Alloreactive Cells Research Articles

Sustained reduction of alloantibody secreting plasma cells and donor specific antibody with proteasome inhibition in mice

The long-lived plasma cells, which develop after alloantigen sensitization, produce donor specific alloantibodies (DSAs) that generate a positive serum cross-match and preclude transplantation. Bortezomib, a proteasome inhibitor, is being investigated in clinical desensitization protocols, however preclinical studies in a transplant model are nonexistent. We hypothesized that sustained treatment with only a proteasome inhibitor would eliminate plasma cells and reduce DSA over time.Cardiac allografts were transplanted into murine recipients. Eight weeks after allograft rejection the proteasome inhibitor, bortezomib, was injected intravenously twice weekly for 60days. Serum alloantibody responses were assayed using flow cross-match. Total and alloreactive plasma cell numbers were enumerated using flow cytometry and ELISPOT.All recipients of cardiac allografts rejected their graft promptly within 16days and demonstrated alloantibody by flow cross-match. DSA was sustained in the control mice while mice treated with bortezomib had sustained elimination of DSA and a marked reduction in plasma cell population. Also, bortezomib was associated with an increased level of BLyS.Within a murine model, proteasome inhibition can eliminate alloantibody secreting plasma cells, and reduce alloantibody. Cessation of bortezomib is not associated with return of DSA.

Successful Tolerance to Fully MHC Disparate Renal Allografts via Donor Hematopoietic Mixed Chimerism in Non-Human Primates

To the Editor: Rodent studies clearly show that sharing of MHC genes between donors and recipients is regularly associated with improved allograft survival. This is essentially attributed to: (1) reduction of early inflammatory direct T cell responses involved in acute allograft rejection and (2) self-MHC molecules expression on donor cells promoting regulatory immunity. The precise contribution of donor/recipient MHC gene matching to transplant rejection or acceptance in clinical transplantation has been somewhat more difficult to define due to the use of intensive immunosuppression in all human allograft recipients. Nevertheless, most authors agree that improved long-term graft survival is generally expected in patients receiving MHC-matched renal allotransplants. Rodent studies also demonstrate that transplant tolerance is easier to accomplish when allogeneic tissues express some or all of the recipient's MHC proteins. Nevertheless, tolerance of fully allogeneic transplants is regularly achieved in laboratory-bred mice using a variety of procedures including donor specific transfusion, donor hematopoietic chimerism and lymphocyte costimulation blockade. In contrast, such tolerance in primates has proven to be much more arduous due presumably to the presence in these recipients of preexisting alloreactive memory T cells (TMEM). These TMEM undergo differentiation and clonal expansion following previous exposure to alloantigens through blood transfusion, pregnancy, a previous transplantation or via cross-reactivity with microbial antigens. A recent article in AJT by Ramakrishnan et al. 1, referring to a recent report from our group 2, suggested that we had achieved tolerance of renal allografts in non-human primates via mixed chimerism only in highly MHC-matched transplant pairs. This interpretation of our experience is not fully accurate. Indeed, we have reported an inverse correlation between the level of IL-2 and γIFN memory alloreactivity and the degree of MHC class II and I gene sharing among monkey pairs 3. However, this is not an absolutely consistent observation with a substantial number of fully MHC disparate donor–recipient pairs displaying low pro-inflammatory memory responses 3. This suggests that tolerance susceptibility vs. resistance in primates depends primarily upon the frequencies of pre-existing donor-specific TMEM rather than upon donor–recipient MHC matching. In support of this view, we have observed that over one third of the monkeys that became tolerant of renal allografts had actually received a highly MHC-mismatched (>4/6 CyLA mismatches) transplant 4, 5. In summary, the beneficial effects of MHC matching with regards to transplantation tolerance are undeniable. In addition, it is likely that harnessing anamnestic T cell alloreactivity in primates will be essential to successful tolerance of allogeneic transplants across MHC barriers in non-human primates and patients. G. Benichou*, O. Nadazdin, A. B. Cosimi, and T. Kawai Transplantation Research Center, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA *Corresponding author: Gilles Benichou, gbenichou@partners.org The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Primary Vascularization of Allografts Governs Their Immunogenicity and Susceptibility to Tolerogenesis

We investigated the influence of allograft primary vascularization on alloimmunity, rejection, and tolerance in mice. First, we showed that fully allogeneic primarily vascularized and conventional skin transplants were rejected at the same pace. Remarkably, however, short-term treatment of mice with anti-CD40L Abs achieved long-term survival of vascularized skin and cardiac transplants but not conventional skin grafts. Nonvascularized skin transplants triggered vigorous direct and indirect proinflammatory type 1 T cell responses (IL-2 and IFN-γ), whereas primarily vascularized skin allografts failed to trigger a significant indirect alloresponse. A similar lack of indirect alloreactivity was also observed after placement of different vascularized organ transplants, including hearts and kidneys, whereas hearts placed under the skin (nonvascularized) triggered potent indirect alloresponses. Altogether, these results suggest that primary vascularization of allografts is associated with a lack of indirect T cell alloreactivity. Finally, we show that long-term survival of vascularized skin allografts induced by anti-CD40L Abs was associated with a combined lack of indirect alloresponse and a shift of the direct alloresponse toward a type 2 cytokine (IL-4, IL-10)-secretion pattern but no activation/expansion of Foxp3(+) regulatory T cells. Therefore, primary vascularization of allografts governs their immunogenicity and tolerogenicity.

CMV-, EBV- and ADV-Specific T Cell Immunity: Screening and Monitoring of Potential Third-Party Donors to Improve Post-Transplantation Outcome

Adoptive immunotherapy with virus-specific T lymphocytes can efficiently reconstitute antiviral immunity against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) without causing acute toxicity or increasing the risk of graft-versus-host disease. To gain insight into antiviral T cell repertoires and to identify the most efficient antigens for immunotherapy, the frequencies of CMV-, EBV- and ADV-specific T cells in 204 HLA-typed healthy donors were assessed using viral peptides and peptide pools. Confirmatory testing for CMV serology by Western blot technique revealed 19 of 143 (13%) false-positive results. We observed highly significant individual and overall differences in T cell frequencies against CMV, EBV, and ADV antigens, whereas antigen-specific T cells were detected in 100% of CMV- seropositive donors, 73% of EBV- seropositive donors, and 73% of ADV-seropositive donors. At least 124 (61%) potential T cell donors were identified for each virus. Among the tested antigens, frequencies for CMVpp65 and EBVBZLF1 peptide pools were highest. Short-term in vitro peptide stimulation revealed that a donor response to a certain ADV- and EBV-derived peptide may not be determined without prior stimulation. A modified granzyme B ELISpot was used to detect T cell specificity and alloreactivity. Treatment with allogeneic virus-specific cytotoxic T lymphocytes from seropositive third-party donors may be a feasible therapeutic option for infections following cord-blood stem cell transplantation or hematopoietic stem cell transplantation from virus-seronegative donors.

Long-term safety and survival outcomes after TK-expressing donor lymphocyte infusion (TK-DLI) in allogeneic hematopoietic stem cell transplantation (HSCT).

7007 Background: Suicide gene therapy (SGT) was firstly applied to allogeneic HSCT, addressing the need for modulation of graft vs host disease (GvHD) reactions while preserving graft vs leukemia (GvL) effect of alloreactive T cells. HSV-TK gene insertion in donor T-cells modulates alloreactivity by selectively destroying dividing alloreactive cells involved in GvHD. Methods: Long-term safety and survival was assessed in 128 pts entering worldwide 10 phase I-II trials that used TK-DLI to improve GvL, immune reconstitution (IR) and GvHD control. In all, 57 pts received TK DLI at our Institution: 23 to treat relapse after HLA-identical HSCT (Ciceri, 2007) and 34 to improve IR after haploidentical HSCT (Ciceri, Bonini, 2009). Results: SGT was feasible, safe and effective in promoting a dynamic and specific modulation of alloreactivity. TK-DLI clinical benefit, defined by chimerism, tumor response and/or IR, was achieved by 65 pts (51%). Grade 2 to 4 GvHD (n=28, 22%) was fully controlled by SGT. TK-DLI engrafted in 51 pts (90%) and, being detectable at low frequency up to 14 yrs, no SGT-related adverse events occurred. In HLA-identical setting (n=23; median follow-up, 15 yrs), 11 pts (48%) had disease response and 2 pts (9%) were alive in complete response (CR). In haploidentical setting (n=34; median follow-up, 7 yrs), 25 pts (73%) had IR and 9 pts (26%) were alive in CR. All pts were monitored according to guidelines on long-term survivors (Majhail, 2012). There were no major infections, while 3 pts had a second tumor. Immunity against TK-DLI was reported exclusively after HLA-identical allo-HSCT indicating that TK-DLI is not limited by SGT-specific immunity after haploidentical HSCT. Conclusions: Long-term follow-up confirms the high benefit to risk ratio of TK-DLI. A phase III trial is ongoing in haploidentical HSCT (NCT00914628). Clinical trial information: NCT00423124.

KIR Gene Mismatching and KIR/C Ligands in Liver Transplantation

Killer immunoglobulin-like receptors (KIRs) bind human leukocyte antigen (HLA) class-I (HLA-I) ligands and regulate functions of natural killer cells and subsets of T cells. KIR/HLA-I interactions allow predicting natural killer cell alloreactivity in hematopoietic stem cell transplantation and in HLA-compatible kidney transplants, but its meaning in liver transplantation remains controversial. KIR and HLA genotypes were studied in 402 liver transplants, using sequence-specific oligonucleotides and primer methods. Recipients and donor KIRs, HLA-C genotypes, KIR gene mismatches (MMs) between recipient-donor pairs, and KIR/HLA-ligand combinations were analyzed in overall transplantations, in the acute rejection (AR; n=110) and non-AR (n=292) groups. KIR gene MMs between recipients and donors, mainly in activating KIRs, and KIR2DL3 and KIR2DS1 of recipients in the presence of donor C2 ligands, significantly enhanced early AR rate (P<0.05), with KIR2DL3 and KIR2DS1 exhibiting a synergic effect in dependence of the donor C2 ligand number (χ2=7.662, P=0.022). KIR2DL3, KIR2DS1, and also KIR2DS4 significantly influenced short-term graft survival, with a benefit for transplantations combining KIR2DL3 recipients and donors having C1 ligands (log rank, P<0.019 at 1 year; hazards ratio [HR], 0.321; 95% confidence interval [CI], 0.107-0.962; P=0.042), whereas KIR2DS1 and KIR2DS4 recipients combined with donors lacking C1 ligands (C2/C2) exhibited a worse graft survival (log rank, P=0.035 at 6 months; HR, 7.713; 95% CI, 2.156-27.369; P=0.002 for KIR2DS1; and log rank, P=0.006 at 1 year; HR, 3.794; 95% CI, 1.267-11.365; P=0.017 for KIR2DS4). This study shows that KIR gene-gene MMs increase AR and that KIRs/C ligands associated to AR and KIR2DS4/C ligands also influence short-term graft survival.

Target‐Induced Natural Killer Cell Loss as a Measure of NK Cell Responses

Natural killer (NK) cells are an important effector cell of innate immunity. Their interaction with susceptible target cells triggers NK cell cytotoxicity and the release of cytokines. Immunofluorescence flow cytometry-based assays are now the preferred methods for measuring NK cell responses. For these assays, assessment is made on NK cells (CD3(-)CD56(+) CD16(+)) within the viable lymphocyte gate, defined by the parameters of size (FSC) and granularity (SSC). Accordingly, NK cells that have not dissociated from target cells at the time of measurement, or that have undergone target cell-induced apoptosis, are excluded from the viable lymphocyte gate and therefore from analysis. This unit describes a protocol for assessing NK cell function in response to various target cells (natural killing, antibody-dependent cell cytotoxicity, and NK cell alloreactivity) based on the loss of NK cells from the lymphocyte gate. This target-induced NK loss (TINKL) should provide a sensitive measure of NK cell responses in a clinical laboratory setting.

Homozygosity for killer immunoglobin-like receptor haplotype A predicts complete molecular response to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients

Several recent reports suggest a possible role for killer immunoglobulin-like receptors (KIR) in the onset of chronic myeloid leukemia (CML) and response to therapy with tyrosine kinase inhibitors (TKIs). To explore this hypothesis, we studied KIRs and their human leukocyte antigen class I ligands in 59 consecutive patients with chronic-phase CML (mean age, 53 years; range, 23-81 years) and a group of 121 healthy control participants belonging to the same ethnic group as the patients. The 2-year cumulative incidence of complete molecular response, obtained after a median of 27 months (range, 4-52 months), was 51.2%. An increased frequency of the activating receptor KIR2DS1 (pm = 0.05) and a reduced frequency of the KIR-ligand combination KIR2DS2/2DL2 absent/C1 present (pm = 0.001) were significantly associated with CML. Moreover, KIR repertoires in patients appeared to influence response to TKI therapy. Homozygosity for KIR haplotype A (pm = 0.01), a decreased frequency of the inhibitory KIR gene KIR2DL2 (pm = 0.02), and low numbers of inhibitory KIR genes (pm = 0.05) were all significantly associated with achievement of complete molecular remission. These data suggest that a decrease in properly stimulated and activated NK cells might contribute to the occurrence of CML and indicate homozygosity for KIR haplotype A as a promising immunogenetic marker of complete molecular response that could help clinicians decide whether to withdraw treatment in patients with CML.

A Color-Coded Reporter Model to Study the Effect of Immunosuppressants on CD8+ T-Cell Memory in Antitumor and Alloimmune Responses

Mammalian target of rapamycin (mTOR) inhibitors possess anticancer properties potentially useful in reducing posttransplantation malignancy. Besides controlling tumor-sensitive proliferative and angiogenic effects, mTOR influences transcription factors T-bet and Eomesodermin (Eomes) in CD8 cytotoxic T cells (Tc), which are key in rejecting tumors, and allografts. To study the role of mTOR in tumor and transplant immunity in an antigen-specific way, we used T-cell receptor transgenic B6.OTI recipients, B6.OVA.TG donors, and OVA-B16F10 melanoma cells. For tracking color-coded OTI-Tc cells associated with antitumor and alloimmunity in vivo, CD8-OTI transgenic reporter mice were created by crossbreeding DsRed-expressing B6.Nagy mice with B6.OTI mice. The role of mTOR in regulating the differentiation and function of alloreactive Tc cells in vitro was explored by stimulating OTI-Tc cells with ovalbumin-transgenic antigen-presenting cells in the presence of rapamycin or tacrolimus. Rapamycin, but not tacrolimus, induced a pro-antitumor phenotypic shift from CD62LCD44 effector memory Tc cells to CD62LCD44 central memory Tc cells, which featured up-regulated levels of T-bet and Eomes and preserved levels of interferon-γ and perforin. For future investigations, an in vivo model was established whereby DsRedOTI-Tc cells adoptively transferred into B6 mice bearing either a ovalbumin-transgenic mouse skin transplant or OVA-B16F10 tumor could be traced by fluorescence-activated cell sorting analysis as effector or memory Tc cells in transplant and tumor tissues. mTOR, but not calcineurin, inhibition spares antitumoral memory Tc cells by distinctively regulating T-bet and Eomes. This finding is now testable in a new tumor transplant model, which incorporates DsRedOTI-Tc cell tracing, opening the way to study the differential effects of immunosuppressants in posttransplantation malignancy.

A Two-Step Approach to Allogeneic Haploidentical Hematopoietic Stem Cell Transplantation

Strategies that exploit natural killer (NK) cell alloreactivity or attenuate rather than deplete T cells have resulted in improved outcomes after haploidentical hematopoietic stem cell transplantation (HSCT). However, no approach has consistently produced the triad of optimal immune reconstitution, avoidance of significant graft-versus-host disease (GVHD), and durable control of malignancy. We developed a two-step approach to haploidentical HSCT in which the lymphoid and myeloid portions of the graft are given in two separate steps in order to control and optimize T-cell dosing. The initial results from these trials have included robust immune reconstitution, low rates of toxicity and significant GVHD, and durable disease control in good-risk patients, as well as insights regarding a threshold for T-cell dosing above which graft-versus-tumor (GVT) effects might be expected. Patients who were not in remission at the time of HSCT had higher rates of relapse requiring efforts to further strengthen GVT effects. Second-generation trials are underway to further exploit changes in the dosing and timing of administration of T cells and to optimize donor selection in an effort to decrease relapse rates in high-risk patients.

Haploidentical Hematopoietic Stem Cell Transplantation With a Megadose T-Cell–Depleted Graft: Harnessing Natural and Adaptive Immunity

For patients with high-risk acute leukemia who do not have a matched donor or who urgently need a transplant, transplantation from a full human leukocyte antigen (HLA) haplotype mismatched family donor should be considered a viable option. Clinical trials have shown that a strategy for haploidentical transplantation based on the infusion of high numbers of T-cell-depleted hematopoietic progenitor cells and no post-transplant immunosuppression controls bi-directional T-cell alloreactivity, ie, graft rejection and graft-versus-host disease (GvHD) in patients with leukemia. Overall, event-free survival compares favorably with reports of transplants using sources of stem cells other than the matched sibling. This transplant modality has highlighted the crucial role of donor-versus-recipient natural killer cell (NK) alloreactivity in the control of leukemia relapse. Current studies are focusing on rebuilding post-transplant immunity to improve clinical outcomes.

Post-Transplantation Cyclophosphamide for Tolerance Induction in HLA-Haploidentical Bone Marrow Transplantation

Allogeneic hematopoietic stem cell transplantation (alloSCT) is a potentially curative therapy for many hematologic and immunologic diseases. Further, partial or full donor hematopoietic chimerism following alloSCT may be sufficient to guarantee immunologic tolerance to solid organs from the same donor, obviating any requirement for prolonged pharmacologic immunosuppression. Despite alloSCT's potential, the procedure is beset by two major limitations. The first relates to the procedure's toxicity, including conditioning regimen toxicity, graft-versus-host disease (GVHD), and infection. The second limitation is the lack of histocompatible donors. A human leukocyte antigen (HLA)-matched sibling or unrelated donor cannot be identified expeditiously for up to 40% of patients. Historically, alloSCT from partially HLA-mismatched, or HLA-haploidentical, relatives has been complicated by unacceptably high incidences of graft rejection, severe GVHD, and non-relapse mortality. Recently, our groups have developed a method to selectively deplete alloreactive cells in vivo by administering high doses of cyclophosphamide in a narrow window after transplantation. Using high-dose, post-transplantation cyclophosphamide (PT/Cy), crossing the HLA barrier in alloSCT is now feasible and donors can be found for nearly all patients. This review discusses the history of HLA-haploidentical SCT, recent clinical results, and immunologic mechanisms of action of high-dose PT/Cy for prevention of graft rejection and GVHD.

Double-Unit Umbilical Cord Blood Transplantation Induces MHC-Matched Single-Unit Dominance with Development of Immunocompetent Lymphocytes in Recipient Mice

Abstract 4097 Background:Double-unit umbilical cord blood cell (dUCBC) transplantation has emerged as an effective strategy for improving the engraftment of umbilical cord blood stem cells in the bone marrow of recipients. Due to a lack convenient animal models, analyses of the differentiation capacity of dUCBC in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In the present study, we evaluated the characteristics of immune reconstitution induced by dUCBC transplantation in mice. Materials and Methods:Natural killer cells were depleted from female C57BL/6 (B6) [H-2b] recipient mice by intraperitoneal administration of rabbit anti-asialo GM1 polyclonal antibody 1 day before transplantation. On the following day, the lethal X-ray-irradiated B6 recipients were given transplants of three different combinations of dUCBC {group (1) GFP-Tg B6 [H-2b] and BALB/c [H-2d]; group (2) GFP-Tg B6 [H-2b] and C3H [H-2k]; group (3) BALB/c [H-2d] and C3H [H-2k]}, each combination containing an equal number of cells. At 16 weeks after transplantation, reconstitution of immune cells was evaluated by flow cytometric analysis utilizing specific antibodies against lineage markers such as CD3 (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes). The donor origin of each lineage population was determined by anti-H-2Kk (for C3H) and/or H-2Kd (for BALB/c) antibody staining. GFP+ lineage cells were identified as being of B6 donor origin. Skin grafting was then performed in all recipients to assess the functional maturity of the newly developed T and B cells induced by dUCBC transplantation. Results:The survival rate at 16 weeks after transplantation was 73% (8/11) for case (1), 92% (12/13) for case (2), and 50% (3/6) for case (3). In the great majority of cases (1) and (2), in which dUCBC were administered as a stem cell source, the MHC-matched single unit from GFP-Tg B6 acts as the sole source of long-term hematopoiesis (75% (6/8) for case (1); 100% (12/12) for case (2)). CD3+ T cell peripheral blood chimerism from BALB/c was observed in two of the eight B6 survival recipient mice in case (1) at an early stage of hematopoiesis, predicting the long-term engrafting unit. On the other hand, hematopoiesis in case (3) with fully allogenenic dUCBC transplantation was reconstituted by the B6 recipients' own X-ray-resistant hematopoietic stem cells (HSC). Our results indicate that MHC-matched UCBC-HSC predominantly engraft in the recipient's bone marrow after dUCBC transplantation. However, the nature of this selective mechanism remains largely unknown. In all cases, alloreactive cytotoxic cells in recipient may participate in such selection. In dUCBC transplantation, the included allogeneic cells probably act as stimulators for promoting the differentiation and maturation of MHC-matched HSC through activation of certain types of signal transduction (for example, through cytokine secretion). Currently, we are investigating the possible presence of alloreactive cytotoxic cells in bone marrow. Functionally, these recipients were tolerant of skin grafted from B6, whereas they rejected skin from BALB/c and C3H within 20 days, indicating that both CD4+ helper and CD8+ killer T cells were functionally mature in the recipient mice. Correspondingly, only the alloantibody to BALB/c and C3H was produced in the recipients. One of two chimeric recipient mice in case (1) reacted to only C3H skin with T and B cells. Conclusions:dUCBC transplantation clearly rescued mice that had been subjected to lethal X-ray irradiation. Furthermore, our observations indicate that T and B cells derived from dUCBCs transplants are immunologically fully competent with the ability to distinguish self from non-self MHC antigens. However, a clear understanding of the mechanisms underlying the predominant engraftment of MHC-matched HSCs in the recipient's bone marrow will be necessary. Disclosures:No relevant conflicts of interest to declare.

The Expansion of Gastrointestinal-associated αβ T Cell Clones in Peripheral Blood Over Time Is a Disease Feature of Severe Acute Graft-Versus-Host Disease

AbstractAbstract 228Donor T cell alloreactivity in hematopoietic stem cell transplantation (HSCT) can result in acute graft-versus-host disease (GVHD) which is a major cause of morbidity and mortality that limits the clinical use of HSCT. Despite the potential large diversity in possible T cells (1015) it is thought that within each individual patient undergoing HSCT, a tractable and defined number of T cell clones mediate GVHD. We hypothesized that the identification of the most frequent T cell clones in the gastrointestinal (GI) tract of human patients with GVHD and subsequent tracking of these clones in the peripheral blood may provide a way to bracket and study disease related clones.To investigate T cell clonal dynamics in human GVHD, we collected endoscopic GI tract biopsy samples and matched blood specimens for twelve patients undergoing myeloblative HSCT with suspected GI GVHID prior to or within one day of corticosteroid therapy. We also collected peripheral blood thirty days after biopsy. Five of these patients were negative for GVHD or had mild grade I/II GI GVHD, whereas seven had severe and fatal grade III/IV GI GVHD. These patients had heterogenous indications, conditioning regimens and GVHD prophylaxis which included cyclosporine, tacrolimus and methotrexate. None of the patients had cytomegolovirus (CMV) colitis or other identifiable GI or significant systemic infectious complications, although four patients had low-level blood CMV positivity by PCR. As a control, we compared endoscopic GI biopsies from patients with inflammatory bowel disease as well as normal healthy screening subjects. We extracted genomic DNA and performed T cell receptor (TCR) β CDR3 repertoire sequencing at Stanford and with GigaGen GigaMune Rep-Seq™, both using the Illumina next generation sequencing MiSeq™ and HiSeq™ platforms. Results:For each tissue sample from each patient, between 1,000–4,000 unique T cell sequences were observed in endoscopic tissue samples (2–6 samples per patient). We sorted the most frequent clones in the GI tissue samples by rank order and fractionated the top 100 clones for analysis. Many of these top clones were found in the matched peripheral blood at the time of diagnosis with most of these occurring at frequencies well below those in the GI tissue. For the patients with negative or mild GVHD at the time of diagnosis, the GI-identified clones that were found in the peripheral blood had a mean frequency and standard deviation of 0.001369 +/− 0.0016. This is indistinguishable from peripheral blood samples from patients with severe GVHD which showed a mean frequency and standard deviation of 0.0013 +/− 0.0009 (P=0.36, 2 tailed t-test).We next tracked the GI-identified clones in the peripheral blood to the obtain fold change of the clones at thirty days after diagnosis to the time of diagnosis. The patients with negative or mild GVHD had a mean, median, and standard deviation in fold change of 1.9, 1.7, and 0.9 respectively. The patient with severe GVHD patients had a mean, median and standard deviation in fold change of 32.2, 30.7, and 17.3. A two tailed t-test comparison of the two groups showed they were statistically distinguishable with p-values of 0.0003 for examination of the mean fold change and 0.0005 for the median fold change.For patients with severe GVHD, the peripheral blood at 30 days after diagnosis showed a highly oligoclonal expansion of individually private GI-identified clones. Many of these clones were identified in their respective donor T cell pool. For those patients evaluated with upper and lower endoscopy, many clones were shared between the colon and duodenum or gastrum, however, the dominant clones between these sites appeared to be different. Conclusion:We found that a comparison of the fold change in the peripheral blood frequencies of the top one hundred clones identified in the GI tract biopsies can statistically distinguish five negative or mild GVHD patients from seven severe and fatal cases. These results support the use of T cell repertoire sequencing and associated approaches in human patients to both clarify the pathophysiology of the disease and eventually perhaps to provide an independent immune biomarker that could guide therapy. A major challenge will be to further phenotype and examine the T cell clones identified in the GI tissue and to investigate ways to determine which clones are highly associated or even causal to GVHD. Disclosures:Meyer:GigaGen Inc: Consultancy, Equity Ownership. Löhr:GigaGen Inc.: Employment, Equity Ownership. Hsu:GigaGen Inc.: Employment, Equity Ownership. Johnson:GigaGen Inc.: Employment, Equity Ownership.

FLT3-Kinase Inhibitors Quizartinib and Midostaurin Do Not Impair T-Cell Reactivity and Activation

AbstractAbstract 1045In patients with FLT3-ITD mutated AML, FLT3-inhibitors have been used successfully as a ‘bridging therapy' before allogeneic transplantation. Inhibitors of other kinases (such as imatinib for BCR-ABL positive CML) have previously been used successfully after allogeneic transplantation – even before discontinuation of immunosuppressive medication. However, it is known that some BCR-ABL inhibitors such as dasatinib exert strong inhibitory effects on primary T-cells through inhibition of Src-kinases relevant for T-cell receptor signaling. Even imatinib and nilotinib - although not affecting Src kinase activity – showed decreased T-cell activation and reactivity to some extent. Thus, the influence of FLT3-kinase inhibitors on T-cell function may be critical in the context of allogeneic bone marrow transplantation for FLT3-ITD-positive AML. Besides inhibition of FLT3-kinase, midostaurin (PKC412) exerts activity against PDGFR, VEGFR or c-KIT. In contrast, second generation inhibitors such as quizartinib (AC220) act in a far more FLT3-specific manner.Therefore, we aimed to investigate the effects of both clinically relevant FLT3-inhibitors on T-cell receptor signaling in comparison to the well characterized and potent BCR-ABL inhibitor dasatinib. Investigating primary T-cells derived from healthy donors, we applied a dose range of 10–50 nM dasatinib, 5–50nM midostaurin and 10–50 nM quizartinib. These dose ranges have been previously described to be achievable as trough levels during inhibitor therapy in early clinical trials. Upon incubation with dasatinib (10nM and 50nM), we found overall reduction in global tyrosine phosphorylation as detected by Western-blotting using the 4G10 antibody. In contrast, treatment with midostaurin left the activation of T-cell receptor signaling pathways unaffected. Comparable to DMSO control, overall phosphorylation was induced almost immediately after stimulation. Western-blotting of LCK and Plcg1 showed similar time dependent activation compared to total phosphorylation. Likewise, quizartinib did not reduce overall tyrosine phosphorylation level and left activation of downstream kinases (ZAP70, MAPK, LCK, Plcg1) largely unaffected.As activation of primary T-cells is a critical step in immune responses against viral and tumor antigens we aimed to investigate the influence of FLT3-kinase inhibitors quizartinib and midostaurin on activation of CD8+ T-cells. T-cells from healthy donors were stimulated using either PHA 0.5% or CD3/CD28 beads to ensure a more T-cell receptor specific stimulation. Using CD3/CD28 stimulation, CD69 expression was almost abrogated following dasatinib treatment. Applying clinically relevant doses of midostaurin or quizartinib to isolated T-cells did not influence CD69 expression. Expression levels upon PHA or CD3/CD28 stimulation were comparable to DMSO-control - even in the presence of 50nM midostaurin or quizartinib. Proliferation of T-cells upon CD3/CD28 stimulation was impaired by dasatinib treatment, while midostaurin and quizartinib left T-cell proliferation largely unaffected – as determined by CSFE staining. In order to investigate the T cell allo-reactivity, mixed lymphocyte culture was performed, where human pan-T-cells are co-cultured with allogeneic antigen presenting cells. T-cell proliferation – as measured by 3H-thymidine incorporation – was significantly impaired by dasatanib but neither midostaurin nor quizartinib treatment. Investigation of leukemia- and virus-antigen-specific T-cell responses are currently under way to gain deeper insight regarding this clinically relevant scenario.Overall, we found FLT3-kinase inhibitors midostaurin and quizartinib to leave T-cell activation, proliferation and function unaffected in-vitro. This information may be useful for the design of up-coming clinical trials testing the safety and efficacy of FLT3-kinase inhibitors in combination with allogeneic stem-cell transplantation. Disclosures:Lipka:Novartis Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heidel:Novartis Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Inhibition of Jak1/Jak2 Is More Effective Than Inhibition of Jak3 in Protecting Mice From Acute Graft-Versus-Host Disease (aGvHD) by Significantly Decreasing Alloreactive CD4+ T-Cells.

AbstractAbstract 2997▪▪This icon denotes a clinically relevant abstract Background:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for patients with hematological malignancies. However, its success is limited by a life-threatening complication: the graft-versus-host disease (GvHD). Although numerous studies have described immunosuppression protocols to mitigate acute GVHD (aGvHD), novel approaches are needed. Chemokines are well known inducers of leukocyte trafficking and activation. Stimulation of the chemokine receptor signaling pathway leads to initiation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway that contributes to the pathogenesis of GvHD. The key role of JAK signaling in normal and abnormal lymphocyte development and function, along with the cytotoxic effects of its inhibitor INCB18424 (Ruxolitinib) on leukemia cells, prompted us to hypothesize that this selective JAK1 and 2 inhibitor could be useful as anti-GvHD agent while maintaining antitumor activity. Since CP-690550, a more selective JAK3 inhibitor, was recently shown to protect against GvHD in mouse models, we also tested whether blocking the JAK1/JAK2 pathway could be more effective in preventing GvHD. Methods:To assess the therapeutic effect of pharmacologic modulation of JAK1 and 2 on GvHD, a major histocompatibility complex (MHC) mismatched HSCT mouse model was used. Recipient BALB/c mice were lethally irradiated and treated either with spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GvHD cohort, n=8), or with spleen and BM cells from B6 donors along with INCB18424 90mg/kg/day at days -1 to 13 (INCB18424 cohort, n=10) or with CP-690550 15mg/kg/day (CP-690550 cohort, n=8) at days -1 to 13. Syngeneic transplants (B6-B6, n=6) and BALB/c recipients treated with B6 BM cells only (control cohort, n=8) were also included as controls. Mice were characterized for GvHD by monitoring overall survival and weight loss. Recipient mice were sacrificed and tissues harvested on day 14 and 30 post transplant and GvHD confirmed by histology. Results:All mice in the GvHD cohort had clinical evidence of GvHD (weight loss, generalized erythema of the skin and poor fur quality) by day 14. The INCB18424 treated mice showed markedly reduced weight loss along the time of observation when compared to the GvHD cohort. Animals in the CP-690550 cohort tended to gain weight during the time of treatment (day-1 to 13), but thereafter they exhibited reduced body weight similar to that observed in the GvHD cohort. The histological examination of the stomach, liver, skin and intestine obtained at day 14 revealed no sign of GvHD in the control group as well as in the INCB18424 group. On the other hand, mild to moderate signs of GvHD were present in the tissues of CP-690550 treated mice and extensive inflammation and disruption of the normal architecture of the tissues was observed in the GvHD group. To determine whether INCB18424 treatment affected alloreactive CD4+ T cells, total spleen T cells were harvested at day 14 from the GvHD cohort and from recipients either of INCB18424 or CP-690550. Total spleen T cells were co-cultured with BM derived BALB/c (recipient-derived) or C57BL/6 (donor-derived) dendritic cells (DCs). After 24h, T cells alloreactivity was determined by IFN-γ production assessed by intracellular staining. As expected, T cells from GvHD mice showed significantly higher alloreactivity against BALB/c DCs compared to the reactivity observed against syngeneic B6 DCs (5.24% and 0.84% respectively, p<0.05). The alloreactivity observed when T cells from INCB18424 treated mice were stimulated with allogeneic BALB/c DCs was significantly lower than that in the GvHD group (0.64% and 5.24% respectively, p<0.05) and eas also lower than that of the CP-690550 group (1.43%). STAT phosphorylation analysis demonstrated that INCB18424 treatment was effective in vitro. Conclusions:The inhibition of Jak/STAT signaling using the sensitive and specific inhibitor of Jak1/Jak2, INCB18424, conferred effective protection from aGvHD in a HSCT mouse model. INCB18424 treatment was more effective than the targeting of JAK3 with CP-690550. In fact, CP-690550 administered during GvHD induction was not completely sufficient to restore the normal weight and to prevent the histological appearance of GvHD whereas INCB18424 was. INCB18424 protected mice against acute GvHD by significantly decreasing alloreactive CD4 T cells. Disclosures:No relevant conflicts of interest to declare.

KIR Haplotype B Donors but Not KIR-Ligand Mismatch Result in a Reduced Risk of Relapse After Haploidentical Hematopoietic Stem Cell Transplantation Using Reduced Intensity Conditioning and a CD3/CD19 Depleted Graft.

AbstractAbstract 3101Natural killer (NK) cell alloreactivity, after allogeneic hematopoietic cell transplantation (HCT) is influenced by the interaction of killer-cell immunoglobulin-like receptors (KIRs) on donor NK cells and human leukocyte antigen (HLA) class I ligands on recipient cells. Recently, a positive influence of KIR haplotype B versus haplotype A donors on the outcome of HLA-matched allogeneic HCT was demonstrated (Cooley et al., Blood 2010). Previously, Ruggeri et al. (Science 2002) reported the positive influence of KIR-ligand mismatch (MM) on outcome of haploidentical HCT (HHCT). Here we investigated the influence of the donor KIR haplotype and KIR-ligand MM on relapse of 57 patients with hematologic malignancies receiving HHCT after reduced intensity conditioning and graft CD3/CD19 depletion. 36 patients with AML, eight with ALL, four with multiple myeloma, four with NHL and one with MCL, CML, CMML, MDS, CLL, respectively (median age 45 years, range 19–61 years) were evaluated. Patients were “high risk” because of relapse (n=8), prior HCT (n=23), refractory disease (n=20) or cytogenetic risk (n=6). At HHCT, 29 patients were in complete remission (CR) and 28 in partial remission (PR). 15 KIR genes were determined by real-time PCR as described (Vilches et al., Tissue Antigens 2007, Alves et al., Tissue Antigens 2009), and donors were assigned the A/A or B/x haplotype. Patients and donors were HLA-typed by high-resolution molecular methods. Of the 57 donors, 17 had KIR haplotype A (29.8%) and 40 KIR haplotype B (70.2%). A KIR-ligand MM was found in 34 of 57 patients (59.6%). Cumulative incidence adjusted for competing risk showed no difference between KIR haplotype A or B patients regarding non-relapse mortality (NRM) (Gray’s test: p=0.200), but a significantly reduced incidence of relapse for patients with a haplotype B donor (p=0.001). In particular, patients in PR benefited more from a haplotype B graft (p=0.008) than patients in CR (p=0.297). This resulted in a trend in the Kaplan-Meier estimated event free (EFS) at 3 years of 26.8 % for KIR haplotype B and 11.7 % for KIR haplotype A (HR=1.33 [CI=0.66–2.70], log rank test: p=0.422). In detail, all patients in PR died within 1.2 years when haplotype A donor cells were transplanted whereas 25% of haplotype B recipients were still alive after 3 years (HR=1.27 [CI=0.49–3.30], p=0.631). In comparison, 16.6% of haplotype A and 28.1% of haplotype B recipients in CR survived for more than 3 years (HR=1.46 [CI=0.54–3.94], p=0681). Surprisingly, KIR-ligand MM cumulative incidence curves were not statistically different for relapse (p=0680) or NRM (p=0.579). In addition, KIR-ligand MM resulted in a trend for decreased EFS rate for MM patients (17.6%) in contrast to matched patients (33.7%; HR=1.47 [CI=0.89–2.75], p=0.230). These effects were even more pronounced when analyzing the patient cohort with AML. Of the 36 donors, 10 showed KIR haplotype A (27.8%), 26 KIR haplotype B (72.2%) and KIR-ligand MM was present in 25 patients (69.4%). EFS at 3 years was prolonged for KIR haplotype B graft recipients (EFS: HR=2.29 [CI=0.88–5.96], p=0.087). In addition, cumulative incidence adjusted for competing risk analysis revealed a reduced incidence of relapse for patients with a haplotype B donor (all AML patients: p=0.079, AML in PR: p=0.049), but not for NRM (all AML patients: p=0.806, AML in PR: p=0.674). Again, KIR-ligand MM cumulative incidence curves were not significantly different for both relapse (p=0.126) and NRM (p=0.535). In line, KIR-ligand MM led to decreased EFS rate for MM patients (16.0%) in contrast to matched patients (53.0%; HR=2.27 [CI=1.08–5.06], p=0.045). Taken together, in the setting of RIC and CD3/CD19 depleted HHCT we could not confirm the positive data with KIR-ligand MM but observed a significant lower risk of relapse with a KIR haplotype B donor. We therefore conclude from our results that a donor KIR B haplotype should be favored as donor for HHCT using RIC and CD3/CD19 depletion in patients with hematological malignancies, particularly if no complete remission has been achieved prior to HHCT. Disclosures:Off Label Use: off lable use of drugs for conditioning.

TK-Cells Safety and Efficacy in Allogeneic Hematopoietic Stem Cell Transplantation: Long-Term Clinical and Molecular Assessment in 128 Treated Patients.

Abstract 3061 Background and methods.Suicide gene therapy (SGT) applied to allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been one of the first clinical applications of gene therapy. In allo-HSCT the antileukemic potential mediated by alloreactive lymphocytes towards patient-specific antigens, such as minor and major histocompatibility antigens, is counterbalanced by the Graft-versus-Host-Disease (GvHD). The risk of GvHD increases with the level of HLA disparity between host and donor, and leads to impaired quality of life and reduced survival, particularly in patients (pts) transplanted from HLA-mismatched donors. In this contest SGT has been applied to modulate alloreactivity, by inserting the HSV-tk gene in donor lymphocytes. This suicide gene/prodrug system requires cell cycle for optimal killing. In the context of allo-HSCT, this characteristic ensures a further level of specificity in GvHD control, by allowing to selectively kill highly proliferating alloreactive cells during GvHD, while sparing resting T cells. We assessed long-term safety in TK cells treated pts. Results.Overall 128 pts have been treated worldwide in 10 phase I-II clinical trials with donor lymphocytes expressing the HSV-tk suicide gene, with the purpose of enforcing the graft-versus-tumor (GvT) effect and/or promoting a functional post-transplant immune reconstitution (IR) while allowing control of GvHD. This approach proved highly feasible, safe and effective in promoting a dynamic and patient-specific modulation of alloreactivity. TK cells engrafted in the majority of pts and a clinical benefit, measured as improvement of hematopoietic chimerism, malignant regression and/or IR, was reported for 65 pts (51%). GvHD grade II-IV was observed in 28 pts (22%) and was always rapidly and completely controlled by the activation of suicide machinery.A selected population of 57 pts, treated at San Raffaele Institution, was studied in more detail for long-term analysis: 23 pts received cells to treat relapse occurring after an HLA-identical allo-HSCT (Ciceri F et al, Blood. 2007: 109; 4698–470) and 34 pts to improve IR after haploidentical HSCT (Bonini C et al, Blood. 2002; 100: 115a; Ciceri F, Bonini C et al, Lancet Oncology. 2009; 10: 489–500). No adverse event correlated to the gene transfer procedure was ever reported.Genetically modified cells engrafted in 90% of treated pts and could be detected in vivo, at low frequencies for up to 14 years (y).In the HLA-identical setting 11 pts obtained clinical response of the malignant disease and 3/11 are alive with a median follow-up of 15y. Two pts are in complete remission (CR), while one pt affected by chronic myelomonocytic leukemia relapsed 15y post transplant. This pt was subsequently treated with a second transplant from an unrelated match donor and is now in CR 1 year after transplant.In the haploidentical setting, 25 pts/34 reached the target of IR and 9 are alive and in CR with a median follow-up of 7y. Four out of 9 experienced GVHD in the early phase post IR, none presented signs, symptoms, complications related to GvHD and none needed pharmacological treatment in long-term.According to international guideline for long-term follow-up (Majhail NS et al, BBMT. 2012; 18: 348–371) all pts underwent regular screening and clinical evaluation. No major infections occurred in the late phase, no ocular, oral, respiratory, hepatic, renal, genitourinary, muscle-skeletal or neurological distress. Noteworthy 3 pts developed second cancer (2 skin cancer non-melanoma, 1 endometrial cancer), 2 pt hypothyroidism, 1 pt coronary artery disease and 3 pts metabolic syndrome. No evidence of psychosocial symptoms was reported.Immunity against the transgene was reported in pts who received TK cells late after an HLA-identical allo-SCT. The quality of IR at the time of TK cells administration was the most predictive variable. On the contrary, no immunity against HSV-tk was ever detected in pts who received TK cells after transplantation of haploidentical CD34-selected cells, indicating that the infusion of TK cells to highly immunosuppressed pts is not limited by the development of transgene-specific immune responses. Conclusions.Long-term assessment confirmed the overall high benefit/risk ratio of the TK-cell approach in allo-HSCT.A phase III multicentric, randomized clinical trial sponsored by MolMed, is currently undergoing in the context of haploidentical HSCT. Disclosures:Bordignon:MolMed SpA: Employment. Bonini:MolMed S.p.A.: Consultancy.

HLA-DR Expressing Endothelial Cell Expansion of Allogeneic CD4+ T Lymphocytes Towards Either Pro-Inflammatory Th17 or Regulatory Populations

Introduction: The major obstacle to organ transplantation remained chronic rejection due to the generation and the persistence of an alloimmune response principally mediated by Major Histocompatibility Complex molecules (MHC) present on donor cells. Graft microvascular endothelial cells (ECs) express MHC class I and II molecules and have therefore a dual role as both a stimulator of, and a target for allo-immune responses. Recently, we have shown that ECs generate either regulatory CD4+ T cells (Tregs) or pro-inflammatory CD4+IL-17+ cells (Th17) but the factors which orient this CD4+ T cell allo-immune response remain to be determinate. Methods: We have established a model of either inducible or constitutive HLA-DR expression in microvascular ECs using lentiviral vector infection. This model was used to study (1) the mechanisms of EC induced activation of allogeneic CD4+ T cells and (2) to determine the effect of EC pre-activation with an HLA-DR antibody on the orientation of the allogeneic CD4+ T cell response. Results: Under inflammatory conditions, HLA-DR expression by human microvascular ECs allows them to orient the CD4+ T cell response towards either a Th17 pro-inflammatory response or a regulatory T cell response. Expansion of Th17 depended upon IL-6 secretion by ECs which led to STAT-3 activation in allogeneic CD4+ T cells associated with their proliferation, while Treg expansion was dependent upon their interaction with ECs mediated by the adhesion molecule ICAM-1 (CD54). HLA-DR antibody induced signaling in ECs leading to an increase in their secretion of IL-6 and their ability to expand alloreactive Th17 cells while lessening the regulatory T cell response. Conclusion: These data reveal that firstly human microvascular EC regulates polarization of the memory CD4+ T cell response towards either cytokine-dependent inflammation or contact-dependent regulation and secondly that HLA-DR antibody pre-activation of EC increases their ability to induce a pro-inflammatory Th17 response. Together these results provide evidence for a link between the roles of EC as a target of HLA-DR antibody binding and as an allo-stimulator of the pro-inflammatory Th17 response implicated in chronic rejection. Moreover, these data may contribute to our understanding of HLA class II allo-antibodies mediated complement independent mechanisms of EC damage.

Mechanisms Underlying T17-Mediated Allograft Rejection

Introduction: IL-17-producing CD4+ and CD8+ T cells (T17) may play a major role in the pathogenesis of allograft rejection and resistance to tolerance induction. This speculation is based on the observation that T17 cells in T-bet-deficient mice (thus lacking Th1) mediate an aggressive pro-inflammatory alloimmune response resulting in accelerated allograft rejection. However, the basic mechanisms underlying T17-mediated alloimmune responses in transplantation are poorly understood, particularly downstream of the key T17 effector cytokine IL-17 and with respect to regulation by the hallmark T17 transcription factor ROR-gamma-t (RORγt). Methods: To precisely explore the role of RORγt in T17 alloimmunity in the absence of Th1, we created RORγt reporter and knockout mice on a T-bet-deficient (T-bet−/−) background by crossbreeding B6.T-bet−/− with B6. Rorγtgfp/wt reporter and B6. Rorγtgfp/gfp knockout (KO) mice, respectively. CD4+ and CD8+ T effector cells from these mice were studied in T17 conversion assays in vitro and in a heterotopic heart transplant (HTx) model of acute allograft rejection in vivo. Results: First, we explored the fate-mapping capacity of our reporter model by polarizing CD4+ and CD8+ T cells from Rorγtgfp/wtT-bet−/− mice towards T17 and measuring GFP and IL-17 expression in vitro. We discovered that the T17 cells from these mice expressed high levels of GFP and were as capable of producing IL-17 as controls, thus demonstrating the efficacy of our reporter model. Next, we tested whether RORγt significantly contributes to the differentiation of alloreactive T17 cells in the absence of Th1 by studying T cells from Rorγt-T-bet double-KO mice under T17-polarizing conditions in vitro. Importantly, IL-17 expression was substantially lower but still present in the double-KO mice when compared to controls, indicating that RORγt plays an important, but not exclusive, role in driving IL-17-producing T cells in the absence of T-bet. Next, we directly addressed the role of RORγt in T17-mediated alloimmunity in the absence of Th1 in vivo by adoptively transferring FACS-sorted CD3+ T cells from Rorγtgfp/wtT-bet−/− reporter or double-KO mice into B6. Rag−/− recipients of fully mismatched Balb/c HTx. We found that recipients with T cells from Rorγtgfp/wt or Rorγtgfp/gfp controls failed to acutely reject their allografts with the majority surviving long term (50% and 63%, respectively). Surprisingly, however, all recipients transferred with Rorγtgfp/wtT-bet−/− reporter or double-KO T cells promptly rejected their allografts alike recipients transferred with T cells from T-bet−/− controls (MST 21d, 22d, and 18d, respectively), indicating that RORγt seems to be sufficient but not necessary for driving acute allograft rejection in the absence of Th1. Conclusion: In conclusion, RORγt may play a critical but redundant role in T17-mediated alloimmunity. In transplantation, our studies point to an important and as yet understudied role of distinct ROR-transcription factors.