Oncoretroviral and Lentiviral Transduction of Donor T Cells to Facilitate Engraftment of Dog Leukocyte Antigen (DLA)-Haploidentical T-Cell-Depleted Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1751-1751
Author(s):  
Dario Sangiolo ◽  
Marina Lesnikova ◽  
Alla Nikitine ◽  
Hans-Peter Kiem ◽  
Rainer Storb ◽  
...  
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
T Cell ◽  
Transduction Efficiency ◽  
Endogenous Virus ◽  
Short Term ◽  
Donor T Cells ◽  
Allogeneic Stimulation ◽  
Short Term Culture

Abstract Several clinical studies of adoptive immunotherapy with genetically modified (GM) donor T cells infused after allogeneic hematopoietic cell transplantation (HCT) showed limited in vivo function of the transduced T cells. Factors that have hindered successful translation to clinical trials include insufficient preclinical data in large animal models and the need for prolonged cell culture - up to 2 to 3 weeks for optimal oncoretroviral (OR) vector transduction and selection of T cells. In preparation for in vivo studies of GM T cells to facilitate engraftment in the preclinical dog model of allogeneic HCT, we compared transduction protocols with OR and lentiviral (LV) vectors that aimed to decrease the duration of ex vivo T cell culture necessary for stable transduction while maintaining T cell alloreactivity. Vectors expressed enhanced yellow fluorescent protein (YFP) under a constitutive promoter. We compared vectors pseudotyped with viral glycoproteins (GP) including vesicular stomatitis virus (VSV)-G (LV only), feline endogenous virus (RD114), and chimeric RD114 envelope GP fused with murine leukemia virus-A cytoplasmic tail (RD114/TR). Although T cells transduced with LV vectors without prior mitogenic or allogeneic stimulation had 14% – 30% transduction efficiency of predominantly CD4+ cells, transgene expression was not sustained in CD8+ cells after allogeneic stimulation (n=3). In order to transduce T cells that could generate GM alloreactive cytotoxic T lymphocytes (CTL), freshly isolated T cells were stimulated with allogeneic dendritic cells (DC) for 4 days prior to transduction. VSV-G, RD114 or RD114/TR pseudotyped LV had primary transduction efficiency of 1.2 to 9% (n=5). Only cells that were transduced with RD114 or RD114/TR pseudotyped vectors maintained stable YFP expression after 2° allogeneic stimulation. Next, OR YFP vector pseudotyped with RD114 transduced 15 to 36% of DC allo-stimulated T cells (n=3). Both CD4+ and CD8+ cell populations were transduced (CD4+: CD8+ ratio 1.4:1) and the mean YFP fluorescence intensity was increased 0.6-log compared to LV vectors (p=0.01). We then evaluated T cells transduced with OR RD114 pseudotyped vector in vivo. To determine if short-term culture and transduction of T cells facilitated engraftment of CD3-depleted marrow in the DLA-haploidentical HCT model, donor T cells were collected on day −7 prior to HCT, cultured with recipient CD34+ derived DC, and on day −4 cells were transduced with RD114 pseudotyped YFP OR vector. To date, one dog was transplanted after 920cGy total body irradiation with 2x108 CD3+ donor cells/kg (1:1 CD4:CD8 ratio) 25% YFP expression and 2-log CD3-depleted marrow (4x108 TNC/kg). No post-grafting immunosuppression was given. Donor YFP transduced T cells were detected in the peripheral blood daily after HCT, and peaked on day +7. After engraftment on day +8, GVHD developed and the dog was euthanized on day +21 with all-donor chimerism. YFP+ T cells were detected in GVHD affected target organs. Previously, transduced donor CTL cultured for 4 weeks and transplanted with CD3-depleted marrow in this HCT model failed to engraft in all 5 dogs studied. These preliminary results support the hypothesis that short-term culture and transduction of donor T cells with RD114 pseudotyped OR vector maintain in vivo alloreactivity and facilitate engraftment of CD3-depleted marrow in MHC-mismatched recipients.

T Cell Trafficking in Acute GVHD: In Vivo Bioluminescence Evaluation To Elucidate the Role of Different Lymphatic Organs.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 593-593
Author(s):  
Andreas Beilhack ◽  
Stephan Schulz ◽  
Jeanette Baker ◽  
Georg F. Beilhack ◽  
Courtney B. Wieland ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymphoid Organs ◽  
The Body ◽  
Peyer's Patches ◽  
Peyer’S Patches ◽  
Alloreactive T Cells ◽  
Donor T Cells ◽  
Specific Priming

Abstract To study the complex pathophysiology of aGvHD in allogeneic hematopoietic cell transplantation (HCT) we transplanted transgenic luciferase expressing T cell populations into lethally irradiated HCT recipients (murine MHC major mismatch model, H-2q into H-2d). Tracking of light emitting donor T cells in living animals and detailed studies by multi color immunofluorescence microscopy (IFM) and FACS revealed the tight links of spatial and temporal evolution in this complex immune process. Donor derived T cells migrate to T cell areas in lymphoid tissues within a period of 12 hours. In the initial periods donor CD4+ T cells appear first with CD8+ T cell infiltration at later time points. Donor T cells start proliferating in lymphatic tissues on day 2 after transfer, as observed by BrdU stainings. Although alloreactive T cells are similarly activated in all lymphoid organs, they only up-regulate gut homing molecules after more than 5 cell divisions (CFSE proliferation analysis by FACS) in certain lymphoid organs (Peyer’s patches, mesenteric LN and spleen). Abruptly on day 4 after HCT, T cells migrate into intestinal sites. These findings strongly suggested, that specific priming sites are required for alloreactive T cells to induce a distinct type of tissue tropism in GvHD. In contrast to previous reports peformed without host conditioning, depletion of certain lymphoid organs (e.g. Peyer’s patches) before HCT or antibody blocking experiments did not control aGVHD. BLI showed, that anti-L-selectin or anti-MAdCAM-1 antibody treatment alone or in combination was effective in blocking donor T cell migration to lymph nodes and Peyer’s patches, while redirecting these cells to liver and spleen. Subsequently cells proliferated predominantly in the spleen until day 3 after HCT. Surprisingly we observed a full picture of gut infiltration on day 4 and skin involvement on day 5–6, similar in dynamics and strength to the aGvHD isotype control group. These findings demonstrated, that other lymphoid organs can functionally compensate for inducing gut and skin homing of alloreactive T cells. Of importance, we demonstrated that T cells that lacked homing molecules for secondary lymphoid organs had alloreactive properties in vitro, yet did not cause aGVHD in vivo. In summary, the activation of alloreactive T cells in specific sites throughout the body is complex and involves the acquisition of homing molecule expression. Transplantation of T cells with defined homing properties therefore, appears to be a promising alternative in conferring protective immunity early after HCT without the risk of aGvHD.

Allograft T Cell Content Influences Early Engraftment after Reduced-Intensity Stem Cell Transplantation (RIST).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3662-3662
Author(s):  
Robert M. Dean ◽  
Daniel H. Fowler ◽  
Nancy M. Hardy ◽  
Jeanne Odom ◽  
Kathleen Castro ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Group Iii ◽  
Donor Chimerism ◽  
Group I ◽  
Gvhd Prophylaxis ◽  
Donor T Cells ◽  
Reduced Intensity

Abstract Allogeneic hematopoietic stem cells (HSC) generally engraft rapidly and completely after myeloablative conditioning. However, with reduced-intensity conditioning (RIC), mixed chimerism and graft failure are more common. Host immune status and HSC number are factors known to affect engraftment after reduced-intensity stem cell transplantation (RIST). In addition, donor T cells within the allograft may also influencethe kinetics of donor engraftment after RIST. To evaluate this, we performed a controlled comparison of engraftment outcomes among 3 groups undergoing RIST, varying by ex vivo T cell depletion (TCD) or in vivo depletion of activated T cells with methotrexate (MTX) to prevent graft-versus-host disease (GVHD). Group I (n = 50) received T cell replete (TCR) peripheral blood stem cells (PBSC) with cyclosporine (CSA) alone for GVHD prophylaxis. Group II (n = 17) received ex vivo TCD PBSC (positive/negative selection with T cell add-back to uniform dose of 1 x 105 CD3+ cells/kg) with CSA alone for GVHD prophylaxis. Group III (n = 31) received TCR PBSC with CSA plus MTX (5 mg/m2 IV x 4 doses) for GVHD prophylaxis. The 3 groups were similarly immunosuppressed from prior therapy before RIST (median absolute lymphocyte counts 330/μL, 260/μL, and 307/μL for Groups I, II, and III, respectively), and received an identical RIC regimen (fludarabine/cyclophosphamide) plus comparable numbers of filgrastim-mobilized PBSC from HLA-matched sibling donors (median 7.9 x 106, 7.6 x 106, and 6.8 x 106 CD34+ cells/kg, respectively; median 3.6 x 108, 1.0 x 105, and 3.2 x 108 CD3+ cells/kg, respectively). Hematopoietic recovery was slowest in Group III, consistent with the myelosuppressive effects of MTX (Table). A greater proportion of patients in Group I achieved complete donor chimerism (≥ 95%) by day +28 than in Groups II or III (P < 0.025), and at day +100, mixed donor chimerism persisted more often in Groups II and III than in Group I patients (P < 0.01). Correspondingly, early (< day +42) occurrence of grade 3–4 acute GVHD, before initiation of planned sequential donor lymphocyte infusions (DLI) in Group II, was more frequent in Group I than in either Groups II or III (p=0.08). Table: Hematopoietic Recovery, Engraftment, and GVHD Group Days to ANC > 500, median (range) Days to plt > 100, median (range) Donor chimerism ≥ 95% Early acute GVHD, grades 3–4 Day +28 Day +100 I 9 (7–13) 15.5 (12-42) 37/44 (84%) 36/38 (95%) 9/50 (18%) II 9 (7–10) 17.5 (11–40) 8/17 (47%) 9/14 (65%) 0/17 (0%) III 14 (7–21) 21.5 (12–85) 23/31 (74%) 21/31 (68%) 2/31 (6%) Thus, the deletion of T cells by either ex vivo TCD or in vivo MTX administration measurably alters the kinetics and degree of donor T cell engraftment after RIST. These observations provide evidence that donor T cells are an independent factor affecting engraftment of allogeneic HSC after RIST by compensating for incomplete host immune ablation. These data also support the hypothesis that a graft-versus-host effect plays a significant role in engraftment after RIST. Manipulation of donor T cells through graft engineering techniques may be a useful strategy to enhance engraftment in the setting of RIST.

Adoptive Transfer of CMV-Specific Donor T Cells Following Allogeneic Transplantation Leads to Rapid and Safe Systemic Reconstitution.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 44-44
Author(s):  
Karl S Peggs ◽  
Kirsty Thomson ◽  
Edward Samuel ◽  
Gemma Dyer ◽  
Julie Armoogum ◽  
...  
Keyword(s):  
T Cells ◽  
Drug Therapy ◽  
T Cell ◽  
Peripheral Blood ◽  
Chronic Gvhd ◽  
Antiviral Drug ◽  
Donor T Cells ◽  
Post Infusion ◽  
Cmv Reactivation

Abstract Abstract 44 Reactivation of CMV remains a significant problem following allogeneic hematopoietic stem cell transplantation. Antiviral drug therapy is effective but toxic, and resistant strains of CMV are increasingly being reported. Virus-specific T lymphocytes are necessary for the control of viral reactivation. Adoptive transfer of donor derived CMV-specific T cells has been reported previously but most methods to produce such cells have involved several weeks of in vitro culture or have produced a therapeutic product restricted to CD8 T cells. The current method involves a short incubation of donor peripheral blood mononuclear cells with either CMV-pp65 protein (20 hours) or a pool of peptides from pp65 (6 hours) with subsequent isolation of interferon-gamma secreting cells by CliniMACS using IFNψ capture microbeads (Miltenyi Biotec). This technique permits rapid isolation of an enriched IFNψ secreting T cell product, manufactured to clinical grade, which is then cryopreserved in dosed aliquots for subsequent infusion. Here we report the outcome of a single arm phase I/II in which CMV-T cells given pre-emptively at first detection (qPCR) of CMV DNA in peripheral blood, or at day +40-60 as prophylaxis. CMV replication was monitored by weekly PCR and reconstitution of CMV-specific T cells by pentamer labelling and/or IFNψ secretion assay. Conventional antiviral drug therapy was instituted if the viral load rose above institutional threshold. 30 recipients of T cell depleted low intensity transplants from HLA-matched CMV-seropositive related donors were enrolled between 2006 and 2008. Donors underwent a second, short apheresis procedure approximately 15 days after collection of the mobilised HPC-A for the collection of CMV-T cells. 26 clinical-grade products were produced to full cGMP standards; four donors were unsuitable or withdrew. The mean yield of cells following enrichment was 41.7% with a median purity of 43.9% (range 1.4-81.8). Adequate CMV-T cells were isolated from all donors. Both pp65 and peptide stimulated products contained both CD4 and CD8 reactive T cells. Median dose of CMV-specific CD4 T cells was 2840/kg and of CMV-specific CD8 was 630/kg. Eighteen patients received a single dose of 1×10^4 CD3+/kg; 13 were CMV seropositive; 11 were treated pre-emptively and 7 prophylactically. 83% had received T cell deplete regimens. Within 2 weeks of infusion in vivo expansion of CMV-T cells was observed in 17 of 18 patients. One patient required 4 weeks to generate detectable CMV-T cell in his peripheral blood. TCR-BV usage of the CMV-T cells post infusion matched that of the cells which had been infused. The 7 patients who had cells infused prophylactically all showed expansions of CMV-T cells in the absence of detectable viral DNA in peripheral blood. Subsequent low level CMV-reactivation was seen in one of these and was associated with rapid CMV-T cell expansion with clearance of virus without anti-viral drug therapy. One developed subsequent extensive chronic GvHD and required antiviral treatment for multiple reactivation episodes following introduction of steroids. Of the 11 patients treated pre-emptively, 9 received antiviral therapy for the initial reactivation, although in 7 patients this was required for only 7-15 days. (compared to a median of 21 days in historical controls). Three patients had a further CMV reactivation event. One followed prednisolone therapy for acute grade II GvHD. The second was the patient who had shown poor T cell expansion post infusion and had required prolonged anti-viral therapy (33 days) for the initial CMV reactivation. The third patient received no treatment and cleared virus following a further in vivo expansion of CMV-reactive T cells, suggesting the presence of a functional memory population. GVHD incidence and severity was no worse than seen in comparable historical controls. 3 patients suffered grade 2-3 acute GvHD. 3/17 evaluable patients developed extensive chronic GvHD (2 were recipients of T replete grafts). 16/18 patients were alive at the end of the 6 month monitoring period and CMV-reactive T cells were detectable in all 16. CMV-specific donor T cells can be readily produced to cGMP compliance which can be safely infused and lead to early immune reconstitution in at-risk patients. Cells expand in response to subsequent CMV-reactivation and patients appear to require fewer anti-viral treatment episodes which is being tested in an ongoing phase III trial. Disclosures: Lowdell: Cell Medica Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Attenuated Acute Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation In the Absence of RORγt.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3742-3742
Author(s):  
LeShara M Fulton ◽  
Michael J Carlson ◽  
James Coghill ◽  
Michelle L. West ◽  
Angela Panoskaltisis-Mortari ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Graft Versus Host Disease ◽  
Allogeneic Stem Cell Transplantation ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract Abstract 3742 CD4+ T helper (Th) cells play a critical role in the development of Graft-versus-Host Disease (GvHD). The relative contributions of particular Th subsets to GVHD pathogenesis, however, are incompletely understood. In order to clarify the contribution of the Th17 subset to GVHD induction, we made use of mice knocked out at the RORgt locus (RORgt−/−), a transcription factor crucial for Th17 polarization. Methods: Haplotype matched and complete MHC mismatched murine HSCT models were used. For the haploidentical model C57BL/6 (H-2b, B6) mice served as donors while C57BL/6 × DBA2 F1 (H-2bxd, B6D2) mice functioned as recipients. Effector T cells (Teffs) were isolated from the spleens of wild type (WT) B6 and RORgt knockout mice backcrossed 7–8 generations onto a B6 background. B6D2 mice were lethally irradiated with 900 rads on day -1 and injected intravenously with 4 × 106 Teffs from WT or RORgt−/− mice supplemented with 3 × 106 WT T cell depleted bone marrow cells (TCD BM) on day 0. For the completely MHC mismatched model, BALB/c mice (H-2d) were lethally irradiated with 800 rads on day -1 and administered 5 × 105 WT or RORgt−/− Teffs supplemented with 5 × 106 B6 TCD BM on day 0. Results: B6D2 mice that received RORgt−/− Teffs displayed significantly attenuated GvHD, recovering from weight loss by day +31 and demonstrating 100% survival on day +60. Conversely, mice that received WT Teffs showed intense disease progression with 100% mortality by day +31 (Figure A, p<0.0001 for survival comparison between WT and RORgt−/− recipients using Fisher's exact test). Similar results were seen using the completely MHC mismatched model, with superior overall survival noted in those animals receiving RORgt −/− Teffs (put in p value here). Recipients of RORgt −/− T cells demonstrated statistically significant decreased TNF in serum compared to WT recipients (Figure B, p=0.001 comparing WT and RORgt−/− recipients using student's t test). Interestingly, despite the decreased severity of GvHD, serum concentrations of IFN-g were increased in recipients transplanted with RORgt −/− T cells. Chimerism studies post-transplant revealed complete donor reconstitution in recipients of both RORgt−/− and WT Teffs. Donor Teffs isolated from recipient livers post-transplant consistently demonstrated an activated phenotype, with low L selectin and high CD25 expression. Conclusions: T cell expression of the Th17 transcription factor, RORgt, is critical for the development of lethal GvHD following allogeneic stem cell transplantation in both the haploidentical and MHC complete mismatch models. GvHD attenuation in the absence of RORgt is not the result of an inability for donor T cells to undergo activation or to engraft in vivo. Interestingly, the absence of RORgt from donor T cells led to enhanced IFN-g in serum. Thus, in vivo, the Th17 pathway is critical for the induction of GvHD. Disclosures: No relevant conflicts of interest to declare.

CD19-Targeted Donor T Cells Exert Potent Graft Versus Lymphoma Activity and Attenuated Gvhd

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 451-451 ◽  
Author(s):  
Arnab Ghosh ◽  
Marco L. Davila ◽  
Lauren F. Young ◽  
Christopher Kloss ◽  
Gertrude Gunset ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Acute Gvhd ◽  
Tcr Signaling ◽  
Car T Cells ◽  
Donor T Cells ◽  
Car T

Abstract Abstract 451 Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells. In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ϵ, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

Opposing Effects of PD-1/PD-L1/L2 Engagement and IFN-γ/TNF-α in the Treatment of AML w/ Anti-CD33 Chimeric Antigen Receptor-Modified T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5891-5891
Author(s):  
Jacob Halum Basham ◽  
Terrence L. Geiger
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Chimeric Antigen Receptor ◽  
Short Term ◽  
Car T Cells ◽  
Tnf Α ◽  
Car T ◽  
Ifn Γ

Abstract Chimeric antigen receptor-modified T lymphocytes (CART cells) have shown benefit as an adjuvant immunotherapy in the treatment of B cell malignancies. This success of re-targeted T cells has not been extended to other hematologic malignancies. We have developed an immunotherapeutic approach to treat acute myeloid leukemia (AML) using CAR T cells re-directed against the myeloid-specific antigen CD33 (CART-33). CART-33 cells are potent and specific in eliminating AML cells in vitro and in vivo. Despite this, CART-33 cells have shown poor in vivo expansion and persistence in NOD-SCID IL2rγ (-/-) (NSG) AML xenograft models. To address the reason for this, we assessed the impact of AML-expressed programmed death ligands 1 & 2 (PD-L1/2) on CART-33 cell activity. PD-L1 inhibits T cell functions upon binding PD-1, which is upregulated with T cell activation. Less is known about PD-L2's effect. Interferon-gamma (IFN-γ), a primary effector cytokine secreted by CD4+ and CD8+ effector T cells, is a known potent inducer of PD-L1 on AML blasts. Using AML cell lines U937, Oci-AML3, CMK, and MV4-11 we show that IFN-γ, TNF-α, and activated CART-33 supernatant can induce up-regulation of PD-L1 and PD-L2 on AML. IFN-γ and TNF-α synergize strongly in up-regulating PD-1 ligands on AML. The kinetics and induction of PD-L2 are distinct from that of PD-L1. Although PD-L1 is well documented to suppress T cell function via ligation of T cell expressed PD-1, induction of PD-L1/L2 had no effect on the cytolytic activity of CART-33 cells against AML in short term (<48 h) cultures. Paradoxically, 24 hr pre-treatment of AML with either IFN-γ or CART-33 supernatant increased AML susceptibility to killing by CART-33 cells despite elevated expression of PD-L1/L2 by AML. Our results highlight the regulatory complexity of AML cytolysis by re-targeted T lymphocytes, and argue that tumor-expressed PD-L1 and PD-L2 impacts the sustainability, but not short-term killing activity, of adoptively transferred CAR T cells in the treatment of AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Piggybac Transposon Mediated CD19 CAR-T Cells Derived from CD45RA-Positive PBMCs Possess Potent and Sustained Antileukemic Function

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2807-2807
Author(s):  
Masaya Suematsu ◽  
Shigeki Yagyu ◽  
Nobuyoshi Nagao ◽  
Susumu Kubota ◽  
Yuto Shimizu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Stress Test ◽  
Transduction Efficiency ◽  
Antigen Stimulation ◽  
Car T Cells ◽  
Car T

Abstract Background: The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. We previously demonstrated that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit memory-rich phenotype that is characterized by a high proportion of CD45RA+/CCR7+ T cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA− peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA− CAR, respectively), and compared their phenotype and antitumor function. Methods: CD45RA+ and CD45RA− PBMCs were isolated by magnetic selection from whole PBMCs, then the CD19-CAR transgene was transduced into these cells using the PB transposon system, as described previously. Transduction efficiency of CD19 CAR transgene was determined 24 hours by flow cytometry after transduction. The phenotype of CD19 CAR-T was evaluated by flow cytometry on day 14. High throughput RNA sequencing was performed to see the T cell activation/exhaustion profile upon antigen stimulation. Sequential killing assays were performed by adding fresh tumor cells into CAR-T cells co-cultured with tumor cells every three days by restoring an effector target ratio of 1:1. To see the durable antitumor efficacy in vivo, we performed in vivo stress test, in which CAR T-cells dosage was lowered to the functional limits, so that these CAR-T cells should be maintained and expanded in vivo, to achieve the antitumor efficacy. We injected 5 x 10 5 of firefly luciferase-labeled CD19+ tumor cells (REH) into NSG mice via tail vein, then these mice were treated with 1 x 10 5 of CD19 RA+ CAR-T, RA− CAR-T, or control CAR-T cells, respectively, at day 6 after the tumor injection. Results: RA+ CAR T cells demonstrated better transient transduction efficiency 24 h after transduction (RA+ CAR-T: 77.5 ± 9.8% vs RA− CAR-T: 39.7 ± 3.8%), and superior expansion capacity after 14 days of culture than RA− CAR-T cells (RA+ CAR-T: 32.5 ± 9.3-fold vs RA− CAR-T: 11.1 ± 5.4-fold). RA+ CAR-T cells exhibited dominant CD8 expression (RA+ CAR-T: 84.0 ± 3.4% vs RA− CAR-T: 34.1 ± 10.6%), less expression of exhaustion marker PD-1 (RA+ CAR-T: 3.1 ± 2.5% vs RA− CAR-T: 19.2 ± 6.4%) and T cell senescence marker CD57 (RA+ CAR-T: 6.8 ± 3.6% vs RA− CAR-T: 20.2 ± 6.9%), and enrichment of naïve/stem cell memory fraction (CAR+/CD45RA+CCR7+ fraction; RA+ CAR-T: 71.9 ± 9.7% vs RA− CAR-T: 8.0 ± 5.3%), which were associated with longevity of CAR-T cells. Transcriptome analysis revealed that RA+ CAR-T cells exhibited the enrichment of naïve/memory phenotype and less expression of canonical exhaustion markers, and these exhaustion profiles even maintained after the antigen stimulation. RA+ CAR-T cells demonstrated sustained killing activity even after multiple tumor rechallenges in vitro, without inducing exhaustion marker expression of PD-1. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, in our study, the expression of CAR molecule on the cell surface following antigen stimulation in RA+ CAR was controlled at a relatively lower level that in RA− CAR-T cells. RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells than RA− CAR-T cells in in vivo stress test (Fig.1A-C). On day15, bone marrow studies in RA+ CAR group exhibited abundant human CD3 positive T cells with less expression of PD-1, and relatively smaller amount of REH cells than RA− CAR group (Fig.1D). Furthermore, in two of long-lived mice in RA+ CAR group, human CD3 positive T cells were expanded even day 50 after treatment as confirmed by sequential bone marrow studies (Fig.1E), which indicated the antigen-induced proliferation and long-term functionality of RA+ CAR-T cells in vivo. Conclusion: Our results suggest that PB-mediated RA+ CAR-T cells exhibit memory-rich phenotype and superior antitumor function, thereby indicating the usefulness of CD45RA+ PBMC as a starting material of PB-CAR-T cells. Figure 1 Figure 1. Disclosures Yagyu: AGC Inc.: Research Funding. Nagao: AGC Inc.: Current Employment. Kubota: AGC Inc.: Current Employment. Shimizu: AGC Inc.: Current Employment. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

scholarly journals Conversion of Anergic T Cells Into Foxp3- IL-10+ Regulatory T Cells by a Second Antigen Stimulus In Vivo

2021 ◽  
Vol 12 ◽  
Author(s):  
Anna Sophie Thomann ◽  
Theresa Schneider ◽  
Laura Cyran ◽  
Ina Nathalie Eckert ◽  
Andreas Kerstan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Time Window ◽  
Common Mechanism ◽  
Short Term ◽  
Subsequent Conversion ◽  
Time Periods ◽  
Anergic T Cells ◽  
Tr1 Cells

T cell anergy is a common mechanism of T cell tolerance. However, although anergic T cells are retained for longer time periods in their hosts, they remain functionally passive. Here, we describe the induction of anergic CD4+ T cells in vivo by intravenous application of high doses of antigen and their subsequent conversion into suppressive Foxp3- IL-10+ Tr1 cells but not Foxp3+ Tregs. We describe the kinetics of up-regulation of several memory-, anergy- and suppression-related markers such as CD44, CD73, FR4, CD25, CD28, PD-1, Egr-2, Foxp3 and CTLA-4 in this process. The conversion into suppressive Tr1 cells correlates with the transient intracellular CTLA-4 expression and required the restimulation of anergic cells in a short-term time window. Restimulation after longer time periods, when CTLA-4 is down-regulated again retains the anergic state but does not lead to the induction of suppressor function. Our data require further functional investigations but at this stage may suggest a role for anergic T cells as a circulating pool of passive cells that may be re-activated into Tr1 cells upon short-term restimulation with high and systemic doses of antigen. It is tentative to speculate that such a scenario may represent cases of allergen responses in non-allergic individuals.

scholarly journals Use of Ex Vivo Graft Manipulation and Posttransplant Cyclophosphamide Result in Low GvHD Rates and Acceptable Engraftment after RIC Regimens in Pediatric Mismatched SCT

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3255-3255
Author(s):  
Peter Lang ◽  
Michaela Döring ◽  
Anne-Marie Lang ◽  
Patrick Schlegel ◽  
Christian M. Seitz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Suppression ◽  
Pediatric Patients ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
Post Transplant ◽  
Donor T Cells ◽  
Organ Dysfunctions

Introduction: There are currently two strategies to prevent Graft-versus-Host Diseases (GvHD) mainly applied in haploidentical transplantation. One is ex-vivo T-cell depletion of TcRa/b T-cells and the other is the T-replete approach, in which the donor T-cells remain in the graft and are tolerized in vivo by post-transplant cyclophosphamide (pCy). The ex-vivo depletion strategy does not require post-transplant immune suppression for GvHD prevention, whereas T-replete transplants require intensive immune suppression. A major obstacle for engraftment is the persistence of patients' T-cells despite intensive and myeloablative condition regimens, thus probably leading to rejection of the graft. We hypothesized that both methods could be combined in a setting of Reduced Conditioning setting (RIC). The ex-vivo T-cell depletion would allow to omit post-transplant immunosuppression and the pCy given at day +3 and +4 could induce in-vivo tolerance of the residual patients' T-cells not eliminated by RIC. Therefore, we applied this strategy in patients who were not eligible based on their poor clinical condition and who were considered to endure only a very reduced conditioning regimen. Results: We report on a cohort of 6 pediatric patients who were not eligible for myeloablative condition regimens due to preexisting organ dysfunctions (lungs, gut or liver) but were in urgent need of an SCT from matched unrelated (n=2) or haploidentical family donors (n=4). Diagnoses were: immune deficiencies (n =4; CARMIL 2, STAT 1, ICF 2, 1 not classified), relapsed metastatic ependymoma, refractory Burkitt´s lymphoma. All patients received a non-myeloablative conditioning regimen (ATG (Thymoglobin) 2mg/kg d-9 to d-7, fludarabine 30mg/m² d-6 to d-2, TBI 4Gy d-1, cyclophosphamide 50mg/kg d+3, d+4; adapted from Aversa, Reisner et al. Blood Adv. 2017). One patient additionally received thiotepa 2x5mg/kg on d-2. The CliniMACS® device was used for TCRab/CD19 depletion of peripheral stem cells; a median number of 14x10E6 CD34+ cells/kg bw with 6.4x10E3/kg bw residual TCRa/b T-cells was infused without any further posttransplant immune suppression. Four patients received a single add back of CD45 RA depleted donor T-cells at d+7. Dosages of 1x10E5/kg, 1x10E6/kg or 5x10E6/kg were administered. Two patients received an additional T-cell depleted stem cell boost after application of pCy Engraftment occurred in 4/6 patients; 2 patients rejected their haploidentical grafts and showed complete autologous reconstitution. Median time to reach ANC>500 was 19 days (range 15-23). Four patients had no signs of GvHD; 1 patient had grade I; the patient who had received the highest dose of CD45RA depleted DLI developed grade III but could be treated successfully. No cGvHD occurred. Immune recovery was rapid. Median numbers of CD3+ T-cells, CD3/CD4+ T-cells, CD19+ B cells and CD56+ NK cells at d30 and d100 were 120/µl, 9/µl, 0/µl, 140/µl and 205/µl, 60/µl, 67/µl and 206/µl, respectively. 3 patients are alive and well with a median follow up of 824 days (43-1100). Last observed donor chimerisms were 95-100%. Causes of death in 3 other patients were: MAS/sepsis (STAT 1 deficiency, d 264) and progression in both patients with malignancies (d282 and d73). The patient with relapsed ependymoma showed a transient tumor regression for 3 months posttransplant whereas the patient with refractory Burkitt´s lymphoma had only a short response for 4 weeks. Conclusions: The combination of TCRa/b depletion and pCy allowed to use a very reduced conditioning regimen which could be administered in pediatric patients even with preexisting significant organ dysfunctions without severe side effects. GvHD could be effectively prevented (except in one patient who received a high number of DLI) together with an acceptable engraftment rate provided by post cy. Thus, this method might offer the possibility to establish a donor-derived hematopoiesis without using pharmacological myeloablation and with minimal toxicity and might be the basis for future strategies to further reduce the conditioning regimen, especially for patients with non-malignant diseases. Disclosures No relevant conflicts of interest to declare.

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