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

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

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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 ◽

10.1182/blood-2019-129654 ◽

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

Blood ◽

10.1182/blood.v118.21.1900.1900 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1900-1900

Author(s):

Emanuela I Sega ◽

Dennis B Leveson-Gower ◽

Mareike Florek ◽

Robert S Negrin

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Treg Function ◽

Donor T Cells ◽

Alloreactive T Cell

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

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Deletion of AMP-Activated Protein Kinase (AMPK) in Donor T Cells Protects Against Graft-Verus-Host Disease through Control of Regulatory T Cell Expansion and Target Organ Infiltration

Blood ◽

10.1182/blood.v128.22.806.806 ◽

2016 ◽

Vol 128 (22) ◽

pp. 806-806 ◽

Cited By ~ 1

Author(s):

Kevin Beezhold ◽

Nathan Moore ◽

Pailin Chiaranunt ◽

Rebecca Brown ◽

Craig A. Byersdorfer

Keyword(s):

T Cells ◽

T Cell ◽

Lower Percentage ◽

Cd4 Cells ◽

Post Transplant ◽

Alloreactive T Cells ◽

Target Organs ◽

Donor Cells ◽

Donor T Cells

Abstract Allogeneic hematopoietic stem cell transplantation (alloHSCT) represents a curative treatment for high-risk leukemia and a number of non-malignant hematologic disorders. However, the therapeutic use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that the alloreactive T cells responsible for GVHD increase their dependence on the oxidation of fat relative to either syngeneic or naive T cells. To explore this adaptation mechanistically, we studied the role of AMPK, an intracellular energy sensor and known driver of fat oxidation, in donor T cells during GVHD. Alloreactive T cells increased phosphorylation of AMPK as early as day 3 post-transplant, with up-regulation in pathways both up- and downstream of AMPK. Changes in phosphorylation were up to 8-fold higher in alloreactive T cells compared to naive T cells or syngeneic controls (p=0.0003). We then investigated the role of AMPK during GVHD pathogenesis using donor cells deficient in AMPK (from AMPKα1fl/flα2fl/fl x CD4-Cre mice). AMPK-/- T cells caused significantly less GVHD in both major-MHC and minor-histocompatibility mismatch models of GVHD (Figure 1A), with a coordinated decrease in the number of donor T cells recovered on day 7 post-transplant (3.15 +/- 0.49x106 versus1.87 +/- 0.53x106, p=0.0006, wildtype (wt) versus AMPK-/- respectively). Importantly, expansion of syngeneic T cells was unaffected by AMPK deficiency (Figure 1B). We next investigated the ability of AMPK-/- T cells to mount effective cytotoxic and anti-leukemia responses. AMPK-/- T cells demonstrated equivalent cytotoxicity against MHC-mismatched targets both in vitro and in vivo and differentiated in similar proportions into cytokine-producing cells (IFN-γ, TNFα, IL-17, and IL-4). We then assessed graft-versus-leukemia (GVL) potential in AMPK-/- cells using a GVL model with high tumor burden. AMPK-/- T cells exhibited equivalent clearance of p815 leukemia cells on day 13 post-transplant (Figure 2A), and extended survival of recipient mice similarly to wt T cells (Figure 2B). To elucidate possible mechanisms underlying this separation of GVL and GVHD responses, we evaluated metabolic pathways in wt and AMPK-/- T cells recovered on day 7. To our surprise, rates of fatty acid oxidation were identical between wt and AMPK-/- T cells and loss of AMPK did not impact alloreactive T autophagy, nor impair signaling downstream of mammalian target of rapamycin. To define the mechanism underlying AMPK-/- benefits, we quantitated levels of regulatory T cells (Treg) on day 7 post-transplant. In contrast to expectation, both the percentage and total number of Treg increased in mice receiving AMPK-/- T cells (0.85 +/- 0.32x104 vs. 1.69 +/- 0.34x104, wt vs. AMPK-/-, p=0.004). Loss of AMPK facilitated donor Treg expansion, as elimination of FoxP3+ cells prior to transplantation abrogated differences between wt and AMPK-/- donors on day 7. Importantly, Treg levels were equal in wt versus AMPK-/- donors prior to transplantation. Finally, we assessed the ability of AMPK-/- T cells to infiltrate into GVHD target organs. As shown in Figure 3, peri-portal infiltration of AMPK-/- cells was significantly reduced compared to wt T cells, and infiltrates in recipients of AMPK-/- cells contained many fewer CD3+ T cells per high-powered field, with CD3+ cells representing a lower percentage of cells overall. Decreased hepatic infiltration correlated with a lower percentage of circulating CD4+ cells and lower levels of the integrin pair α4β7 (55.2 +/- 1.4% versus 47.2 +/- 2.8% α4β7Hi cells, p=0.0017, wt vs. AMPK-/-). In conclusion, deletion of AMPK in donor T cells decreases GVHD severity but spares anti-leukemia responses and preserves homeostatic immune reconstitution. Mechanistically, this occurs through a decrease in pathogenic T cell numbers, an increase in the number and percentage of Treg cells, fewer circulating CD4+ cells, and decreased infiltration of donor cells into target organs. From these findings, we conclude that AMPK represents a clinically relevant target in donor T cells pre-transplant and are actively exploring ways to translate this exciting therapy into clinical practice. Disclosures No relevant conflicts of interest to declare.

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Donor Dendritic Cells Home to Lymphoid Organs in Allogeneic Transplant Recipients, Enhance Donor T-Cell Chimerism, and Polarize Donor T-Cell Activation.

Blood ◽

10.1182/blood.v114.22.62.62 ◽

2009 ◽

Vol 114 (22) ◽

pp. 62-62

Author(s):

Kimberly T. Fan ◽

Jian-Ming Li ◽

Ying Lu ◽

Wayne Harris ◽

Kataryna A. Darlak ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lymphoid Organs ◽

Transplant Recipients ◽

Allogeneic Transplant ◽

Post Transplant ◽

Mhc Ii ◽

Donor T Cells ◽

Secondary Lymphoid Organs

Abstract Abstract 62 Background: The fate of donor dendritic cells (DC) contained in the allograft, and their role in regulating immunity following allogeneic transplant has not been fully explored. We have previously demonstrated that donor BM CD11b− DC added to purified hematopoietic stem cells (HSC) and T-cells polarize donor T-cells to Th1 immunity, and improve leukemia survival among tumor-bearing transplant recipients compared with recipients of allogeneic CD11b+ donor antigen presenting cells (APC) or grafts with no donor APC (Li, et al Blood 2008, 112:1252). Objective: In this study, we studied the homing of donor DC in the primary and secondary lymphoid organs of transplant recipients in murine allogeneic HSC transplantation. Methods: In this study we transplanted between 5 × 104 and 1 × 106 FACS-sorted lineage− CD11c+ CD11b− DC (predominately plasmacytoid DC (pDC) and pDC precursors) and lineage− CD11c+ CD11b+ APC and APC precursors (CD11b+ APC) purified from GFP+ or luciferase+ transgenic donors in combination with 3 × 103 lineage− c-kit+ Sca-1+ HSC and 1 × 106 CFSE stained donor T-cells in B6□B10BR, B6□BALB/C, and FVB□B10BR allogeneic transplant models to explore homing, maturation, and persistence of donor DC in the primary and secondary lymphoid organs of transplant recipients. Homing and short-term maturation of donor DC was assessed 3 days and 10 days post-transplant by FACS analysis gating on GFP+ donor DC. In vivo maturation of DC was assessed by FACS gating on GFP+ donor APC progeny in transplant recipients and compared with DC subsets cultured with CD40L (1μg/mL) or LPS (10ng/mL). Proliferation and IFN-ψ production of CFSE-labeled donor T-cells isolated from recipients' peripheral blood, lymph nodes, and spleen was assessed by flow cytometric analysis of CFSE and intracellular cytokine staining. Persistence of donor DC was assessed by bioluminescence imaging 7, 14, 21, and 28 days post-transplant in FvB□B10BR allogeneic transplant model using 5 × 104 pDC and CD11b+ APC from FvB luciferase+ transgenic donors transplanted with 3 × 103 HSC and 3 × 105 T-cells from FvB wild-type donors. Results: The frequencies of GFP+ pDC and CD11b+ APC recovered from transplant recipients at day 10 post-transplant were 2.2 ± 0.2% and 1.8 ± 0.3%, respectively in BM and 4.9 ± 1.5% and 1.9 ± 0.5%, respectively in recipient spleen. Absolute numbers of GFP+ donor DC were measured in the sections of spleen by confocal microscopy and were similar comparing recipients of pDC and CD11b+ APC (9.0/μL and 7.3/μL spleen, respectively, at day 10). The GFP+ CD11b+ APC had a scatter profile consistent with myelo-monocytic differentiation while GFP+ pDC retained mononuclear properties. The expression of MHC-II and CD80/CD86 on GFP+ CD11b+ APC recovered from transplant recipients was minimal, indicating predominant differentiation to myeloid-derived suppressor cells rather than to DC. FACS analysis of lineage expression patterns in donor GFP+ pDC and CD11b+ APC recovered from recipient BM were similar compared with sorted APC cultured with CD40L or LPS. PDC up-regulated MHC-II, CD80 and CD86 expression, while CD11b+ APC expressed high levels of PDL1 and PDL2 and up-regulated F4/80 and Gr-1-associated markers. In contrast, the same donor APC populations recovered from the spleen expressed lower levels of DC-associated markers. Bioluminescent imaging showed that luciferase+ donor DC persisted and expanded in the lymph nodes and spleen of transplant recipients for up to one month. The immunological effect of transplanting pDC led to increased donor T-cell proliferation, IFN-ψ production and CD25 expression compared with transplanting grafts containing HSC, T-cells and CD11b+ APC, or grafts containing only HSC and T-cells in both MHC-mismatched murine HSCT models. In conclusion, the results indicate that different subsets of donor APC and DC home to lymphoid tissues and up-regulate a pattern of co-stimulatory and co-inhibitory molecules consistent with their observed immunological immune activity in vivo. Maturation of pDC in vivo was slower than that in vitro, and pDC maturation was slower in the recipients' BM compared with recipient's spleen suggesting that the post-transplant microenvironment may impair DC differentiation. Less mature pDC in the stem cell graft that home to the BM may facilitate donor engraftment and immune reconstitution, while more mature pDC that home to the spleen may polarize donor T-cell Th1 responses. Disclosures: Waller: NIH-National Heart, Lung, and Blood Institute, NHLBIP01H1086773: Research Funding.

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T-Cell Derived Interferon Gamma Directly Targets Intestinal Epithelium to Induce Stem Cell Apoptosis in GI Gvhd

Blood ◽

10.1182/blood-2019-130811 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 585-585

Author(s):

Shuichiro Takashima ◽

Maria L. Martin ◽

Suze A. Jansen ◽

Ya-Yuan Fu ◽

Anastasiya Egorova ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lamina Propria ◽

Intestinal Epithelium ◽

Ex Vivo ◽

Reporter Mice ◽

Donor T Cells ◽

Kinetics Of

Crypt base intestinal stem cells (ISCs) marked by Lgr5 and Olfm4 maintain the intestinal epithelium, and Paneth cells (PCs) provide an epithelial niche for ISCs in the small bowel. ISCs are reduced during gastrointestinal (GI) GVHD, but the precise mechanisms including the role of niche injury are unknown. Additionally, the specific effects of Interferon-γ (IFNγ) on intestinal epithelium in GVHD remain ill-defined. We evaluated kinetics of ISC loss by histology after allogeneic (allo) BMT using Lgr5-LacZ reporter mice. In both MHC- and miHA-mismatched models (LP>B6 and B6>BDF1), ISC numbers quickly recovered from pretransplant TBI conditioning in recipients of T-cell-depleted (TCD) BMT by day +10, but ISCs failed to recover in recipients of allo T cells. T-cell-induced ISC reduction was functionally validated by genetic marking of stem cell progeny and by culturing intestinal organoids from crypts isolated post-BMT. Similar to the kinetics of ISC loss, ISC-dependent organoid-forming capacity was impaired in recipients of allo T cells compared with TCD BMT recipients on day +10 (p<0.05). Likewise, BMT into Olfm4 reporter mice showed significantly reduced lineage tracing from ISCs in recipients of allo T cells (Fig. 1). To better understand the potential for T cells to interact with the ISC compartment, we performed whole-mount 3-D microscopy to distinguish T cell localization within the intraepithelial and lamina propria compartments post-transplant. We found that donor T cells invading the small bowel after BMT were mostly in the crypt region, and infiltration within the lamina propria adjacent to the ISC compartment was much greater than invasion within the epithelium itself (Fig. 2). We next established an ex vivo co-culture system, to model interactions between intestinal epithelium and donor T cells and investigate mechanisms of T-cell-mediated ISC injury. Screening of effector pathways revealed no impact of perforin or FasL, but identified IFNγ as a principal mediator of ISC injury. Culture with allo T cells significantly reduced viable human and mouse intestinal organoid numbers, and this was inhibited by IFNγ neutralization. IFNγ receptor knockout (IFNγR-/-) organoids were resistant to T cells. IFNγ increased expression of Bak1 and decreased expression of Bcl2 in organoids, and induced ISC apoptosis defined by Annexin+Dapi-Lgr5+ phenotype. ISC killing was mediated by intraepithelial JAK/STAT signaling, as JAK1- and STAT1-deficient organoids were resistant, and it was inhibited by Ruxolitinib. Investigating the role of IFNγ in vivo, FACS analysis confirmed donor T cells to be the primary producers of IFNγ in crypt lamina propria, and BMT with IFNγ-/- donor T cells reduced crypt apoptosis, and preserved ISC frequencies. Moreover, BMT with recipients lacking IFNγR specifically in the intestinal epithelium significantly protected ISCs, reduced crypt apoptosis, and ameliorated GI GVHD pathology (Fig.3). Furthermore, ISCs were also protected by epithelial deletion of STAT1 and by Ruxolitinib treatment. As specific genetic manipulation of ISCs in vivo is not possible because genetic targeting of ISCs results in the same changes in their progeny, we utilized ex vivo models to determine if IFNγ kills ISCs by directly inducing their apoptosis or by damaging the PC niche. Manipulation of the niche by culturing wild-type ISCs with IFNγR-/- PCs was not protective to allo T cells. Using a niche-independent high-purity Lgr5+ ISC culture system based on combined GSK3β and HDAC inhibition, IFNγ directly induced cleaved-caspase-3+ ISC apoptosis and substantial ISC colony death, which were inhibited by Bak/Bax deficiency and by the pan-caspase inhibitor QVD. These results confirmed that IFNγ can directly induce ISC apoptosis independent of other cytotoxic effector molecules and independent of injury to the PC niche. In summary, T cells migrating to the GI tract primarily infiltrated the lamina propria adjacent to the ISC compartment, and T-cell-derived IFNγ directly targeted intestinal epithelium via JAK1/STAT1 signaling to induce ISC apoptosis in a PC-independent manner. ISC reduction and GI GVHD pathology were prevented by inhibiting the IFNγR/JAK1/STAT1 axis within the intestinal epithelium, indicating that in addition to their effects on T cells, JAK inhibitors may treat GVHD by inhibiting pathologic cytokine signaling within target organs and shielding them from allo T cells. Disclosures Hanash: Nexus Global Group LLC: Consultancy.

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Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer

Blood ◽

10.1182/blood-2007-02-073304 ◽

2007 ◽

Vol 110 (4) ◽

pp. 1132-1140 ◽

Cited By ~ 148

Author(s):

Ou Cao ◽

Eric Dobrzynski ◽

Lixin Wang ◽

Sushrusha Nayak ◽

Bethany Mingle ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Antibody Formation ◽

Coagulation Factor ◽

Gene Replacement ◽

Factor Ix ◽

In Vivo Gene Transfer

Abstract Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (Treg)–deficient model (Rag-2−/− mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4+CD25+ Tregs by in vivo gene transfer. Hepatic gene transfer–induced Tregs express FoxP3, GITR, and CTLA4, and suppress CD4+CD25− T cells. Tregs are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4+CD25+GITR+ that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4+CD25+ Tregs leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4+CD25+ Tregs in preventing immune responses to the transgene product in gene transfer.

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T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Gut ◽

10.1136/gutjnl-2015-309453 ◽

2015 ◽

Vol 66 (3) ◽

pp. 454-463 ◽

Cited By ~ 24

Author(s):

Daniele Mennonna ◽

Cristina Maccalli ◽

Michele C Romano ◽

Claudio Garavaglia ◽

Filippo Capocefalo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

High Throughput ◽

High Throughput Sequencing ◽

Cell Epitope ◽

Patient Specific ◽

Cancer Genes ◽

Healthy Donors

ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.ConclusionsThese results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

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T cell-intrinsic role of IL-6 signaling in primary and memory responses

eLife ◽

10.7554/elife.01949 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 73

Author(s):

Simone A Nish ◽

Dominik Schenten ◽

F Thomas Wunderlich ◽

Scott D Pope ◽

Yan Gao ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Suppressive Effect ◽

Immune Recognition ◽

Th17 Response ◽

Cell Responses ◽

Underlying Mechanisms ◽

Specific Deletion

Innate immune recognition is critical for the induction of adaptive immune responses; however the underlying mechanisms remain incompletely understood. In this study, we demonstrate that T cell-specific deletion of the IL-6 receptor α chain (IL-6Rα) results in impaired Th1 and Th17 T cell responses in vivo, and a defect in Tfh function. Depletion of Tregs in these mice rescued the Th1 but not the Th17 response. Our data suggest that IL-6 signaling in effector T cells is required to overcome Treg-mediated suppression in vivo. We show that IL-6 cooperates with IL-1β to block the suppressive effect of Tregs on CD4+ T cells, at least in part by controlling their responsiveness to IL-2. In addition, although IL-6Rα-deficient T cells mount normal primary Th1 responses in the absence of Tregs, they fail to mature into functional memory cells, demonstrating a key role for IL-6 in CD4+ T cell memory formation.

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

Blood ◽

10.1182/blood.v104.11.593.593 ◽

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.

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Donor DC Subsets Polarize Donor T-Cell Immune Responses in Allogeneic BMT.

Blood ◽

10.1182/blood.v106.11.573.573 ◽

2005 ◽

Vol 106 (11) ◽

pp. 573-573

Author(s):

Jian-Ming Li ◽

Cynthia Giver ◽

Doug McMillan ◽

Wayne Harris ◽

David L. Jaye ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Immune Responses ◽

Peripheral Blood ◽

Immune Reconstitution ◽

Hematopoietic Cell ◽

Post Transplant ◽

Donor T Cells ◽

Ifn Γ

Abstract Introduction: Impaired or inappropriate immune reconstitution after allogeneic bone marrow transplantation (BMT) can lead to infection, graft-versus-host disease (GvHD) and leukemia relapse. We have previously reported that BM contains two populations of dendritic cell (DC) subsets, CD11b+ DC and CD11b− DC, and that CD11b depleted donor BM promoted increased donor T-cell chimerism and increased graft-versus-leukemia (GvL) activity in C57BL/6 → B10BR transplants [BBMT, 2004, 10: 540]. To explore the mechanism by which CD11b-depletion improved allo-reactivity, we performed allogeneic hematopoietic cell transplants using defined populations of donor stem cells, DCs, and T-cells in a MHC mis-matched BMT model. Methods: We transplanted FACS purified populations of 50,000 GFP+ CD11b- DC or CD11b+ DC in combination with 5,000 FACS purified Lin- Sca-1+ c-kit+ hematopoietic stem cells (HSC) and 300,000 or 1,000,000 congenic spleen T-cells from C57BL/6 donors into C57BL/6[H-2Kb], B10BR[H-2Kk] and PL/J[H-2Ku] recipients. Proliferation of CFSE stained donor T-cells was measured at 72 hours post-transplant. FACS cytometric bead array and intracellular cytokine staining measured serum and intracellular cytokines in donor T-cells. Results: The initial proliferation and Ki-67 expression of CFSE labeled donor T-cells in allogeneic recipients were much higher than in syngeneic recipients (homeostatic proliferation). Confocal microscopy showed co-localization of donor DC subsets with donor T-cells in the recipient spleens at 3 and 10 days post-transplant. In the allogeneic transplant settings, donor T-cells co-transplanted with CD11b- DC showed increased IFN-γ synthesis at 3 and 10 days post-transplant compared to donor T-cells co-transplanted with HSC plus CD11b+ DC or HSC alone. Increased proliferation of donor T-cells led to increased donor T-cell chimerism at day 10, 30, 60, and day105 post-transplant among recipients of CD11b- DC compared to recipients of HSC alone or HSC plus CD11b+ DC (Figure 1). Transplantation of spleen T-cells and CD11b- DC did not increase GvHD, but was associated with full donor chimerism. In contrast, transplantation of allogeneic CD11b+ DC led to persistence and expansion of residual host T-cells (Figure 2), increased numbers of donor CD4+CD25++Foxp3+ T-cells, and higher serum level of IL-10 supporting early post-transplant expansion of donor T regulatory cells (Treg). Conclusions: Donor CD11b- DC promoted immune reconstitution by polarizing donor T-cells to Th1 immune responses associated with increased IFN-γ synthesis and donor T-cell proliferation, while donor CD11b+ DC suppressed immune reconstitution by inhibiting donor T-cell allogeneic immune responses. These data support a novel paradigm for the regulation of post-transplant immunity and suggest clinical methods to test the hypothesis that manipulation of the DC content of a hematopoietic cell allograft regulates post transplant immunity in the clinical setting. Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells]

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