Mechanisms of GVL Against a Murine Blast Crisis CML.

Abstract Donor T cells mediate a graft-versus-leukemia effect that is responsible for much of the efficacy of allogeneic hematopoietic stem cell transplantation (alloSCT) in treatment of hematologic malignancies. Chronic phase chronic myeloid leukemia (CP-CML) is the most GVL-sensitive neoplasm. Unfortunately, most other malignancies are relatively GVL-resistant. A striking example is blast crisis CML (BC-CML) which, although sharing its genetic etiology with CP-CML, is nearly refractory to alloimmune T cells. A detailed understanding of GVL-resistance has been hindered by the absence of GVL-sensitive and GVL-resistant murine leukemias that are similar to their human counterparts and are inducible on different mouse strains. In particular, generating gene-deficient leukemias is important for mechanistic experiments. To address these limitations, we have adopted murine models of CP-CML (mCP-CML) and BC-CML (mBC-CML) that share pathology and genetic etiology with their human counterparts. mCP-CML is generated by retroviral transduction of murine bone marrow (BM) with the bcr-abl fusion cDNA (p210), the defining genetic abnormality in human CP-CML. As is the case with human CP-CML, mCP-CML is extremely GVL-sensitive at least in part due to the redundant immune mechanisms sufficient for GVL (Matte et al, Blood 2004). mBC-CML is induced by the retroviral transduction of BM with both p210 and the fusion cDNA NUP98/HOXA9 (Dash, PNAS, 2002), a translocation found in human BC-CML and AML. Relative to mCP-CML, mBC-CML is GVL-resistant. In the MHC-matched C3H.SW→B6 (H-2b) strain pairing, 30–40% of recipients of 4–6 million donor CD4 or CD8 cells die from mBC-CML. This dose is nearly 10-fold higher than required for a similar survival from mCP-CML, even though recipients of mBC-CML and no donor T cells die nearly a week later than recipients of only mCP-CML. Having established that mBC-CML is GVL-resistant, we investigated mechanisms of T cell killing and the roles of donor and recipient antigen presenting cells (APCs). Direct T cell:mBC-CML cognate interactions were required as MHCI− and MHCII− mBC-CML cells (generated in β2microglobulin (β2M) or IAb β chain knockout (KO) BM) were completely insensitive to CD8 and CD4-mediated GVL, respectively. In contrast, neither CD8 nor CD4-mediated GVL was impaired against mBC-CML generated from TNF-receptor1/2 double KO or Faslpr BM. These are the same basic mechanisms of cytotoxicity we observed in GVL against mCP-CML. CD8-mediated GVL against mBC-CML required functional recipient APCs as we observed no GVL when recipients were MHCI− β2M KOs. As was the case with GVL against mCP-CML (Matte, N.Med. 2004), donor APCs were not required as GVL was equivalent in recipients of wild type and β2M KO C3H.SW donor BM. We observed no GVL in MHCII− recipients demonstrating that CD4-mediated GVL also requires functional recipient APCs. In sum, the basic rules of immunogenicity for GVL against mCP-CML and mBC-CML are similar, suggesting that other pathways are responsible for GVL-resistance. One possibility is differential sensitivity to TRAIL-mediated killing and we are currently generating TRAILR-deficient mBC-CML. Another candidate is PD-L1, a B7 family member that can suppress T cell responses. PD-L1 is highly expressed on mBC-CML relative to mCP-CML. We have already generated PD-L1-deficient mBC-CML and GVL experiments with it are underway.

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Memory T cells from minor histocompatibility antigen–vaccinated and virus-immune donors improve GVL and immune reconstitution

Blood ◽

10.1182/blood-2011-07-367011 ◽

2011 ◽

Vol 118 (22) ◽

pp. 5965-5976 ◽

Cited By ~ 34

Author(s):

Ning Li ◽

Catherine Matte-Martone ◽

Hong Zheng ◽

Weiguo Cui ◽

Srividhya Venkatesan ◽

...

Keyword(s):

T Cells ◽

Immune Reconstitution ◽

Memory T Cells ◽

Blast Crisis ◽

Chronic Phase ◽

Histocompatibility Antigen ◽

Leukemia Cells ◽

Minor Histocompatibility Antigen ◽

Hematopoietic Stem ◽

Donor T Cells

AbstractDonor T cells contribute to the success of allogeneic hematopoietic stem cell transplantation (alloSCT). Alloreactive donor T cells attack leukemia cells, mediating the GVL effect. Donor T cells, including the memory T cells (TM) that are generated after infection, also promote immune reconstitution. Nonetheless, leukemia relapse and infection are major sources of treatment failure. Efforts to augment GVL and immune reconstitution have been limited by GVHD, the attack by donor T cells on host tissues. One approach to augmenting GVL has been to infuse ex vivo–generated T cells with defined specificities; however, this requires expertise that is not widely available. In the present study, we tested an alternative approach, adoptive immunotherapy with CD8+ TM from donors vaccinated against a single minor histocompatibility antigen (miHA) expressed by leukemia cells. Vaccination against the miHA H60 greatly augmented TM-mediated GVL against mouse chronic-phase (CP-CML) and blast crisis chronic myeloid leukemia (BC-CML). TM-mediated GVL was antigen specific and was optimal when H60 expression was hematopoietically restricted. Even when H60 was ubiquitous, donor H60 vaccination had a minimal impact on GVHD. TM from lymphocytic choriomeningitis virus (LCMV)–immune and H60-vaccinated donors augmented GVL and protected recipients from LCMV. These data establish a strategy for augmenting GVL and immune reconstitution without elaborate T-cell manipulation.

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Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) Differentiates GVL and GVHD in Allo-HSCT

Frontiers in Immunology ◽

10.3389/fimmu.2020.593863 ◽

2020 ◽

Vol 11 ◽

Author(s):

Mahinbanu Mammadli ◽

Weishan Huang ◽

Rebecca Harris ◽

Aisha Sultana ◽

Ying Cheng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Interleukin 2 ◽

Treatment Strategies ◽

Receptor Expression ◽

Hematopoietic Stem ◽

Target Organs ◽

Donor T Cells ◽

Inducible T Cell Kinase

Allogeneic hematopoietic stem cell transplantation is a potentially curative procedure for many malignant diseases. Donor T cells prevent disease recurrence via graft-versus-leukemia (GVL) effect. Donor T cells also contribute to graft-versus-host disease (GVHD), a debilitating and potentially fatal complication. Novel treatment strategies are needed which allow preservation of GVL effects without causing GVHD. Using murine models, we show that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving GVL effects. Both CD8+ and CD4+ donor T cells from Itk-/- mice produce less inflammatory cytokines and show decrease migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). Itk-/- T cells exhibit reduced expression of IRF4 and decreased JAK/STAT signaling activity but upregulating expression of Eomesodermin (Eomes) and preserve cytotoxicity, necessary for GVL effect. Transcriptome analysis indicates that ITK signaling controls chemokine receptor expression during alloactivation, which in turn affects the ability of donor T cells to migrate to GVHD target organs. Our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.

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Donor BM Dendritic Cells Expressing IDO Limit Graft-Versus-Host Disease After Allogeneic Bone Marrow Transplant.

Blood ◽

10.1182/blood.v114.22.1331.1331 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1331-1331

Author(s):

Ying Lu ◽

Wayne Harris ◽

Jian-Ming Li ◽

Edmund K. Waller

Keyword(s):

T Cells ◽

T Cell ◽

Marrow Transplant ◽

Allogeneic Bone ◽

Transplant Recipients ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

Donor T Cells ◽

Ifn Γ ◽

Th1 Polarization

Abstract Abstract 1331 Poster Board I-353 Background In contrast to the essential role of host dendritic cells (DC) in the initiation of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) reactions, less is known about the effects of donor DC on T cells in these processes. We have previously reported that adding donor BM plasmacytoid DC (pDC) progenitors to a murine graft composed of purified hematopoietic stem cells (HSC) and T-cells increased donor activation and Th1 polarization leading to enhanced GVL activity without increasing GVHD (Li et al. 2007 Blood 110:2181), while larger numbers of human donor pDC were associated with less GVL activity following allogeneic bone marrow transplant (BMT) (Waller et al. 2001 Blood 97:2948). To explore the dissociation of GVHD from GVL we tested the hypothesis that activation of donor T-cells by donor pDC leads to reciprocal induction of indoleamine 2,3-dioxygenase (IDO) expression and immune counter-regulatory activity by donor DC that limits donor T-cell allo-reactivity. Methods pDC precursors were purified by high-speed FACS from un-stimulated BM harvested from wild type (WT) and IDO knock-out (IKO) mice. T-cell proliferation and immune polarization in response to indirect antigen presentation by syngenic DC was measured in mixed lymphocyte reaction (MLR) and by recovery of CFSE-labeled donor T-cells from allogeneic transplant recipients. IDO expression in DC was measured by FACS and intracellular staining using pDC from IKO BM as a negative staining control. FACS-purified 5 × 104 pDC either from WT mice or from IKO mice in combination with 3 × 103 c-kit+ Sca-1+ hematopoietic stem cells (HSC) and 3 × 105 T-cells were transplanted in MHC mismatched C57BL/6→B10.BR model following lethal irradiation. Results FACS-purified lineage−CD11cloCD11b− pDC expressed B220 (72%), CD90 (51%), and CD317 (PDCA-1) (93%), had low levels of MHC-II, partial expression of CD4, and lacked expression of CD24, CD80, CD86 and NK cell or granulocytic markers. IDO expression in purified pDC was up regulated by IFN-γ produced by syngenic T-cells in vitro in one-way MLR. In vivo proliferation of CFSE-labeled donor T-cells was enhanced in mice that received pDC from either WT or IKO mice. Co-transplantation of IKO pDC led to higher proliferation rates of CD8+ T-cells but not CD4+ T-cells compared with the proliferation of corresponding donor T-cell subset co-transplanted with WT DC. The incidence and severity of GVHD (weight loss and GVHD score) were markedly increased in recipients receiving pDC from IKO mice as compared with mice receiving WT pDC. The enhanced GVL activity of donor T-cells induced by transplanted donor WT pDC was abolished when IKO pDC were transplanted into tumor-bearing recipients. Transplanting WT donor pDC led to larger numbers of donor-derived CD4+CD25+Foxp3+ T-reg cells in the spleens of transplant recipients compared with mice receiving IKO pDC (p<0.01) in combination with purified HSC and T-cells. Conclusions Taken together, our data suggest IDO expression in pDC as a critical downstream event that inhibits continued T-cell activation and GVHD. We propose a feedback model in which donor pDC initially induce Th1 polarization of activated donor CD8+ T-cells that secret high levels of IFN-γ. IDO expressed by donor pDC in response to local IFN-γ subsequently induces a counter-regulatory effect including the generation of T-reg and down-modulation of CD8+ T-cell allo-reactivity and proliferation, limiting GVHD while preserving the GVL activity of donor T-cells. Disclosures No relevant conflicts of interest to declare.

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Flagellin, a TLR5 Agonist, Reduces GvHD in Allogeneic HSCT Recipients While Enhancing Anti-Viral Immunity: A Novel Therapeutic Approach

Blood ◽

10.1182/blood.v118.21.144.144 ◽

2011 ◽

Vol 118 (21) ◽

pp. 144-144

Author(s):

Mohammad S Hossain ◽

David L Jaye ◽

Brian P Pollack ◽

Alton B Farr ◽

John Roback ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd8 T Cells ◽

Viral Immunity ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Post Transplant ◽

Radiation Chimeras ◽

Donor T Cells

Abstract Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p<0.05), and less MCMV in the liver on day 10 post infection (p<0.02) compared with the PBS-treated control recipients. Overall immune reconstitution after flagellin-treatment was robust and associated with larger numbers of CD4+CD25+foxp3+ regulatory T cells in the thymus. To further define the role of flagellin-TLR5 agonistic interactions in the reduction of GvHD, we next generated B6 ^ TLR5 KO (KO) and KOB^6 radiation chimeras by transplanting 10 × 106 BM cells from wild-type (WT) B6 or TLR5 KO donors into the congenic CD45.1+ B6 or KO recipients conditioned with 11Gy (5.5Gyx2) TBI. The radiation chimeras were irradiated again with 9.0Gy (4.5Gy × 2) on 60 days after the first transplant and transplanted with 3 × 106 splenocytes and 5 × 106 TCD BM from H-2K congenic donors. Two 50mg doses of flagellin were administered 3 hrs before irradiation and 24 hrs after HSCT. All flagellin-treated B6 ^ B6 radiation chimeras survived with only 12% weight-loss by 80 days post transplant compared with 50% survival among recipients of flagellin-treated B6 ^ KO and 40% survival among KO ^ B6 radiation chimeras. All flagellin-treated KO^ KO and PBS-treated radiation chimeras died within 65 days post transplant. These data suggested that interaction of flagellin with the TLR5 expressing host gut epithelium and donor hematopoietic cells are both required for the maximum protective effect of this TLR5 agonist on GvHD in allogeneic HSCT recipients. Together our data demonstrate that peritransplant administration of flagellin effectively controls acute and chronic GvHD while preserving enhanced post-transplant donor anti-opportunistic immunity. Since flagellin has been found to be safe for use in humans as vaccine adjuvant in a number of clinical trials, the clinical use of flagellin in the setting of allogeneic HSCT is of interest. Disclosures: No relevant conflicts of interest to declare.

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Interruption of IFNγR Signaling Results in Altered T Cell Trafficking in Vivo and Abrogation of GvHD While Maintaining a Robust Gvl Response

Blood ◽

10.1182/blood.v120.21.455.455 ◽

2012 ◽

Vol 120 (21) ◽

pp. 455-455

Author(s):

Jaebok Choi ◽

Edward Dela Ziga ◽

Julie Ritchey ◽

Lynne Collins ◽

Julie Prior ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell ◽

Cell Trafficking ◽

Hematopoietic Stem ◽

T Cell Trafficking ◽

Target Organs ◽

Donor T Cells

Abstract Abstract 455 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with relapsed/refractory leukemia, and marrow failure states such as myelodysplasia and aplastic anemia. However, allo-HSCT is complicated by allogeneic donor T cell-mediated graft-versus-host disease (GvHD) which can be life-threatening especially in recipients of unrelated or HLA-mismatched hematopoietic stem cell products. These same alloreactive donor T cells also mediate a beneficial graft-versus-leukemia (GvL) effect. Thus, the clinical goal in allo-HSCT is to minimize GvHD while maintaining GvL. Recent studies have suggested that this might be achieved by infusing regulatory T cells (Tregs) which in some preclinical models suppress GvHD-causing alloreactive donor T cells but have only limited effects on GvL-promoting alloreactive donor T cells. Unfortunately, Tregs exist in low frequency in the peripheral blood, are costly to purify and expand, and after expansion are difficult to isolate due to the lack of cell surface markers, all of which prevent their routine use in the clinic. Thus, alternative therapeutic approaches that do not require Tregs are needed. We have found that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells induce significantly less GvHD in both a MHC fully-mismatched (B6 (H-2b) → Balb/c (H-2d)) and a minor-mismatched (B6 (H-2b) → B6×129(H-2b)) allo-HSCT models compared to WT T cells. In addition, IFNγR−/− donor T cells maintain a beneficial GvL effect, which has been examined in both systemic leukemia and solid tumor models using luciferase-expressing A20 cells derived from Balb/c. We find that IFNγR−/− T cells migrate primarily to the spleen while WT T cells to GI tract and peripheral lymph nodes (LNs) using bioluminescence imaging (BLI), suggesting that altered T cell trafficking of IFNγR−/− T cells to GvHD target organs might be the major reason for the reduced GvHD. We further demonstrate that the IFNγR-mediated signaling in alloreactive donor T cells is required for expression of CXCR3 which has been implicated in trafficking of T cells to areas of inflammation and target organs, commonly known to be the sites of GvHD. Indeed, CXCR3−/− T cells recapitulate the reduced GvHD potential of IFNγR−/− T cells. In addition, forced overexpression of CXCR3 in IFNγR−/− T cells via retroviral transduction partially rescues the GvHD defect observed in IFNγR−/− T cells. We next examine if inhibition of IFNγR signaling using a small molecule inhibitor can recapitulate the anti-GVHD effects seen in IFNγR−/− T cells. We find that INCB018424, an inhibitor of JAK1/JAK2 which are the mediators of IFNγR signaling, blocks CXCR3 expression in vitro. Most importantly, in vivo administration of INCB018424 after allo-HSCT alters T cell trafficking and significantly reduces GvHD. Thus, the IFNγR signaling pathway represents a promising therapeutic target for future efforts to mitigate GvHD while maintaining GvL after allo-HSCT. Moreover, this pathway can be exploited in other diseases besides GvHD such as those from organ transplantation, chronic inflammatory diseases and autoimmune diseases. Disclosures: DiPersio: genzyme: Honoraria.

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A novel anti-CD19 chimeric T cell therapy platform ET019002 was safe and effective in inducing CR in blast crisis of CML: A case study.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e18540 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e18540-e18540

Author(s):

Pengcheng He ◽

Hong Liu ◽

Haibo Liu ◽

Qijing Li ◽

Mina Luo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Blast Crisis ◽

Leukemic Cells ◽

Maximum Temperature ◽

T Cell Therapy ◽

Nasal Sinus ◽

Hematopoietic Stem ◽

The Subject

e18540 Background: CML can progress to blast crisis in which the disease behaves like an acute leukemia. A novel chimeric T-cell therapy ET019002 is built upon Eureka Therapeutics ARTEMIS platform and co-expressed with an additional anti-CD19 scFv fused to the CD28 protein. ET019002 demonstrated superior preclinical anti-tumor activities to the classical anti-CD19 CAR-T cells. Herein we describe the first-in-human clinical study of ET019002 in a blast crisis of CML subject with T315I mutation, breast, skin, nasal sinus, liver, CNS involvement and heavily previous lines of therapy. Methods: The subject received infusions of ET019002 T cells at the dose of 1x106 receptor+ T cells/kg on Day 0, 66 and 122. The primary objective is safety and the secondary objectives include ET019002 T-cell engraftment and anti-tumor efficacy. Results: The subject developed a transient fever and chills with a maximum temperature of 40.2°C 4 days post the 1st infusion. Fever was resolved within 3 days after giving NSAIDs. No inflammatory cytokines except IL-6 were elevated during the study. IL-6 elevated 4 days post the 1st infusion, reached peak level at 507.18 pg/ml on Day 6 and decreased to 37 pg/ml on Day 8. This was not seen after the 2nd and the 3rd infusion. Lumps from breast and skin disappeared and the enlarged liver and spleen were back to normal size. Bone marrow biopsy at day 12 showed lymphoblast cells were significantly decreased from baseline of 38% to the level of 1%. The leukemic cells in cerebrospinal fluid were cleared on Day 14. The copy number of tumor-related fusion gene BCR-ABL/ABL was reduced from 1.489 x 100 to 3.942 x 10-5. However, 2 months post the 1st infusion, BCR-ABL/ABL rebounded to 3.795 x 10-4. The 2nd infusion showed no effect on the relapse of the leukemia in molecular level. The 3rd infusion resulted in CR which relapsed 1 month post the infusion. Flow cytometry analysis showed the loss of CD19 on the leukemic cells. Conclusions: Repeated infusion of autologous ET019002 was safe and achieved a three-month CR after the ET019002 T cells therapy, which could provide a window for the patient to receive curative therapies such as bridging allogeneic hematopoietic stem cells. Clinical trial information: NCT03642496.

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Division rate and phenotypic differences discriminate alloreactive and nonalloreactive T cells transferred in lethally irradiated mice

Blood ◽

10.1182/blood.v98.10.3156 ◽

2001 ◽

Vol 98 (10) ◽

pp. 3156-3158 ◽

Cited By ~ 38

Author(s):

Sébastien Maury ◽

Benoı̂t Salomon ◽

David Klatzmann ◽

José L. Cohen

Keyword(s):

T Cells ◽

T Cell ◽

Division Rate ◽

Hematopoietic Stem ◽

Phenotypic Differences ◽

Graft Versus Host ◽

Alloreactive T Cells ◽

Donor T Cells ◽

Life Threatening ◽

T Cell Transfer

Abstract After non-T-cell–depleted allogeneic hematopoietic stem cell transplantation (HSCT), both alloreactive and homeostatic signals drive proliferation of donor T cells. Host-reactive donor T cells, which proliferate on alloantigen stimulation, are responsible for the life-threatening graft-versus-host disease. Non–host-reactive donor T cells, which proliferate in response to homeostatic signals, contribute to the beneficial peripheral T-cell reconstitution. The elimination of alloreactive T cells is a major therapeutic challenge for HSCT and would greatly benefit from their specific identification. After T-cell transfer in lymphopenic recipients, the present results show that alloreactive T cells rapidly divided; up-regulated CD69, CD25, and CD4 molecules; and down-regulated CD62L. In contrast, nonalloreactive T cells started to divide later and did not up-regulate CD69, CD25, and CD4. Thus, these 2 cell populations can be effectively discriminated. This should facilitate the specific depletion of alloreactive T cells in allogeneic HSCT.

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Characteristics of the TCR Vbeta Repertoire and Identical Clonally Expanded T Cells in Chronic Myeloid Leukemia Patients in Advanced Phase with ABL Mutations

Blood ◽

10.1182/blood.v126.23.5136.5136 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5136-5136

Author(s):

Ling Xu ◽

Yuhong Lu ◽

Jing Lai ◽

Wei Yu ◽

Zhenyi Jin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Myeloid Leukemia ◽

Natural Science ◽

Myeloid Leukemia ◽

Research Funding ◽

Blast Crisis ◽

Chronic Phase ◽

Cytotoxic Lymphocyte ◽

Advanced Phase

Abstract Abstract Tumor specific or related antigen cytotoxic lymphocyte (CTL) have been identified in chronic myeloid leukemia patients, however, whether they are constituted by specific type of T cell receptor chains has not been illustrated so far. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR Vβ T cells in chronic myeloid leukemia in chronic phase (CP-CML). In this study, we investigated the distribution and clonality of the TCR Vβ repertoire in 5 CML patients in blast crisis (BC-CML) and one in acceleration phase (AP-CML) with ABL kinase domain mutations (KDMs) including T315I, E255K, F317L+S417Y, Y-253F and L387M+T-315A. Examination of TCR Vβ expression and clonality was performed by reverse transcription-polymerase chain reaction (RT-PCR) combined with GeneScan analysis. Significantly skewed TCR Vβ repertoires were observed in those patients, and 4 to 8 oligoclonally expanded TCR Vβ subfamilies could be identified in each sample, which distributed in 15/24 different subfamilies (TCR Vβ4, Vβ5, Vβ6, β8, Vβ9, Vβ10, Vβ15, Vβ16, Vβ17, Vβ18, Vβ19, Vβ21, Vβ22, Vβ23, Vβ24). Intriguingly, a relatively highly expanded Vβ9 clone with the same length as CDR3 (139 bp) was found in all three CML patients in lymphoid blast crisis (LBC-CML) who had different KDMs, but the clone was not detected in the other two CML patient in myeloid blast crisis (MBC-CML) or the one CML patients in accelerated phase. In conclusion, restricted TCR Vβ repertoire expression and decreased clone complexity was a general phenomenon in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency status of these patients, and clonally expanded Vβ9 T cell clones may represent a specific immune response to leukemia-associated antigens in LBC-CML patients. Disclosures Li: The Foundation for High-level Talents in Higher Education of Guangdong, China ([2013]246-54),and the Guangzhou Science and Technology Project Foundation (201510010211): Research Funding; National Natural Science Foundation of China (81270604, U1301226, and 81400109), the Guangdong Natural Science Foundation (S2013040016151 and S2013020012863): Research Funding.

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Generation of CMV Specific T Cells From CMV Seronegative Donors by T Cell Receptor RNA Transfer.

Blood ◽

10.1182/blood.v114.22.3577.3577 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3577-3577

Author(s):

Simone Thomas ◽

Sebastian Klobuch ◽

Mirjam H.M. Heemskerk ◽

Diana Stolle ◽

Katrin Besold ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Human Fibroblasts ◽

Expression Level ◽

Specific Lysis ◽

Hematopoietic Stem ◽

Peptide Epitope ◽

Retroviral Transduction ◽

Ifn Γ

Abstract Abstract 3577 Poster Board III-514 Reactivation of latent human cytomegalovirus (CMV) infection is a frequent complication in CMV seropositive patients after allogeneic hematopoietic stem cell transplantation (HSCT). Although antiviral drug therapy is successfully used to reduce the risk of CMV disease, long-term virus control requires the re-establishment of protective antiviral T cell immunity in the host. The latter is challenging, particularly if the donor is CMV seronegative and thus, no CMV specific T cells are being transferred from donor to recipient during HSCT. Grafting nonreactive T cells of CMV seronegative donors by virus-antigen specific T cell receptors (TCR) may be an efficient means to transfer CMV specific T cell function into allogeneic HSCT recipients. In this study, we intended to reprogram T cells of CMV seronegative donors with human TCR recognizing the immunodominant HLA-A*0201 binding peptide epitope NLVPMVATV (495-503) derived from the CMV pp65 protein. A common approach for TCR gene transfer into T cells is retroviral transduction bearing the risk of insertional mutagenesis which hampers clinical translation. In addition, heterologous recombination between introduced and naturally expressed TCR chains might lead to the induction of harmful autoimmunity. Herein we used in vitro transcribed RNA encoding the CMV pp65/HLA-A*0201-specific TCR for electroporation of anti-CD3 stimulated T cells in peripheral blood mononuclear cells (PBMC) of CMV seronegative donors. This procedure resulted in transient surface expression of the introduced TCR chains up to 5 days as shown by flow cytometry. Maximum expression level was observed at 4 to 24 h after electroporation, with up to 70% of total CD8+ and CD4+ T cells staining positive for the vβ13.1 subfamily domain of the TCRβ chain. After introduction of TCR RNA, the intensity of CMV pp65/HLA-A*0201 tetramer staining was 60% and 50% of total CD8+ and CD4+ cells, respectively. In IFN-γ ELISPOT and 51Chromium-release assays, TCR RNA transfected T cells recognized HLA-A*0201 expressing T2 cells pulsed with titrated amounts of CMV pp65 (495-503) peptide. Minimal peptide concentration triggering specific lysis was 0.1 nM to 1 nM at a CD8+ to target (CD8+:T) ratio of 2:1. The EC50 value (0.2 nM) was in the same range of avidity compared to that of a retrovirally transduced counterpart construct of this TCR. Most importantly, TCR recipient CD8+ T cells gained the ability to lyse HLA-A*0201 positive human fibroblasts upon infection with CMV. Specific lysis between 20% and 100% was observed at a CD8+:T ratio of 1:1 or higher. We next sorted CD8+ T cells from PBMC of CMV seronegative donors into naïve and memory cells according to expression of the differentiation markers CD45RA and CD45RO. Although 90% of naïve CD8+ T cells stained positive for the CMV pp65/HLA-A*0201 tetramer after electroporation of TCR RNA, they mediated only marginal lysis toward CMV infected fibroblasts. In contrast, TCR RNA transfected memory CD8+ T cells showed strong lysis against CMV infected fibroblasts at a CD8+:T ratio of 0.7:1 or higher. Specific lysis was detected for at least 3 days after electroporation. In summary, our data demonstrate that nonreactive human T cells can be successfully redirected with CMV pp65 TCR RNA. The expression level of the introduced TCR is sufficient to trigger IFN-γ production and cytolytic activity toward CMV infected human fibroblasts. Electroporation of TCR RNA is comparably easy and eliminates the risk of retroviral transduction. We therefore believe that CMV pp65 TCR RNA has the potential to be further developed as a therapeutic “off-the-shelf” reagent for patients who undergo drug-resistant CMV reactivation after HSCT. Disclosures: No relevant conflicts of interest to declare.

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Interruption of the IFN γR/CXCR3 Axis Results in Altered T Cell Trafficking In Vivo and Abrogation of GvHD While Maintaining a Robust Gvl Response

Blood ◽

10.1182/blood.v118.21.2971.2971 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2971-2971

Author(s):

Jaebok Choi ◽

Edward Dela Ziga ◽

Julie Ritchey ◽

Julie Prior ◽

Lynne Collins ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell ◽

Neutralizing Antibodies ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Target Organs ◽

Donor T Cells

Abstract Abstract 2971 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with relapsed/refractory leukemia, and marrow failure states such as myelodysplasia and aplastic anemia. However, allo-HSCT is complicated by allogeneic donor T cell-mediated graft-versus-host disease (GvHD) which can be life-threatening especially in recipients of unrelated or HLA-mismatched hematopoietic stem cell products. These same alloreactive donor T cells also mediate a beneficial graft-versus-leukemia (GvL) effect. Thus, the clinical goal in allo-HSCT is to minimize GvHD while maintaining GvL. Recent studies have suggested that this might be achieved by infusing regulatory T cells (Tregs) which in some preclinical models suppress GvHD-causing alloreactive donor T cells but have only limited effects on GvL-promoting alloreactive donor T cells. Unfortunately, Tregs exist in low frequency in the peripheral blood, are costly to purify and expand, and after expansion are difficult to isolate due to the lack of cell surface markers, all of which prevent their routine use in the clinic. Thus, alternative therapeutic approaches that do not require Tregs are needed. Using a MHC-mismatched GvHD model, B6 (H-2b) → Balb/c (H-2d), we demonstrated that infusion of IFN γR deficient allogeneic donor T cells induce significantly less GvHD, compared to WT T cells, determined by survival (74% vs. 0 % in overall survival; p =0.0004), weight and percentages of B220+ B cells (12.4% vs. 3.8%; p =0.0205), CD3+ T cells (14.3% vs. 4.3%; p =0.0025) in blood. Of note was that the IFN γR deficient donor T cells maintained a beneficial GvL effect, which was examined in both a systemic leukemia and a solid tumor model using luciferase-expressing A20 cells derived from Balb/c. We found that IFN γR deficient donor T cells responded normally to allogeneic antigens as measured by in vitro mixed lymphocyte reaction analyses, and express similar levels of granzyme B, compared to WT T cells. However, IFN γR deficient T cells trafficked predominantly to the spleen while WT T cells trafficked to gastrointestinal tract and peripheral lymph nodes, which are major GvHD target organs, based on in vivo bioluminescence imaging. All of these findings suggest that the reduced GvHD was not due to reduced function, altered subsets or relative deficiency of allogeneic donor T cells but from modification of in vivo trafficking of IFN γR deficient donor T cells compared to WT T cells. We further demonstrated that the IFN γR-mediated signaling in alloreactive donor T cells was required for expression of CXCR3 which has been implicated in trafficking of T cells to areas of inflammation and target organs, commonly known to be the sites of GvHD. CXCR3−/− T cells demonstrated a reduction in GvHD while maintenance of the same robust GvL effect using the same MHC mismatched transplant model. Thus, the IFN γR-CXCR3 axis represents a promising therapeutic target for future efforts to mitigate GvHD while maintaining GvL after allo-HSCT. Current studies are focused on 1) whether forced expression of CXCR3 rescues the GvHD-inducing potential of IFN γR deficient donor T cells and 2) if inhibition of IFN γR signaling (IFN γR, JAK1 and/or JAK2, CXCR3 and STAT1) using both neutralizing antibodies and small molecule inhibitors can recapitulate the anti-GvHD and pro-GvL effects seen in IFN γR−/− and CXCR3−/− T cells. Disclosures: No relevant conflicts of interest to declare.

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