Donor APCs Promote Gvhd in MHC-Mismatched Transplants by Indirectly Presenting Host Minor Histocompatibility Antigens.

Abstract Abstract 689 In MHC-mismatched allogeneic bone marrow/stem cell transplantation (alloBMT), T cells that at least in-part recognize the allogeneic MHC are likely the dominant mediators of acute GVHD. This would explain why far fewer T cells induce acute GVHD in MHC-mismatched as opposed to MHC-matched alloBMT. However, despite this difference, in human recipients of haploidentical allografts, the organ distribution and histologic appearance of GVHD, especially chronic GVHD, are similar. Moreover, late-onset GVHD in MHC-mismatched alloBMT occurs when recipient antigen presenting cells (APCs) have been replaced by donor APCs. We therefore investigated how indirect presentation of host minor histocompatibility antigens (miHAs) by donor APCs contributes to GVHD. To test this, we employed the MHC/miHA mismatched B6 (H-2b) (right arrow) BALB/c (H-2d) strain pairing. We compared GVHD in irradiated BALB/c mice that were reconstituted with B6 CD4 cells and either B6 BM or B6 MHCII-/- BM. We reasoned that if BALB/c miHAs presented by donor B6 APCs were important, GVHD would be reduced in recipients of MHCII-/- BM. As measured by weight loss and survival, this was indeed the case. However, it was possible that receiving MHCII-/- donor BM reduced GVHD for reasons unrelated to the presentation of BALB/c miHAs. To address this we compared GVHD in irradiated B6.C (H-2d) recipients of B6 CD4 cells and either B6 or B6 MHCII-/- BM. This strain pairing shares the same MHC mismatch as does the B6(right arrow)BALB/c model, but lacks miHAs except those encoded by the congenic H-2d region. In contrast to the prior experiments, B6.C recipients of MHCII-/- or wild type BM developed similar GVHD. To further define indirect-presentation of miHAs in MHC/miHA-mismatched alloBMT, we used B6 Marilyn CD4+ T cell receptor transgenic T cells that recognize an IAb-restricted peptide derived from the male DBY protein. Male or female BALB/c mice were irradiated and reconstituted with female B6 BM and female polyclonal B6 CD4 cells along with graded numbers of Marilyn T cells. At day 7 and 14 post transplant Marilyn cells expanded at least 1000-fold greater in male than in female recipients. Marilyn T cell expansion was similar when male antigen was restricted to hematopoietic or nonhematopoietic cells. There was little expansion of Marilyn cells in male BALB/c mice if donor BM was MHCII-/-, confirming that expansion of Marilyn T cells depended on donor APCs presenting DBY. Indirectly primed Marilyn cells were functional as measured by IFN-γ production after restimulation by the DBY peptide in vitro. As we saw reduced GVHD in the B6 (right arrow) BALB/c model in recipients of MHCII-/- BM, we reasoned we should be able to detect polyclonal CD4 cells responding to host miHA presented by donor APCs. That was the case as we found an increase in DBY-reactive polyclonal CD4 cells by ELISPOT in male recipients as compared to female recipients at day 7 (1.5 fold for IFN-γ and TNF-αa). This difference was lost when donor BM was MHCII-/-. In summary, indirect presentation of host miHAs by donor APCs in MHC-mismatched alloBMT is surprisingly efficient and alloreactive CD4 cells that target host miHAs presented by donor APCs contribute to GVHD in MHC/miHA-disparate alloBMT. The generation of T cells that target host miHAs presented by donor APCs allows GVHD to persist even when host APCs that prime T cells that recognize the allogeneic MHC have been eradicated. This provides a mechanistic explanation for why late onset GVHD, including chronic GVHD, in MHC-mismatched alloBMT resembles that in MHC-matched alloBMT as both may target host miHAs indirectly presented by donor-derived APCs. These data also provide further rationale for targeting donor-derived APCs as a method for preventing and treating GVHD. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Anti-CD25 Antibody Daclizumab Delays Treg Reconstitution, Promotes CD4 Memory, and Does Not Prevent Acute or Chronic Gvhd After Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v120.21.4195.4195 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4195-4195 ◽

Cited By ~ 1

Author(s):

Frederick L Locke ◽

Joseph Pidala ◽

Barry Storer ◽

Paul J. Martin ◽

Michael A Pulsipher ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Binding Sites ◽

Acute Gvhd ◽

Disease Risk ◽

Chronic Gvhd ◽

Central Memory ◽

Cd4 Cells ◽

Grade Ii

Abstract Abstract 4195 Background: Daclizumab, a humanized monoclonal antibody, binds CD25 and blocks formation of the high affinity IL-2 receptor on T cells. A multi-center, randomized, double-blind clinical trial using daclizumab to perturb IL-2 signaling as a way of reducing T cell mediated graft versus host disease (GVHD) after allogeneic unrelated bone marrow transplantation (BMT) was completed 18 years ago. We report long term outcomes and biological correlates. Methods and Patients: Between April, 1993 and December, 1994, 209 adult or pediatric patients receiving marrow from an unrelated donor for hematologic malignancies or severe aplastic anemia at 12 sites in Europe and North America were randomized to one of three arms: 5 weekly doses of placebo (arm A, n=64); 0.3 mg/kg daclizumab (arm B, n=69); or 1.2 mg/kg daclizumab (arm C, n=76) to a maximum of 100 mg beginning on the day before BMT. Conditioning included total body irradiation (TBI) (1200–1440 cGy). All patients received methotrexate (15 mg/m2 day 1; 10 mg/m2 days 3, 6, 11) plus cyclosporine for at least 180 days. The study was designed to provide 80% power to detect a reduction in the incidence of acute GVHD (grade II-IV, requiring treatment with corticosteroids to day +100) from 80% to 55%. Randomization was stratified by donor HLA (matched vs. mismatched) and age (<20 vs. >=20). Arms were well balanced for HLA matching, age, disease risk, TBI dose, and diagnosis. Diagnoses were 31 ALL, 30 AML, 118 CML, 16 MDS, and 14 other. Median age was 31 years (range 1–54); 61% were male; 45% had high risk disease (14% AML with active disease or beyond 2nd remission, 20% CML beyond first chronic phase); 29% had a HLA-A, -B or -DR mismatched donor. T cells from a subset of patients (n=107) were collected at days 11–35, days 36–80, and days 81–100 and analyzed by flow cytometry for expression of total CD25, daclizumab binding, and free CD25 binding sites. Samples from arm A and arm C, including those from 1 year long term follow up (LTFU) were also evaluated for T cell phenotype. Results: The incidence of grade II-IV acute GVHD for arms A, B, and C were 34%, 42%, and 45%, and results did not differ between arm A vs. arm B (p=0.60) or arm C (p=0.38). Since other clinical outcomes did not differ significantly between arm B and arm C, the two daclizumab dose arms were combined for post-hoc exploratory analysis. There was a suggestion that the daclizumab arms showed decreased risk of relapse (HR 0.57, 95% CI: 0.32–1.00, p=0.06) and increased risk of chronic GVHD (HR 1.49, 95% CI: 0.96–2.31, p=0.07) compared to the placebo arm. Daclizumab did not increase OS (HR 0.89, 95% CI: 0.61–1.29, p=0.54) compared to placebo, and there was no interaction between disease risk and OS. Daclizumab administration reduced the total numbers of T cells expressing CD25 at days 11–35 (arm A=28% vs. arm B=16%, p<0.0001; or arm C=18%, p=0.0002); at days 36–80 (arm A=26% vs. arm B=19%, p=0.03; or arm C=19%, p=0.03); figures were equivalent at days 81–101. Daclizumab was bound to CD25 in vivo, at days 11–35 (arm A=3% vs. arm B=74%, p<0.0001; or arm C=83%, p<0.0001) and persisted at days 81–101 (arm A=4% vs. arm B=17%, p=0.05; or arm C=39%, p<0.0001). Daclizumab administration decreased the numbers of cells with free CD25 binding sites at days 11–35 (arm A=45% vs. arm B=7%, p<0.0001; or arm C=3%, p<0.0001); at days 36–80 (arm A=49% vs. arm B=25%, p<0.0001; or arm C=11%, p<0.0001); and was equivalent at days 81–101. Available samples from arm A (n=18) and arm C (n=40) were tested for Treg (CD4+ CD127- CD25+ FOXP3+) and central memory (CD4+ CD45RA- CCR7+) phenotype. Compared to placebo (arm A), daclizumab administration (arm C) decreased the proportion of CD4 cells that were Tregs at days 11–35 (12% arm A vs. 7% arm C; p=0.008), but not at days 81–101 and LTFU. Daclizumab increased the proportion of CD4 cells exhibiting central memory phenotype at LTFU (10% arm A vs. 21% arm C, p=0.02). Conclusion: This randomized phase II/III study shows that daclizumab does not prevent GVHD. There was suggestion for increased chronic GVHD and decreased relapse. Daclizumab delayed Treg reconstitution while increasing CD4 central memory at LTFU. Based on these results, further trials should test whether anti-CD25 therapy can promote anti-tumor immunity by impairing Treg reconstitution after transplant, in the appropriate clinical setting. Disclosures: Off Label Use: Daclizumab administration following hematopoietic cell transplantation. Walker:Baxter Corporation: Research Funding. Light:Bristol Myers Squibb: Employment.

Download Full-text

Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application

Haematologica ◽

10.3324/haematol.13067 ◽

2008 ◽

Vol 93 (10) ◽

pp. 1535-1543 ◽

Cited By ~ 22

Author(s):

M. Griffioen ◽

H.M. E. van Egmond ◽

H. Barnby-Porritt ◽

M. A.W.G. van der Hoorn ◽

R. S. Hagedoorn ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Genetic Engineering ◽

Clinical Application ◽

T Cell Receptors ◽

Histocompatibility Antigens ◽

Cell Receptors ◽

Minor Histocompatibility Antigens

Download Full-text

Genome Wide Single Nucleotide Polymorphism Typing for Identification of Putative Minor Histocompatibility Antigens in Graft Versus Host Disease.

Blood ◽

10.1182/blood.v108.11.447.447 ◽

2006 ◽

Vol 108 (11) ◽

pp. 447-447 ◽

Cited By ~ 1

Author(s):

Ann Mullally ◽

Cheng Li ◽

Haesook Kim ◽

Mehrdad Mohseni ◽

Edwin P. Alyea ◽

...

Keyword(s):

Stem Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Histocompatibility Antigens ◽

Single Nucleotide ◽

Hematopoietic Stem ◽

Minor Histocompatibility Antigens ◽

Sibling Pairs ◽

Graft Versus Host ◽

Genome Wide

Abstract Previous studies have demonstrated that disparity across minor histocompatibility antigens (mHA) can cause graft versus host disease (GVHD) in patients who receive hematopoietic stem cell grafts from HLA-identical donors. mHA are peptide epitopes derived from normal cellular proteins presented by self MHC. Most autosomal mHA are generated as a result of non-synonymous coding single nucleotide polymorphisms (cSNPs), which lead to differences in the amino acid sequences of homologous proteins between donor and recipient cells. Although it is estimated that several hundred mHA exist in humans, only 16 have been definitively characterized to date. Using the Affymetrix 20K cSNP array we performed SNP typing on 97, HLA-A2+ hematopoietic stem cell transplant (HSCT) recipients and their sibling donors. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) obtained from patients and their donors. All patients were in remission at the time of sampling, all had undergone HSCT at the Dana-Farber Cancer Institute between 1998 and 2005 and all samples were drawn prior to transplantation. The transplants included myeloablative and non-myeloablative conditioning regimens, T cell depleted and non-T cell depleted grafts and sex matched and sex mis-matched donors. Using dChip software, we evaluated each of the 20,000 non-synonymous cSNPs on the array for mismatch between sibling pairs and for an association between mismatch in the GVHD direction and the development of acute or chronic GVHD. Mismatch in the GVHD direction was defined as a homozygous donor (AA or BB) and a heterozygote recipient (AB) or a homozygous donor (AA or BB) and a homozygously mismatched recipient (BB or AA). We ranked the cSNPs on the array in order of the strength of the association between mismatch in the GVHD direction and the development of either acute or chronic GVHD. There was no overlap between the 40 mismatched cSNPs most strongly associated with acute GVHD and the 40 most tightly associated with chronic GVHD. Mismatch at the SNP rs12407003 in the OMA1 gene was most highly associated with acute GVHD with mismatch in the GVHD direction occurring in 13 of 41 pairs with acute GVHD and 2 of 56 without (p=0.0003 by Fisher’s Exact Test). OMA1 encodes a mitochondrial membrane-bound metallopeptidase. 65 sibling pairs were assessable for chronic GVHD. Mismatch at the SNP rs2740349 in the GEMIN4 gene was most strongly associated with chronic GVHD with mismatch in the GVHD direction occurring in 10 of 26 pairs with chronic GVHD and 1 of 39 without (p=0.0002). GEMIN4 is part of a cytoplasmic multiprotein complex. This study demonstrates a novel, genome-wide method of identifying putative mHA using a cSNP array. It reveals that mismatch of non-synonymous cSNPs in the GVHD direction occurs at an appreciable frequency in sibling pairs consistent with the hypothesis that the number of mHA in humans is large. Interestingly, the pattern of mismatch differs between acute and chronic GVHD. The study also identifies individual non-synonymous cSNPs for which mismatch in the GVHD direction is highly associated with the development of GVHD. Further evaluation of these cSNPs in larger independent cohorts will be undertaken to validate this association and targeted immunologic analysis of peptides derived from these cSNPs will examine their role as putative mHA.

Download Full-text

CD28-Directed T Cell Costimulation Blockade with Abatacept to Prevent GvHD During High-Risk Unrelated HSCT: A First-in-Disease Feasibility Trial,

Blood ◽

10.1182/blood.v118.21.4078.4078 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4078-4078

Author(s):

Leslie S. Kean ◽

Amelia Langston ◽

Muna Qayad ◽

H. Jean Khoury ◽

Divya Tiwari ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Hematologic Malignancies ◽

Feasibility Trial ◽

Effector Memory ◽

Unrelated Donor ◽

Gvhd Prophylaxis ◽

The Mean

Abstract Abstract 4078 Background: Acute GvHD remains the major cause of complications and death following unrelated-donor HSCT. In a non-human primate model, we have previously shown that in vivo costimulatory blockade of donor T-cells could provide effective protection against GVHD. To begin to explore its clinical utility, we are conducting a trial (Clinical Trials.Org # NCT01012492) to determine the feasibility of combining abatacept (CTLA4-Ig) with cyclosporine and methotrexate as acute GVHD prophylaxis for patients undergoing unrelated marrow and peripheral blood stem cell transplants for hematologic malignancies. Methods: Patients older than 12 with advanced hematologic malignancies, conditioned with either TBI/Cytoxan, Busulfan/Cytoxan or Fludarabine/Melphalan are eligible. Abatacept is administered IV on days −1, +5, +14, and +28 at 10 mg/kg in addition to standard GvHD prophylaxis consisting of cyclosporine (day −2 to day 100), and methotrexate (15 mg/m2 on day +1 and 10 mg/m2 on days +3, 6 and 11). Patients are then followed for clinical outcomes and immunologic reconstitution through day +365. Results: 9 patients (planned enrollment = 11 patients) have thus far been enrolled on the study of which 5 are evaluable for engraftment, toxicity and acute GvHD. The other four patients consist of 2 who are currently receiving abatacept, 1 who was discovered to have an ongoing viral infection at the start of the first abatacept infusion so was removed from the treatment regimen, and 1 who is awaiting transplant. The median age for the 5 evaluable patients is 47 years (17–74 years). 3 patients had AML and 2 had ALL. Patients were conditioned with Bu/Cy (n=1), TBI/Cy (n=2) and Flu/Melphalan (n=2). 4 donor-recipient pairs were allele matched at 9 of 10 loci (A, B, C, DRB1 and DQB1), while 1 was fully matched. Four of the 5 patients are currently alive and in remission and 1 relapsed at day +98 (and died on day +121 with refractory AML). The four other patients are surviving without relapse with a follow-up of 155–313 days. All 5 patients received the 4 scheduled abatacept doses. No infusional side effects were noted. All patients achieved neutrophil engraftment (median day +20 (11–47). 4 of 5 patients have achieved platelet engraftment (median day +27 (14–35). Donor engraftment (100% CD33 and 99–100% CD3 at Day +30) occurred in all cases. All patients have demonstrated rapid lymphocyte engraftment, with the mean ALC reconstituting to >500 cells/μL by day +21 post-transplant. At day +100, the mean CD3+ count was 673 +/− 251 cells/μL. Both CD8+ and CD4+ T cells reconstituted by day 100, with the mean CD8+ count = 384 +/− 148 cells/μL and the mean CD4+ count = 229 +/− 119 cells/μL. T cell reconstitution was accompanied by a shift away from naïve (Tn, CCR7+/CD45RA+) toward a CCR7-/CD45RA- effector memory (Tem)-predominant phenotype. Thus, the average proportion of CD4+ Tem cells in the recipient increased from 22 +/− 6% pre-transplant to 46 +/− 7% at day +100 with a concomitant loss of CD4+ Tn cells. Likewise, the proportion of CD8+ Tem also significantly increased, from an average of 15 +/− 4% pre-transplant to 32 +/− 7% at day +100, also with a reciprocal decrease in CD8+ Tn cells. One patient developed steroid responsive grade 3 acute GVHD involving the skin and the liver, followed by steroid responsive liver chronic GvHD. This patient is currently weaning corticosteroids. Another patient developed steroid responsive late-onset (day +217) acute GVHD (liver and GI) during cyclosporine weaning, which was also steroid responsive, and is also currently weaning corticosteroids. No other systemic acute or chronic GvHD has occurred. No unexpected complications or life-threatening infections were observed. 3 patients have experienced 5 episodes of CMV reactivation, all responsive to antiviral therapy. One patient developed polyclonal EBV-related PTLD (plasmacytic hyperplasia) in the absence of EBV viremia, which regressed without intervention. No other EBV-related disease has occurred. Conclusions: These preliminary data suggest that abatacept can be safely added to cyclosporine and methotrexate for GVHD prophylaxis in recipients of hematopoietic grafts from unrelated donors, with encouraging rates of acute GVHD. As such, they support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept: It is an immunosuppressive agent that targets the CD28/B7 T cell costimulation pathway. It is approved for use in Rheumatoid arthritis.

Download Full-text

Cytokine production and cytolytic mechanism of CD4+cytotoxic T lymphocytes in ex vivo expanded therapeutic Epstein-Barr virus–specific T-cell cultures

Blood ◽

10.1182/blood.v99.9.3302 ◽

2002 ◽

Vol 99 (9) ◽

pp. 3302-3309 ◽

Cited By ~ 49

Author(s):

Qi Sun ◽

Robert L. Burton ◽

Kenneth G. Lucas

Keyword(s):

Cell Culture ◽

T Cells ◽

T Cell ◽

Cytotoxic T Lymphocytes ◽

Epstein Barr Virus ◽

Ex Vivo ◽

Cd4 Cells ◽

Barr Virus ◽

Ifn Γ ◽

Epstein Barr

Abstract Ex vivo expanded Epstein-Barr virus (EBV)–specific T cells have been successfully applied clinically for adoptive immunotherapy. However, the role of CD4+ T cells in the therapeutic T-cell culture has not been established for the reconstitution of EBV-specific immunity. We isolated and characterized CD4+ T-cell lines from the ex vivo T-cell cultures. Monoclonal line PD-F4 and oligoclonal lines ND-R4 and TD-B4 were CD3+CD4+CD8−. Cytolytic tests with targets of mismatched major histocompatibility complex (MHC) and anti-MHC antibodies confirmed that the cytotoxicity of these CD4+ cells was restricted by MHC class II. Single cells of ND-R4 expressed interferon-γ (IFN-γ, or interleukin 4 (IL-4), but rarely coexpressed these 2 cytokines. In contrast, PD-F4 coexpressed IFN-γ, IL-2, and IL-4. Kinetic studies with PD-F4 showed that expression of the 3 cytokines plateaued 5 hours upon stimulation and was then drastically reduced, with a pattern consistent with independent modulation and differential off-cycle signal requirements. The cytotoxicity of these CD4+ cells was largely resistant to brefeldin A, an inhibitor for cytolytic pathways by Fas-ligand family molecules. Although sensitive to concanamycin A and ethyleneglycotetraacetic acid, which inhibit cytotoxicity by granule exocytosis, the CD4+ cytotoxic T lymphocytes (CTLs) did not express perforin, suggesting a cytotoxic mechanism independent of perforin although involving exocytosis. Flow cytometric analysis showed that the CD4+ CTLs expressed granulysin, a recently identified cytolytic molecule associated with exocytotic cytolytic granules. These data suggested that CD4+ T cells in the therapeutic B-lymphoblastoid cell lines–primed T-cell culture are diverse in producing TH1 and TH2 cytokines, and may exert specific cytotoxicity via exocytosis of granulysin.

Download Full-text

Up-regulated expression in nonhematopoietic tissues of the BCL2A1-derived minor histocompatibility antigens in response to inflammatory cytokines: relevance for allogeneic immunotherapy of leukemia

Blood ◽

10.1182/blood-2004-09-3749 ◽

2005 ◽

Vol 106 (12) ◽

pp. 3955-3957 ◽

Cited By ~ 10

Author(s):

Freke M. Kloosterboer ◽

Simone A. P. van Luxemburg-Heijs ◽

Ronald A. van Soest ◽

H. M. Esther van Egmond ◽

Roel Willemze ◽

...

Keyword(s):

T Cells ◽

Inflammatory Cytokines ◽

Inflammatory Responses ◽

Interferon Γ ◽

Hematopoietic Tissue ◽

Histocompatibility Antigens ◽

Minor Histocompatibility Antigens ◽

Tnf Α ◽

Relative Specificity ◽

Ifn Γ

T cells directed against hematopoietic-restricted minor histocompatibility antigens (mHags) may mediate graft-versus-leukemia (GVL) reactivity without graft-versus-host disease (GVHD). Recently, the HLA-A24–restricted mHag ACC-1 and the HLA-B44–restricted mHag ACC-2 encoded by separate polymorphisms within the BCL2A1 gene were characterized. Hematopoietic-restricted expression was suggested for these mHags. We demonstrate BCL2-related protein A1 (BCL2A1) mRNA expression in mesenchymal stromal cells (MSCs) that was up-regulated by the inflammatory cytokines tumor necrosis factor α (TNF-α) and/or interferon γ (IFN-γ). Analysis of cytotoxicity and IFN-γ production illustrated that ACC-2–specific T cells did not recognize untreated MSCs or IFN-γ–treated MSCs but showed specific recognition and killing of MSCs treated with TNF-α plus IFN-γ. We hypothesize that under steady-state circumstances BCL2A1-specific T cells may exhibit relative specificity for hematopoietic tissue, but reactivity against nonhematopoietic cells may occur when inflammatory infiltrates are present. Thus, the role of BCL2A1-specific T cells in differential induction of GVL reactivity and GVHD may depend on the presence of inflammatory responses that may occur during GVHD.

Download Full-text

Itacitinib, a JAK1 Selective Inhibitor Preserves Graft-Versus-Leukemia (GVL), Enhances Survival and Is Highly Efficacious in a MHC-Mismatched Mouse Model of Acute GvHD

Blood ◽

10.1182/blood-2018-99-112144 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4522-4522 ◽

Cited By ~ 1

Author(s):

Ashish Juvekar ◽

Bruce Ruggeri ◽

Sindy Condon ◽

Andrew Borkowski ◽

Reid Huber ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Inflammatory Cytokine ◽

Acute Gvhd ◽

Colon Tissue ◽

Graft Versus Leukemia ◽

Tnf Α ◽

Dosing Regimens ◽

Ifn Γ

Abstract Introduction: Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK1/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (GRAVITAS-301: NCT03139604; GRAVITAS-119: NCT03320642). Methods: Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the selective JAK1 inhibitor, itacitinib (60 mg/kg or 120 mg/kg twice daily). Engraftment was analyzed for the proportion of donor and host leukocytes (CD45+, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analysis. Colon samples were sectioned and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3 and CD3+phosphoSTAT3 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of itacitinib on preservation of Graft-versus-Leukemia (GVL) were evaluated by injecting BALB/c mice with A20 lymphoma cells that are of H-2Kd phenotype along with combination of splenocytes and T cell depleted bone marrow from C57BL/6 (H-2Kb) mice. Results: Itacitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss and improving GvHD scores. Itacitinib did not significantly impact donor engraftment as determined by CD45+/H-2Kb quantification by flow cytometry. Similar efficacy was observed with 60 mg/kg versus 120 mg/kg twice daily dosing regimens. Oral itacitinib administration achieved JAK1 IC50 coverage for 4 h and 12 h at 60 mg/kg twice daily and 120 mg/kg twice daily, respectively. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Itacitinib (120 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen with itacitinib treatment, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with significant JAK1/STAT3 inhibition as measured by reductions in normalized pSTAT3 in T cells and colonic epithelial cells. Itacitinib treatment did not negatively impact GVL responses, as evidence by T cell mediated reduction of tumor burden. Furthermore, itacitinib treatment enhanced the survival of the recipient BALB/c mice in comparison to the vehicle treated animals. Conclusions: Itacitinib, a selective JAK1 inhibitor ameliorated GvHD severity when administered prophylactically or therapeutically and had no detrimental effects on engraftment and preservation of GVL. Furthermore, itacitinib inhibited JAK1/STAT3 activation in diseased colon tissue and infiltrating T-cells, and reduced disease burden and improved survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. Disclosures Juvekar: Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

Download Full-text

Development of Chronic Lymphocytic Leukemia (CLL) Reactive Cytotoxic T Lymphocytes after Non-Myeloablative Hematopoietic Stem Cell Transplant Correlates with Anti-Leukemia Response.

Blood ◽

10.1182/blood.v108.11.413.413 ◽

2006 ◽

Vol 108 (11) ◽

pp. 413-413

Author(s):

Tetsuya Nishida ◽

Ana Kostic ◽

David G. Maloney ◽

Rainer F. Storb ◽

Stanley R. Riddell

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Histocompatibility Antigens ◽

Antitumor Response ◽

Hematopoietic Stem ◽

Minor Histocompatibility Antigens ◽

Cell Responses ◽

T Cell Lines

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) following non-myeloablative (NM) conditioning is a promising approach for treating patients with advanced fludarabine refractory CLL. In this setting, a graft versus leukemia (GVL) effect mediated by donor T cells is critical for tumor eradication. We have evaluated the development of alloreactive and CLL-reactive cytotoxic T lymphocyte (CTL) responses in patients after NM-HSCT to determine if the generation of detectable T cell responses was associated with an antitumor response. Seven patients with fludarabine refractory CLL were conditioned with fludarabine (30mg/m2 x 3 doses) and total body irradiation (2 Gy) prior to receiving G-CSF mobilized peripheral blood stem cells from an HLA matched donor. Peripheral blood mononuclear cells (PBMC) were obtained from the recipient pretransplant and at intervals after NM-HSCT. When chimerism showed a major proportion of donor CD3+ T cells, the postransplant PBMC were stimulated in vitro with recipient CLL cells from the pretransplant collections. CLL cells lack or express low levels of co-stimulatory and adhesion molecules, and are poor stimulators of T cells in vitro. Thus, prior to their use as stimulators and targets, the CLL cells were activated with CD40 ligand (CD40L), which upregulates costimulatory, adhesion, and MHC molecule expression, and turns CLL cells into effective antigen presenting cells. The cultures were stimulated weekly and supplemented with IL2 and IL7. After two stimulations, the T cell lines were tested for cytotoxicity against donor and recipient target cells including recipient CLL. T cell lines generated from four patients with a good antitumor response after NM-HSCT exhibited cytotoxicity against recipient CLL and EBV transformed B cells (B-LCL), but not against donor B-LCL. By contrast, T cell lines generated from three patients with persistent or progressive disease after NM-HSCT did not have cytotoxicity against recipient CLL, despite the development of GVHD in all patients. Multiparameter flow cytometry and IFN-g secretion assay of T cell lines from patients with an antitumor response showed that both CD8+ and CD4+ T cells produced INF-g in response to recipient CLL. We sorted and expanded CD8+ INF-g+ and CD4+ IFN-g+ T cells and both subsets were able to lyse CLL cells. The cytotoxicity of CD4+ and CD8+ T cells was inhibited completely by concanamycin A, suggesting perforin is the major mechanism for leukemia cell lysis. Twenty-one CD8+ T cell clones specific for distinct minor histocompatibility antigens expressed on CLL were isolated from T cell lines of the four responding patients. Multiple specificities were recognized in three of the four patients. Screening a cDNA expression library has identified the genes encoding two minor histocompatibility antigens recognized by CD8+ T cells, and their characterization is in progress. These findings suggest that the development after NM-HSCT of early, diverse, alloreactive T cell responses specific for antigens expressed by CLL may be an important predictor of outcome. The identification of the antigens recognized may facilitate the development of strategies to evoke an effective antitumor response in a larger fraction of patients.

Download Full-text

Functionally Altered GPI-Anchor Negative Treg Following Alemtuzumab-Based T-Cell Depletion Are Associated with Acute Gvhd.

Blood ◽

10.1182/blood.v120.21.3059.3059 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3059-3059

Author(s):

Eva M Wagner ◽

Lukas A Schaefer ◽

Tobias Bopp ◽

Matthias Theobald ◽

Wolfgang Herr ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Cell Depletion ◽

Gpi Anchor ◽

Activation Markers ◽

T Cell Depletion ◽

Hematopoietic Stem

Abstract Abstract 3059 Introduction: The monoclonal anti-CD52antibody Alemtuzumab is frequently used for T-cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT) to prevent graft versus host disease (GVHD). We previously demonstrated the long term persistence of functionally impaired glycosylphosphatidylinositol (GPI)-anchor negative effector T-cells in patients receiving high dose (100mg) Alemtuzumab in combination with a dose reduced conditioning regimen (Fludarabin + Melpahlan) (Meyer, Wagner et al. BMT 2010). Despite of Alemtuzumab-mediated TCD, half of our patients developed acute GVHD. Since regulatory T cells (Treg) play a major role for controlling GVHD, we asked whether GPI-anchor negative Treg are present in patients with or without GVHD. Methods: We analyzed peripheral blood samples of 12 patients with acute GVHD (aGVHD), 7 patients with chronic GVHD (cGVHD), and 10 patients who never developed GVHD after Alemtuzumab-mediated TCD. To analyze Treg-subsets, we stained for CD3, CD4, CD25, CD127, FoxP3, CD52 as well as for the activation-markers GARP, HLA-DR and CD45RA. Treg were identified as CD3+CD4+CD25+CD127- or CD3+CD4+CD25+FoxP3+ cells and subdivided according to their CD52-expression. We used FLAER staining to confirm that the loss of CD52 on Treg resulted from the loss of the GPI-anchors themselves. We were able to study Treg subpopulations in the time course of patients who recovered from acute GVHD in comparison to patients with persisting late acute GVHD. In individual patients, we isolated GPI-anchor positive and negative Treg by FACS-Sort, expanded them and performed Treg suppression assays. Results: GPI-anchor negative Treg were observed in all patients, independent of the development of GVHD. However, the frequency of GPI-anchor negative Treg varied considerably between patients with acute GvHD and those with chronic GVHD or without GvHD. The percentage of GPI-anchor negative Treg was significantly elevated in patients with aGVHD: median 80.35% (range 56,2–96,8%) in comparison to 17,4% (range 0–57,8%) in patients with cGVHD or without GVHD. Activated Treg were almost exclusively detected among GPI-anchor positive Treg-subpopulation. Patients who resolved from aGVHD restored GPI-anchor positive Treg and the amount of activated Treg rose. The percentage of GPI-anchor negative Treg populations remained high in patients with ongoing aGVHD. In addition, these patients had no GARP-positive activated Treg even under long term immunosuppressive treatment. Preliminary experiments with sorted and expanded Treg populations suggest that GPI-anchor negative Treg were unable to suppress T-cell proliferation upon IL-2 stimulation. Summary: We demonstrate for the first time the reconstitution of GPI-anchor negative Treg in patients following Alemtuzumab-mediated TCD. These T cells were functionally altered and were less likely to exhibit an activated phenotype in vivo. Ongoing acute GVHD was associated with high percentages of GPI-negative Treg suggesting that their functional alteration might play a role in aGVHD pathophysiology. This is in line with the finding that only in patients who resolved aGVHD, the frequency of GPI-anchor positive Treg increased significantly. Further functional analyses are ongoing to estimate the cellular consequence of missing GPI-anchored proteins. In addition, correlating the reconstitution of GPI-anchor negative T-cell populations with further clinical events is ongoing. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

In Allogeneic Transplant Recipients Th17 Cells Originate From Donor Stem Cells and Reside In Target Organs of Chronic Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v122.21.5425.5425 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5425-5425

Author(s):

Antonia MS Mueller ◽

Mareike Florek ◽

Natascha J Kuepper ◽

Jessica Poyser ◽

Judith A. Shizuru

Keyword(s):

T Cells ◽

T Cell ◽

Th17 Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Cell Infiltration ◽

Host Disease ◽

T Cell Infiltration ◽

Target Organs ◽

Donor T Cells

Abstract Graft-vs-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). Acute GVHD results from activated donor T cells that infiltrate and damage target organs, producing an inflammatory state. In contrast, the pathophysiology of chronic graft-vs-host disease (cGVHD) remains poorly understood. cGVHD can follow acute GVHD or emerge de novo (>d+100). The clinical picture varies and manifestations can resemble autoimmune disorders. Because IL-17 has emerged as a principal cytokine involved in autoimmunity, Th17 cells have attracted much attention in the transplant community. While IFNg-producing Th1 cells appear to drive acute GVHD, the role of Th17 cells in the pathophysiology of GVHD has not been fully clarified. Here, we used an established minor-antigen disparate mouse model of acute and chronic GVHD to examine the emergence of IL-17+CD4+ Th17 cells post-HCT. Lethally irradiated BALB.B mice received pure hematopoietic stem cells (HSC; cKIT+Thy1.1loSca1+Lin-) or HSC plus splenic T cells from C57BL/6 donors (HSC: GFP; TC: CD45.1+). At several time points lymphoid and GVHD target organs were analyzed for donor T cell infiltration and T cell IL-17 expression. In this model recipients of HSC + T cells developed acute GVHD with intestinal involvement (diarrhea, weight loss) and a mortality of ∼30%, while mice given pure HSC remained healthy. Survivors stabilized around d45, but developed clinically evident chronic GVHD after 6-12 mo manifested by sclerodermatous skin excoriations and liver fibrosis/cirrhosis. Donor T cell infiltration of tissues (spleen, lymph nodes (LN), liver, intestines) was high at 2 and 4 wks post-HCT, but there was no detectable IL-17 production by CD4 cells during acute GVHD. The degree of donor T cell infiltration decreased (as acute GVHD improved) in these tissues. However, at 2 mo post-HCT higher percentages CD4+IL-17+ cells were observed, first in intestines and mesenteric LN, followed by liver and skin. At all time points post-HCT proportions of Th17 cells were higher in HCT recipients (of HSC +/- T cells) as compared to normal wild-type (WT) tissues. To summarize, our key findings are: (i) In our model acute GVHD was driven by adoptively transferred mature (CD4+) T cells that acquired a Th1 phenotype, whereas IL-17 producing donor cells were not detectable during this period. IFNg and T-bet are negative regulators of RORgT, the master regulator of Th17. Thus, this observation is consistent with the idea that in the presence of donor Th1 cells the development of Th17 cells is suppressed. (ii) The effect of Th1-related suppression of Th17 persisted beyond the acute phase: recipients of T-cell replete grafts that survived acute GVHD but later developed chronic GVHD did not demonstrate increased CD4+IL-17+ cells. In these mice, organ-infiltrating donor T cells were primarily adoptively transferred T cells, supporting the postulation that no plasticity exists between committed Th1 and Th17 cells. (iii) Signs of chronic GVHD were observed in animals that had not suffered from severe acute GVHD. In particular, in groups without acute GVHD we observed CD4+IL-17+ cells starting at 2 mo, peaking around 6 mo and which stabilized >1 yr post-HCT. In spleen and peripheral LN of these mice only low levels of CD4+IL-17+ cells were detectable, but their proportion was high in GVHD target organs (liver, intestines, skin). The susceptibility of organs appeared to change post-HCT with high proportions of CD4+IL-17+ cells in the intestines at 2 mo post-HCT that decreased over time. In contrast, CD4+IL-17+ cells in the liver increased later in the time course (Figures). (iv) A centrally important observation was that CD4+IL-17+ cells primarily originated from donor HSC, even in recipients of mature donor T cells. Likewise, recipients of pure HSC showed increasing proportions of Th17 cells over time, and could also manifest signs of cGVHD. From our model we conclude that IL-17 does not contribute to acute inflammatory GVHD. However, IL-17 can be involved in an alternative pathophysiologic mode of chronic GVHD development in the absence of acute inflammation. Since CD4+IL-17+ cells derive from donor HSC and undergo maturation in the host this form of GVHD is delayed, and the emergence and activity of these cells appears to constitute a true autoimmune phenomenon. Our novel hypothesis may explain parts of the complex and obscure pathophysiology of chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

Download Full-text