murine bone marrow transplantation model resembling human ALCL. Therefore we use an inducible Cre/loxP system where NPM-ALK expression is restricted to early T cells. We infected bone marrow of Lck-Cre transgenic mice with our MSCV-Stop-NPM-ALK-IRES-EGFP vector and transplanted it into lethally irradiated recipient mice. With a latency of 4-5 months, these mice developed Thy1.2-positive lymphomas and died from neoplastic T cell infiltration of bone marrow and lymphatic organs. Immunophenotypic analysis confirmed T cell origin of the lymphomas with the characteristic high CD30 expression. Staining of the T cell subpopulations demonstrated high NPM-ALK expression in immature CD4-/CD8- double negative T cells and undifferentiated CD4+/CD8+ double positive T cells. Interestingly, FACS-staining for the proliferation marker Ki-67 as well as the activation marker CD30 revealed highest expression in the CD4-/CD8- double negative T cells. Therefore we hypothesized that the lymphoma-initiating cell must be within this early T cell population. To substantiate our hypothesis we performed secondary transplantations with sorted T cell subpopulations and indeed, only the CD4-/CD8- double negative population was able to initiate T cell lymphoma in the recipient mice. Immunophenotypic characterization of the lymphoma population of these secondary transplanted mice revealed undifferentiated T cells of all CD4/CD8 subtypes, which argues for the existence of a lymphoma initiating cell population, which can still partly differentiate. Interestingly the CD4-/CD8- double negative lymphoma population aberrantly expressed the T cell receptor alpha/beta chain, which may allow these early T cells to establish a systemic lymphoma. Further analysis of the lymphoma population showed lymphatic precursors (CLP) as well as multipotent progenitors (MMP) and haematopoetic stem cells (LSK), which suggests early bone marrow or thymic progenitor cells as the pool of the lymphoma-initiating cell population. We therefore were able to prove the existence of lymphoma initiating stem cells in a highly relevant NPM-ALK positive CD30 expressing mouse model of ALCL. Further analysis will give insides into eradication of the identified lymphoma stem cell population by clinical relevant NPM-ALK inhibitors and CD30 immunotoxins. Disclosures No relevant conflicts of interest to declare.

scholarly journals Examining the Role of CD30 in an Anaplastic Large Cell Lymphoma Mouse Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2542-2542
Author(s):  
Teresa Poggio ◽  
Linda Graessel ◽  
Stefanie Kreutmair ◽  
Cornelius Miething ◽  
Dietmar Pfeifer ◽  
...  
Keyword(s):  
Immune System ◽  
T Cell ◽  
Mouse Model ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Disease Development ◽  
Large Cell Lymphoma ◽  
The Impact

Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of T-cell non-Hodgkin lymphoma (NHL) mainly affecting children and young adults. About 70% of systemic ALCLs are associated with a characteristic chromosomal translocation, t(2;5)(p23;35) which fuses the anaplastic lymphoma kinase (ALK) gene on chromosome 2 with the nucleophosmin (NPM) gene on chromosome 5, resulting in the NPM-ALK fusion gene, its over-expression and constitutive kinase activity. Immunophenotypic characterization of human ALCL revealed highly CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical trials with CD30-coupled antibodies. However, the impact of CD30 on disease development remains unclear and the relationship between NPM-ALK and CD30 has been investigated mostly using cell line models. Syngeneic mouse models of cancer can serve as useful models since the tumors develop in situ where the contribution made by the immune system and the extracellular matrix can be investigated. Here, we focus on the involvement of CD30 in a retroviral murine bone marrow transplantation model of ALCL. In this model, the BM of Lck-Cre-transgenic mice is infected with a MSCV-Stop-NPM-ALK-IRES-EGFP vector leading to expression of NPM-ALK in early T-cells. With a latency of 3-4 months, mice develop lymphomas and die from neoplastic T-cell-infiltration of BM and lymphatic organs. To investigate the impact of abrogation of CD30 signals on the development of NPM-ALK+ ALCL in our model, CD30 knockout mice were crossed with Lck-Cre mice. Both Lck-Cre NPM-ALK CD30 wt and Lck-Cre NPM-ALK CD30 ko recipients develop a human ALCL-like lymphoma with a pure T-cell phenotype characterized by Thy1.2+ cells infiltrating the thymus, lymph nodes, spleen and BM. First results from Lck-Cre NPM-ALK CD30 ko transplanted mice showed impaired disease induction and prolonged survival compared to CD30 wt animals. Moreover, secondary transplantation of NPM-ALK thymic lymphomas led to distinct deceleration of disease development upon CD30 deletion. Microarray analyses have shed some light on the mechanisms underlying the delayed lymphoma progression of CD30 deleted tumors with an upregulation of inflammatory pathways and proteins that are master players in inflammation and immune responses. Further characterization of the role of CD30-mediated immune response in disease progression using this mouse model and immunocompromised mice is ongoing. An improved understanding of how the immune system affects tumor progression will extend the rationale in translational strategies to use immunotherapies for patients with T-NHLs. Disclosures No relevant conflicts of interest to declare.

Double Negative T Cells Influence TCR VB Variablity to Induce Allotolerance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 759-759
Author(s):  
Zachariah A. McIver ◽  
Marcin Wlodarski ◽  
Jennifer Powers ◽  
Christine O’Keefe ◽  
Tao Jin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Peripheral Tolerance ◽  
Double Negative ◽  
Clonal Deletion ◽  
Tcr Repertoire ◽  
Double Negative T Cells

Abstract Immune alloresponsiveness following allogeneic HSCT is influenced by the dynamics of immune reconstitution and development of allotolerance. In general, tolerance is induced by thymic clonal deletion (central) and apoptosis or suppression of alloresponsive lymphocytes by regulatory T cells in the periphery. We have recently demonstrated that the size of the TCR repertoire within the CD4 and CD8 compartments can be assessed using VB spectrum by flow cytometry, and expansions/losses of individual TCR VB families can be used as a surrogate marker of TCR variability. (Exp. Hem.32: 1010–1022; Br. J. Haematol.129:411–419). Additionally, regulatory T cells can also impact the clonal contractions and expansions within the TCR VB repertoire. Various types of regulatory T cells have been described including CD4+CD25+, CD8+, NK T−cells, and CD3+CD4/CD8− double negative T cells (DN Tregs). In our current study we investigated the role of DN Tregs on the restoration of immune repertoire diversity. We hypothesized that alloresponsiveness clinically detected as a manifestation of GvHD may be associated with oligoclonal T−cell expansions, and in this context decreased numbers of regulatory T cells suggest deficient tolerizing function by regulatory T cells including DN Tregs. Here we studied a cohort of 60 HSCT recipients (AML, CML, CLL, NHL, AA, and PV), of which 25 patients received matched unrelated donor grafts and 35 received matched sibling donor grafts. Blood was sampled between 2003–2006 at monthly intervals after HSCT, and flow cytometry for TCR repertoire in CD4 and CD8 cells as well as the numbers of DN cells were recorded. Additionally, separate samples were collected for measurement of chimerism and were included in analysis when donor lymphoid chimerism was > 60%. A subset analysis was performed based on the presence/absence of GvHD. For the 27/60 (45%) patients with episodes of GvHD, results were obtained at the time of diagnosis of GvHD (grade > 2), while for patients in whom notable GvHD was not captured, the steady−state values at corresponding times were used for analysis. For all patients serial evaluations were available. For the purpose of this study, significant VB expansions/contractions were defined as +/− 2 standard deviation over the average VB family size. Using Cox proportional hazards analysis to identify univariate risk factors for GVHD, CD8 VB TCR contractions > 14 VB families (58.3% contraction of entire CD4 VB repertoire) constituted a strong indicator for increased risk (HR=7.61, p=0.011). This observation is consistent with the fact that oligoclonality of alloreactive T cell clones is frequently accompanied by a significant contraction/loss of remaining VB families and may herald heightened alloresponsiveness as a manifestation of GvHD. Estimation for correlation by Pearson’s correlation coefficient also demonstrated that percentage of DN cells strongly correlated with a normalization of CD4 VB TCR repertoire (lower number of expansions; N=57, R= −0.51, p=0.027), supporting our hypothesis that DN cells participate in peripheral tolerance and suppress proliferative, alloresponsive CD4 clones. In summary, our results further characterize TCR variability post HSCT and define the role of DN cells in the induction of allotolerance.

A Critical Role for Innate Immunity In Allogeneic Hematopoietic Cell Rejection Via the TLR4/TRIF Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 77-77
Author(s):  
Hong Xu ◽  
Jun Yan ◽  
Ziqiang Zhu ◽  
Yiming Huang ◽  
Yujie Wen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Innate Immunity ◽  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Innate Immune System ◽  
Critical Role ◽  
Innate Immune ◽  
Wild Type

Abstract Abstract 77 Adaptive immunity, especially T cells, has long been believed to be the dominant immune barrier in allogeneic transplantation. Targeting host T cells significantly reduces conditioning for bone marrow cell (BMC) engraftment. Innate immunity has been recently shown to pose a significant barrier in solid organ transplantation, but has not been addressed in bone marrow transplantation (BMT). Using T cell deficient (TCR-β/δ−/−) or T and B cell deficient (Rag−/−) mice, we found that allogeneic BMC rejection occurred early before the time required for T cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. Therefore, we hypothesized that by controlling both innate and adaptive immunity, the donor BMC would have a window of advantage to engraft. Survival of BMC in vivo was significantly improved by depleting recipient macrophages and/or NK cells, but not neutrophils. Moreover, depletion of macrophages and NK cells in combination with co-stimulatory blockade with anti-CD154 and rapamycin as a novel form of conditioning resulted in 100% allogeneic engraftment without any irradiation and T cell depletion. Donor chimerism remained stable and durable up to 6 months. Moreover, specific Vβ5½ and Vβ11 clonal deletion was detected in host CD4+ T cells in chimeras, indicating central tolerance to donor alloantigens. Whether and how the innate immune system recognizes or responds to allogeneic BMCs remains unknown. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. The signaling function of TLR depends on intracellular adaptors. The adaptor MyD88 transmits signals emanating from all TLR, except TLR3 while TRIF specifically mediates TLR3 and TLR4 signaling via type 1 IFN. To further determine the innate signaling pathways in allogeneic BMC rejection, B6 background (H2b) MyD88−/− and TRIF−/− mice were conditioned with anti-CD154/rapamycin plus 100 cGy total body irradiation and transplanted with 15 × 106 BALB/c (H2d) BMC. Only 33.3% of MyD88−/− recipients engrafted at 1 month, resembling outcomes for wild-type B6 mice. In contrast, 100% of TRIF−/− mice engrafted. The level of donor chimerism in TRIF−/− mice was 5.1 ± 0.6% at one month, significantly higher than in MyD88−/− and wild-type B6 controls (P < 0.005). To determine the mechanism of innate signaling in BMC rejection, we examined whether TRIF linked TLR3 or TLR4 is the key pattern recognition receptor involved in BMC recognition. To this end, TLR3−/− and TLR4−/− mice were transplanted with BALB/c BMC with same conditioning. None of the TLR3−/− mice engrafted. In contrast, engraftment was achieved in 100% of TLR4−/− mice up to 6 months follow up. Taken together, these results suggest that rejection of allogeneic BMC is uniquely dependent on the TLR4/TRIF signaling pathway. Thus, our results clearly demonstrate a previously unappreciated role for innate immunity in allogeneic BMC rejection. Our current findings are distinct from prior reports demonstrating a critical role of MyD88 in rejection of allogeneic skin grafts and lung, and may reflect unique features related to BMC. The findings of the role of innate immunity in BMC rejection would lead to revolutionary changes in our understanding and management of BMT. This would be informative in design of more specific innate immune targeted conditioning proposals in BMT to avoid the toxicity. Disclosures: Bozulic: Regenerex LLC: Employment. Ildstad:Regenerex LLC: Equity Ownership.

Promoting Regulation Via the Inhibition of DNAM-1 After Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 338-338
Author(s):  
Motoko Koyama ◽  
Rachel D Kuns ◽  
Stuart D Olver ◽  
Katie E Lineburg ◽  
Mary Lor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
T Cell ◽  
Median Survival ◽  
Cd4 T Cells ◽  
Cell Expansion ◽  
Acute Gvhd ◽  
Cd4 T Cell

Abstract Abstract 338 Graft-versus-host disease (GVHD) is the major limitation of allogeneic hematopoietic bone marrow transplantation (BMT). Donor T cells play pivotal roles in GVHD and graft-versus-leukemia (GVL) effects and following BMT all T cell fractions, including regulatory T cells (Treg) express the DNAX accessory molecule-1 (DNAM-1, CD226) and T cell Immunoglobulin and ITIM domain (TIGIT) molecule. DNAM-1 is a co-stimulatory and adhesion molecule, expressed mainly by NK cells and CD8+ T cells at steady state to promote adhesion to ligand (CD155, CD112)–expressing targets and enhance cytolysis. TIGIT is a regulatory ligand expressed predominantly by Treg as steady state which competes for CD155 binding, We have analyzed the role of this pathway in GVHD and GVL. Lethally irradiated C3H/Hej (H-2k) mice were injected with bone marrow cells and T cells from MHC disparate wild-type (wt) or DNAM-1–/– C57Bl6 (H-2b) mice. Recipients of DNAM-1–/– grafts were protected from GVHD (survival 67% vs. 7%, P < .0001). We also confirmed the role of DNAM-1 in GVHD in a MHC-matched BMT model (B6 → BALB/B (H-2b)) where GVHD is directed to multiple minor histocompatibility antigens. Next we examined the donor populations expressing DNAM-1 which mediate this effect. DNAM-1 had little impact on acute GVHD severity in the B6 → bm1 BMT model where GVHD is directed against an isolated MHC class I mismatch and is CD8-dependent. In contrast, recipients of wt bone marrow and DNAM-1–/– CD4 T cells survived long-term (compared to recipients of wt CD4 T cells, survival 81% vs. 25%, P = .003) in the B6 → B6C3F1 BMT model, confirming the protection from GVHD is CD4-dependent. Donor CD4 T cell expansion and effector function (Th1 and Th17), and CD8 T cell expansion and cytotoxic function were equivalent in recipients of wt and DNAM-1–/– grafts. However the percentage and number of Treg were significantly increased in recipients of DNAM-1–/– grafts compared to those of wt grafts. The depletion of Treg from donor grafts eliminated the protection from GVHD seen in the absence of DNAM-1 signalling (median survival 16 days vs. 15.5 days, P = 0.53). Adoptive transfer experiments using FACS-sorted Treg were undertaken to compare the relative ability of B6.WT and B6.DNAM-1–/– Treg to suppress GVHD. The majority of recipients of DNAM-1–/– Treg survived beyond day 50 (median survival; day 56), demonstrating a superior ability to suppress acute GVHD relative to wt Treg where the median survival was day 36 (survival 47% vs. 0%, P = .001). These data demonstrate that donor DNAM-1 expression promotes GVHD in a CD4+ T cell-dependent manner via the inhibition of donor Foxp3+ Treg. Finally, the absence of donor DNAM-1 did not influence leukemia-specific mortality in multiple GVL models, regardless of whether the tumor expressed CD155 or not. Thus we demonstrate that the DNAM-1 pathway promotes GVHD, putatively due to competition with TIGIT on Treg, thereby inhibiting regulatory function. This provides support for therapeutic DNAM-1 inhibition to promote tolerance not only after transplant but also in relevant inflammatory based diseases characterized by T cell activation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Critical role of OX40 in the expansion and survival of CD4 T-cell-derived double-negative T cells

2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Guangyong Sun ◽  
Xiaojing Sun ◽  
Wei Li ◽  
Kai Liu ◽  
Dan Tian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Critical Role ◽  
Cd4 T Cell ◽  
Double Negative ◽  
Double Negative T Cells

scholarly journals Interval of IL-2 Administration Has a Major Impact on Treatment Efficacy for Regulatory T Cell Expansion and Homeostasis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1928-1928
Author(s):  
Yuriko Kishi ◽  
Yusuke Meguri ◽  
Miki Iwamoto ◽  
Takeru Asano ◽  
Takanori Yoshioka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cell ◽  
Dose Interval ◽  
Daily Administration ◽  
Optimum Dose ◽  
Spleen Cells ◽  
Ki 67 ◽  
Significant Difference

Abstract IL-2 has a critical role in the immune homeostasis by expansion and maintenance of regulatory T cell (Treg). We previously demonstrated that low-dose IL-2 administration preferentially increased Treg in patients with active chronic GVHD and resulted in clinical improvement of the symptoms. In these years, the tolerogenic effects of IL-2 have been tested in the setting of various autoimmune-based diseases by many clinical trials. However, the schedules of IL-2 administration in each trial are different and the optimal strategy for expansion and maintenance of Treg is still unclear. To tackle this issue, we examined the impact of IL-2 dose intervals on Treg and the optimization for the induction and maintenance therapy by using murine IL-2 therapy model. CD4+CD25+Foxp3+ Treg were analyzed comparing with CD4+CD25-Foxp3- conventional CD4 T cells (Tcon) and CD8+ T cells. The expressions of Ki-67 and Bcl-2 in each subset were also examined. First, we performed the experimental allogeneic BMT, in which model 1x10E6 spleen cells (CD45.2) and 5x10E6 T cell depleted bone marrow cells (CD45.1) from C57BL/6 donors were transplanted into lethally-irradiated B6D2F1 recipients (CD45.2). To explore the optimum dose-interval for the increase of Treg from the baseline level as the induction therapy, we administered 5000 IU of IL-2 to recipients subcutaneously once (Induction-A), twice (Induction-B), four (Induction -C) or seven (Induction-D) times a week from day 35 to 49 after transplant. On day 50 after transplant, peripheral blood and spleen cells of mice from each group were harvested and CD45.1+H-2Kd- Donor bone-marrow derived lymphocytes were evaluated. The absolute number and %Treg of CD4 T cells, and the Ki-67 and Bcl-2 expression in Treg were compared with each frequency of administration. To increase Treg from baseline, the daily administration (Induction-D) provided the best Treg response among the tested groups and there was significant difference between control group and group I-D (%Treg: 10.5 % vs. 16.7 %, p<0.05). Treg proliferation was positively related to the frequency of IL-2 administration (%Ki-67+ cells: 17.7%, 17.2 %, vs. 9.9%, 9.0% and 7.8%, respectively, p<0.05). Secondly, we investigated the optimum dose-interval for the maintenance of expanded Treg level after the initial inductive IL-2 administration. We expanded Treg by daily administration of IL-2 to B6 mice for 2 weeks, thereafter we administrated IL-2 twice (Maintenance-A), four (Maintenance-B), or seven (Maintenance-C) times a week from day 14 to 21 for the maintenance. Of interest, to maintain expanded Treg level after the induction IL-2 therapy, Treg in cohorts of Maintenance-B and Maintenance-C were significantly higher than in cohort Maintenance-A (mean 9.7%, 10.2% vs 7.1%, respectively) and there was no significant differences between cohorts Maintenance-B and Maintenance-C, suggesting daily administration is not necessary and intermittent administration within the threshold may work for the maintenance of expanded Treg level after the intensive IL-2 administration. Treg proliferation of cohort Maintenance-C was significantly higher than that of the other cohorts (mean %Ki-67: 19.8 %, 20.5 % vs. 23.9 %, p<0.05). There were no significant difference in Bcl-2 expression in Treg among these cohorts but seemed negatively related to the frequency of IL-2 administration. These data suggested that daily administration of IL-2 seems to be optimal for expansion of Treg for induction therapy. In contrast, to maintain the expanded Treg, daily administration may not be required but less frequent times of administration within the threshold could be preferable. Taken together, the interval of IL-2 administration should be a major factor for Treg homeostasis as well as IL-2 dosage. Our data provide important information for developing therapeutic strategies to modulate Treg homeostasis in vivo and promote immune tolerance after transplant. Disclosures No relevant conflicts of interest to declare.

scholarly journals Damage to Thymic Stroma Results in Increased Production of Pathogenic Dual Receptor T Cells Associated with Chronic Graft Versus Host Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1156-1156
Author(s):  
Amritha Balakrishnan ◽  
Burhan Jama ◽  
Nicholas Joseph Gloude ◽  
Eric Jon Anderson ◽  
Edward D. Ball ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Graft Versus Host Disease ◽  
Graft Versus Host ◽  
Post Transplant ◽  
Thymic Stroma

Abstract Evidence from clinical investigations and animal models indicate that chronic graft versus host disease (cGVHD) results from defective thymic generation of functional and self-tolerant T cell populations following hematopoietic stem cell transplantation (HSCT). We have previously demonstrated that the rare subset of T cells that naturally express 2 T cell receptors (TCRs) on the cell surface as a result of incomplete allelic exclusion are predisposed to respond to auto- and alloantigens. Dual TCR T cells disproportionately participate in pathologic alloreactivity in HSCT patients and mouse models of acute GVHD. These findings, combined with observations demonstrating that dual TCR T cells represent a physiologic reservoir of unique TCRs that evade negative selection, prompted us to examine the role of thymic selection and dual TCR T cells in cGVHD. To study the role of post-transplant thymopoiesis in generation of potentially pathogenic dual TCR T cells, we used a mouse model of syngeneic bone marrow transplantation into lethally-irradiated recipients. Radiation-induced damage to the thymic stroma was characterized by disruption of thymic architecture and loss of cortical and medullary thymic epithelial cells (TECs). This damage resulted in significantly increased generation of dual TCR T cells following transplantation of congenically-marked syngeneic T cell-depleted bone marrow. Two-fold increased production of dual TCR T cells persisted for at least 20 weeks after transplantation. These data demonstrate the hazard for production of T cells predisposed to pathogenic reactivity in the post-transplant environment, and suggest that dual TCR T cells could be a source of T cells causing cGVHD. To examine involvement of dual TCR T cells in cGVHD, we analyzed peripheral blood samples from patients after allogeneic HSCT (> 12 months post-transplant) using our previously utilized pair-wise TCRVa labeling flow cytometry approach. Flow cytometry analysis revealed that dual TCR T cells were present at increased frequencies in patients with cGVHD (n = 10, 8.3% + 1.1%, P = 0.028) compared to patients without cGVHD (n = 3, 2.5 + 1.1%) or healthy age-matched controls (n = 5, 1.9 + 0.4%). Dual TCR T cells from patients with cGVHD had an activated CD69+ phenotype as compared to T cells expressing only a single TCR from the same patient. Single-cell TCRa/TCRb sequencing confirmed the increased frequencies of dual TCR T cells specific to activated T cells in patients with cGVHD. Repertoire analysis of TCRs sequenced from single cells indicated that the increase in dual TCR T cells was polyclonal. The single-cell sequencing approach enabled multiplexed examination of T cell lineage-associated transcription factors and cytokines. Single-cell transcriptional profiling demonstrated that dual TCR T cells demonstrated predominantly pro-inflammatory and cytotoxic phenotypes with expression of Tbet and perforin. This is in contrast to T cells expressing only a single TCR from the same patient, or dual TCR T cells from healthy control patients, which had a quiescent phenotype. These data indicate a role for dual TCR T cells in mediating cGVHD. Together, these results suggest that dual TCR T cells may be an important link between post-transplant T cell development and cGVHD. Disclosures No relevant conflicts of interest to declare.

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 ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 65-65 ◽  
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)

scholarly journals Glucagon-like Peptide-2 (GLP-2) Treatment Reduces Gvhd-Related Mortality in a Pre-Clinical Transplant Model Via Expansion of Macrophages with Tolerogenic Potential

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-28
Author(s):  
David W Harle ◽  
Rodney J Macedo Gonzales ◽  
Felix D Rozenberg ◽  
Alexandra Matschiner ◽  
Rajat Bansal ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Progression ◽  
Research Funding ◽  
Cell Phenotype ◽  
Color Flow ◽  
Treatment Groups ◽  
Glucagon Like Peptide ◽  
The Impact

The gastrointestinal (GI) tract is a major target in GVHD. Conditioning-induced damage and mucosal barrier disruption are important factors in GVHD, however therapies targeting these processes have not been identified. Glucagon-like-peptide 2 (GLP-2) is an enterocyte-specific growth factor produced by L cells that has regenerative potential in models of GI damage. Its impact on the mucosal immune system has not been elucidated. We sought to examine the therapeutic and immunologic effect of GLP-2 in murine GVHD. We employed a major MHC-mismatched GVHD model (C57BL/6J → BALB/cJ). Mice were treated with 800nmol/kg/day of Elsiglutide (a GLP-2 analogue, provided by Helsinn) or vehicle beginning on D+1 for 30 days. Treatment with GLP-2 significantly improved survival and GVHD scores (Fig. 1A), while increasing small intestine mass and villi length (Fig 1B). GLP-2 also reduced T-cell infiltration into the jejunum (Fig. 1C). Analysis of intestinal immune cells by 28-color flow cytometry revealed dramatic differences between treatment groups in both myeloid- and T-cells. On D+14, GLP-2 led to an increased proportion of donor CSF-1R+ macrophages in the lamina propria (LP) (Fig. 2A) - cells that support the maintenance of the intestinal stem cell niche (Sehgal, Nat Commun, 2018). On D+21 the LP donor myeloid compartment was further altered, especially in MHC IIlow F4/80+ CD64+ macrophages (Fig. 2B, C). Here GLP-2 treatment expanded macrophages with lower expression of the co-stimulatory molecules CD80 and CD86 as well as the phagocytic marker CD206, whilst increasing the inhibitory molecule SIRPα, consistent with a tolerogenic phenotype. GLP-2 treatment also increased CX3CR1 expression on MHC IIlow macrophages with reduced Ly6C - a phenotype associated with physiologic macrophage maturation and linked to the resolution of colitis (Zigmond, Immunity, 2012). Vehicle-treated mice, conversely, had predominance of Ly6Chigh MHC IIlow LP macrophages reminiscent of an early infiltrating phenotype and near absence of mature macrophages, suggesting an impaired monocyte-macrophage transition that was restored by GLP-2. In addition, GLP-2 treatment led to significant changes in donor intraepithelial lymphocytes on D+21 (Fig. 2D), where CD8 T cells exhibited decreased CD27, CD103 and CXCR3 expression but higher PD-1, suggesting less activation. To assess potential mechanisms for the differences in macrophage and T-cell phenotype, we examined the impact of GLP-2 on the intestinal microbiota. A syngeneic BALB/cJ model was used to explore the effects of GLP-2 independent of GVHD. Stool samples from D+0, D+14, and D+28 were subjected to 16S rRNA sequencing. Vehicle-treated mice had distinct β-diversity clusters at all time-points, showing a transplant effect on the microbiota (Fig. 3A). GLP-2-treated mice had near-complete cluster overlap between D+0 and D+14, suggesting attenuation of the impact of conditioning. GLP-2 treated mice were significantly enriched for Akkermansia muciniphila and Bacteroidales S24-7 family at D+14 and D+28 (Fig. 3B). These taxa have been associated with anti-inflammatory properties and A. muciniphila abundance is linked to epithelial mucin production, which is increased by GLP-2. We then assessed the role of microbial communities in the protective effect of GLP-2 by conducting an allogeneic transplant with 3 caging conditions; 1) vehicle and GLP-2 treated mice caged together, 2) caged separately, or 3) caged separately plus oral antibiotics. We observed a clear cage effect where co-housing the treatment groups improved the survival of vehicle treated mice (Fig. 3C), suggesting transferal of the therapeutic effect via the microbiome. Antibiotic administration also dampened the beneficial effect of GLP-2. Finally, we conducted a GvL experiment by co-transplanting Luc-A20 and monitoring tumor progression via bioluminescence imaging. Both GLP-2 and vehicle-treated mice eliminated the tumor, whereas mice receiving T-cell depleted bone marrow showed tumor progression (Fig. 3D). In summary, our results demonstrate high therapeutic potential of GLP-2 in GVHD. GLP-2 administration led to reduced mortality, modified the microbiome and altered the intestinal immune response to a more tolerogenic state. This novel mechanism sheds light on the role of the enteroendocrine system in maintaining gut homeostasis and sets the stage for therapeutic clinical trials. Figure 1 Disclosures Uhlemann: Allergan: Research Funding; GSK: Research Funding; Merck: Research Funding. Reshef:Gilead: Consultancy; Magenta: Consultancy; Novartis: Consultancy; Monsato: Consultancy; Atara: Consultancy; BMS: Consultancy.

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