ALBERT

Description: Immune checkpoint (IC) therapy provides substantial benefits to cancer patients but can also cause distinctive toxicities termed immune-related adverse events (irAEs). Biomarkers to predict toxicities will be necessary to improve management of patients receiving IC therapy. We relied on serological analysis of recombinant cDNA expression libraries to evaluate plasma samples from patients treated with IC therapy and identified autoantibodies, both in pretreatment and on-treatment samples prior to the development of irAEs, which correlate with the development of immune-related hypophysitis (anti-GNAL and anti-ITM2B autoantibodies) and pneumonitis (anti-CD74 autoantibody). We developed an enzyme-linked immunosorbent assay and tested additional patient samples to confirm our initial findings. Collectively, our data suggest that autoantibodies may correlate with irAEs related to IC therapy, and specific autoantibodies may be detected early for the management of irAEs.

Description: We give an Ulam type stability result for the following functional equation: under a suitable condition. We also give a concrete stability result for the case taking up as a control function.

Description: Administration of the immunosuppressive drug cyclosporine A (CsA) following autologous stem cell transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with autoreactive CD8+ T cells that recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain. To investigate the potential role of cytokines and chemokines in autologous GVHD, interleukin 2 (IL-2), IL-4, IL-10, interferon γ (IFN-γ), and macrophage inflammatory protein-1α (MIP-1α) gene expression in peripheral blood mononuclear cells (PBMCs) was determined in 36 patients treated with CsA following transplantation and correlated with the induction of cytolytic activity against autologous phytohemagglutinin-stimulated lymphocytes (PHA-blasts) and the breast cancer cell line (T47D). The determination of gene expression by real-time polymerase chain reaction (PCR) revealed that IL-10 mRNA levels by PBMCs in patients with autologous GVHD were 29-fold higher than in healthy individuals. IFN-γ (4-fold), IL-2 (3-fold), and MIP-1α (44-fold) mRNA levels were also increased in GVHD-induced patients compared with healthy individuals. The ability of PBMCs to lyse autologous PHA-blasts and T47D tumor cells exhibited an identical temporal relationship with expression of IL-10 and IFN-γ during autologous GVHD. Moreover, the susceptibility to autologous GVHD as assessed in 75 patients was significantly associated with the IL-10−1082 G/G polymorphic alleles, allelic variants in the promoter region that govern IL-10 production. These findings indicate that IL-10 may play an unexpected but critical role in autologous GVHD and could be utilized to enhance a graft-versus-tumor effect after transplantation. Interestingly, polymorphisms in the IL-10 promoter region may also explain differences in the susceptibility of patients to autologous GVHD induction.

Description: Blood stream infection (BSI) is a major problem after cord blood transplantation (CBT). However we have little information on it after reduced-intensity cord blood transplantation (RICBT). This study aims to investigate clinical characteristics of BSI within 100 days of RICBT, and to identify its risk factors in the patient who received RICBT between January 2002 and March 2004 in Toranomon hospital. We reviewed medical records of 77 patients with advanced hematologic diseases who underwent RICBT at Toranomon Hospital between January 2002 and June 2004. Median age of the patients was 55 years (17-79). Median number of infused cells and CD34+ cells were 0.86 x 10E7 (range; 1.73–4.31) and 0.8x10E7/kg (range; 0.01–46.1), respectively. Conditioning regimen consisted of fludarabine (125–150 mg/m2), melphalan (80–140 mg/m2), and TBI 2–8 Gy. GVHD prophylaxis was cyclosporine or tacrolimus. BSI must have met at least one of the following criteria previously described. (O’Grady, NP et al., Infect Control Hosp Epidemiol 2002) Mortality was considered to be directly attributable to a bloodstream pathogen if the patient died within 7 day after the last positive blood culture without any other probable cause of death, The patient’s characteristics were compared between those with or without BSI by univariate analysis. 149 episodes of BSI were observed in 31 patients. Cumulative incidence within 100 days was 0.40. Median onset was day 13 (1–98) after RICBT. The causative organisms were P. aeruginosa (47), S. epidermidis (44), E. faecium (13), T. beigelii (6), S. maltophilia (6), E. faecalis (5), E. coli (3), A. xylosoxidans (3) and others (22). 8 patients died of septicemia directly associated with BSI. A mortality rate was 26%. The causative organism included P. aeruginosa, A. xylosoxidans, S. maltophilia, S, aureus and gram-positive rod. Use of corticosteroid (p=0.012) and time to engraftment (p=0.003) were associated with the development of BSI. BSI is a major complication in our series of RICBT. Use of corticosteroid and prolonged neutropenia might have increased the risk of incidence of BSI.

Description: Syngeneic graft-vs-host disease (SGVHD) is a T cell dependent autoaggression syndrome induced by administering Cyclosporine following syngeneic bone marrow transplantation. The SGVHD autoreactive T cells recognize the MHC class II-invariant chain peptide complex (MHC class II-CLIP) and can be separated into functional subsets based on their differential dependence on the N- and C-terminal peptide flanking domains of CLIP. The present studies were undertaken to determine whether the N- or C-terminal flanking domain dependent subsets of CLIP reactive T cells reside within the CD4+CD25+ regulatory compartment. Multi-color flow cytometry was used to identify and isolate CD4+ (FITC) CD25+ (PE) T cells. Antigen-specific T cells within this compartment were identified with a soluble MHC class II-Ig chimeric construct (bioinylated, Cychrome avidin counterstaining) loaded with variants of CLIP containing the MHC class II binding domain and having either the N- or C-terminal flanking regions (N-CLIP, CLIP-C). Approximately 8.5% of the cells within the normal CD4+ lymphocyte population were CD25+. Both N-CLIP (1.1%) and CLIP-C (4.8%) reactive T cells coould be detected in the CD4+CD25+ population. Assessment of CD28, CTLA4, B7.1 and B7.2 mRNA expression levels by quantitative PCR directly ex vivo, revealed remarkable differences between the N-CLIP and CLIP-C specific CD4+CD25+ T cells. Although CD28 mRNA levels were comparable for both subsets, B7.2 and CTLA4 mRNA transcript levels were significantly increased (〉50 fold) in the CLIP-C+CD4+CD25+ T cells compared to the N-CLIP specific subset. On the other hand, levels of B7.1 mRNA were increased 〉10 fold in N-CLIP+CD4+CD25+ T cells. Additional studies assessing mRNA transcript levels for the regulatory transcription factor Foxp3, also revealed a disparity between the N-CLIP and C-CLIP specific subsets. mRNA transcript levels for Foxp3 were markedly increased (〉35 fold) in the CLIP-C dependent subset compared to the levels detected in N-CLIP+CD4+CD25+ T cells. Low levels of cytokine (IL-2, IL-4, interferon-γ) mRNA transcripts were detected in both subsets. Interestingly, intradermal immunization of normal animals with the peptides presented on dendritic cells increased mRNA transcript levels for type 1 cytokines in the N-CLIP reactive subset and type 2 cytokines in the CLIP-C dependent subset. Taken together, the results indicate that the CLIP-C antigen specific CD4+CD25+ cells have a profile consistent with regulatory T cells whereas the profile of the N-CLIP+CD4+CD25+ lymphocytes is more characteristic of activated T helper cells. The ability to identify and isolate regulatory T cells ex vivo and to modify their activity by immunization provides opportunities to both enhance and monitor the re-establishment of self-tolerance.

Description: Graft-versus-host disease (GVHD) is a serious, life-threatening complication that occurs following allogeneic (allo) bone marrow transplantation (BMT). The use of non-specific immunosuppression or T cell depletion has reduced the incidence of GVHD but at the expense of increased rates of infection and leukemic relapse. Modulation of the major costimulatory pathway (CD28/CTLA4:B7) involved in T cell activation and regulation may lead to specific immune tolerance in the absence of global non-specific immunosuppression. The identification of mRNA splice variants encoding for soluble forms of CD28, CTLA4 and GITR suggests that costimulation of T cells is complex and is not limited to cell-cell contact. The present studies examined the hypothesis that the onset of GVHD and the re-establishment of immune tolerance correlate with the expression levels of these costimulatory molecules. mRNA transcript levels for the soluble (s) and full-length (fl; cell surface associated) variants assessed by quantitative PCR, were temporally examined in peripheral blood lymphocytes (PBLs) from patients undergoing alloBMT (n=38) or autologous (auto) BMT (n=39) with the induction of autoGVHD by cyclosporin A treatment post-transplant. Levels of s and fl CD28 mRNA transcripts in PBLs were significantly increased (〉1.5 fold, P2.3-fold, P2.1-fold). sCTLA4 expression in patients with alloGVHD was significantly decreased than patients without alloGVHD. Interestingly, temporal analysis revealed that the levels for sCTLA4 paralleled the recovery from GVHD implicating an active process in the establishment of non-responsiveness. CD28, CTLA4 and GITR s and fl mRNA levels in CD4+CD25+ T regulatory (Treg) cells from allo and autoBMT patients were significantly increased (7-, 41- or 22-fold, P4 fold reduction of 3H-thymidine incorporation). However, pretreatment of the Treg subset with short interfering RNA (siRNA) to knockdown sCTLA4 gene (confirmed by quantitative PCR) significantly reduced the ability of these cells to suppress the response (minimal suppression was detected, 6%). In vitro siRNA studies also indicated that Treg cells with inhibited sCTLA4 expression were unable to suppress the response of IL-2-stimulated autoreactive CD8+ T cells. Taken together, the results indicate that increased expression of CTLA4 (soluble and cell-surface associated) and the “negative” signal delivered by this molecule to the T cell may regulate the development of GVHD and help to re-establish self tolerance after BMT. Defining the role of costimulation and the modulation of this pathway on immune recognition and regulation not only provides opportunities to enhance the re-establishment of tolerance but also may help to intensify anti-tumor immunotherapeutic strategies.

Description: [Introduction] The effectiveness of allogeneic hematopoietic stem cell transplantation (allo-HSCT) against autoimmune disease has been suggested by studies in animal models and patients who had undergone transplantation for hematological disorders. However, its significant treatment-related mortality (TRM) has limited its application to life-threatening cases. Reduced-intensity stem cell transplantation (RIST) is a rapidly expanding treatment strategy and diminishes TRM by decreasing conditioning regimen toxicity compared to conventional myeloablative transplants. The benefit of RIST for autoimmune disease still remains unclear. [Purpose] The purpose of this study is to analyze outcomes of patients with autoimmune diseases treated with RIST for coexisting hematological diseases. [Patients and Methods] Between April 2001 and March 2004, 19 patients (median age, 53 y; range, 20-70) underwent RIST from allogeneic donor. All of the patients were considered inappropriate for conventional allo-HSCT due to age 〉 50 years and/or organ dysfunction (generally attributable to autoimmune disease-related). Stem cell sources were unrelated CB (n=10), unmanipulated related PB (n=7) and unmanipulated unrelated BM (n=2). The preparative regimens were Flu/Mel-based (n=11), Flu/Bu-based (n=7), and Flu/CY (n=1). The primary disease were NHL (n=7), MDS (n=4), AML (n=3), HD (n=2), ATL (n=2), and SAA (n=1). Concurrent autoimmune diseases were ulcerative colitis (n=4), psoriasis (n=3), rheumatoid arthritis (n=2), interstitial pneumonitis (n=2), atopic dermatitis (n=1), ITP (n=1), CIDP (n=1), polychondritis (n=1), SLE (n=1), and MCTD (n=1). Eleven out of 19 patients were active in autoimmune disease, and seven needed immunosuppressive therapy. [Results] Median follow up period is 174 days (range, 33–987). All patients achieved neutrophile engraftment (median 22 days; range 12–42) and sustained 100% donor chimerism without DLI. 13 patients (68%) developed grade II-IV acute GVHD, and six (31%) developed chronic GVHD. Nine patients (47%) who were treated with steroid, died of transplant-related toxicity; sepsis (n=7), GI bleeding (n=1), and multiple organ failure (n=1). One patient died of leukemia relapse. As of August 2004, ten patients are alive at median follow-up of 402 days, and the actuarial overall survival rate is 51 % at 2 years. Regarding the clinical response for autoimmune disease, no patients experienced flares of the disease: complete remission is observed in seven patients (70%) and they become therapy-free. In the other three patients (30%), disease remains stable. [Conclusion] Our results suggest that a strategy that incorporates RIST for autoimmune disease may be worth considering for further intense evaluation. It may have a considerable graft-versus-autoimmunity (GVA) effect due to the recipient lymphoablation and reconstitution of donor T cells. Organ failure due to autoimmune disease, impaired immune response, and GVHD may contribute to high TRM. Management of regimen-related toxicity and the control of GVHD will be the focus of future investigation.

Description: Background: Graft failure, graft rejection, or disease relapse post allogeneic hematopoietic stem cell transplantation(HSCT) is life-threatening and serious complication that necessitate consideration for a second transplantation. Graft failure has been more frequently reported in patients with aplastic anemia with T-cell depletion of the graft, in cord blood transplants, or when unrelated or HLA-mismatched related donors were used. In second allogeneic stem cell transplant setting, current questions include suitable stem cell source, additional conditioning, immunosuppression, and the use of a different donor, still remain unknown. We performed a feasibility study of reduced intensity umbilical cord blood transplantation (RI-UCBT) in adult patients with graft failure or disease relapse after first allogeneic transplantation. Patients and Methods: Nine patients (median age, 53 years; range 17–68) with advanced hematological diseases [AML (n=4), ALL (n=2), MDS (n=2), ATL (n=1)] who showed relapse or graft failure after first allogeneic transplantation underwent RI-UCBT with single cord blood unit at Toranomon Hospital between May 2003 and February 2004. Eight cases were in non-CR at transplant. A median time from first to second transplant was 226 days (range 31–475). The median number of mononuclear cells transfused was 2.3 x 107 /kg (range 1.8–3.5). HLA disparities were as follows; 5/6 in two cases, 4/6 in seven cases. Conditioning regimen mainly consisted of fludarabine 25 mg/m2 on days -7 to −3, melphalan 40mg/m2 (n=8) or busulfan 4mg/kg(n=1) day −3 to −2, and 4 Gy total body irradiation on day −1. Graft-versus-host disease (GVHD) prophylaxis was composed of ciclosporin alone (n=6) and tacrolimus alone (n=3). Results: All of them achieved primary neutrophil engraftment (〉500/μL) after a median of 22 days (range, 15–32) and achievement of donor T-cell chimerism was confirmed. Four cases developed grade II-IV GVHD, and one case developed limited chronic GVHD. Of all the nine cases received RI-UCBT, seven died from relapse (n=4), sepsis (n=2), and TMA (n=1). Two survived without relapse for +232 and +81 days, respectively. Discussion: Although the number of patients is small and the follow-up period is short, our results corroborate that RI-UCBT for graft failure or relapse post first transplant is tolerated and may be worth considering for further evaluation. Neutrophil engraftment was achieved, however, disease relapse rate and treatment-related mortality is high. The management of disease status and development of ideal conditioning regimen will be the focus of future investigation.

Description: Backgrounds: Cytomegalovirus (CMV) infection is a major complication after allogeneic transplantation; however clinical significance of CMV reactivation after cord blood transplantation remains unclear. Objective: We retrospectively investigated the incidence of CMV antigenemia, and CMV diseases, and its prognosis in adult patients who underwent reduced-intensity cord blood transplantation (RI-CBT) Patients/ Methods: We reviewed medical records of 77 patients who received RICBT at Toranomon Hospital between January 2002 and March 2004. Median age of the patients was 55 years (range 17–79). Underlying diseases were chemorefractory hematologic diseases (n=76) and severe aplastic anemia (n=4). Conditioning regimen comprised fludarabine (125 mg/m2), melphalan (80 mg/m2), and TBI 4-8Gy. GVHD prophylaxis was cyclosporine (n=69) or tacrolimus (n=11). Median total nucleated cells and CD34+ cells was 2.4×106 cells/kg (0.39–4.3), and 0.81×105 cells/kg (0.05–5.7) respectively. HLA disparity was 6/6 (n=3), 5/6 (n=12), 4/6 (n=63), and 3/6 (n=2). All patients were monitored CMV-antigenemia weekly and received pre-emptive gancyclovir or foscarnet. Results CMV antigenemia tested positive in 47 patients on a median of day 32 (range, 12–55) after RICBT. The cumulative incidence of CMV reactivation at day 100 was 0.70. Seven and 29 patients were treated with preemptive ganciclovir and foscarnet, respectively. Adverse events of them were myelosuppression in 3 patients given ganciclovir, and mild hyponatremia in a patient given foscarnet. CMV diseases developed in 15 patients on a median of day 39 (range 15–92); enterocolitis (n=13), pneumonia (n=1), and encephalitis (n=1). Seven of 15 patients were resolved with antiviral treatment, and the other patients were fatal with CMV infection. Univariate analysis showed any risk factors for CMV reactivation. Discussion CMV reactivation and diseases develop early after cord blood transplantation. Opitimal strategy for preventing CMV disease should be established in RICBT.

Description: The potential role of RICBT in adults remains unclear. This study reports the results of RICBT for adults with hematological diseases. The 1st purpose of this report was to investigate the risk factors of non-relapse mortality (NRM) within day 100 of RICBT and the 2nd was to identify its prognostic factors. We reviewed medical records of 107 patients with advanced hematological diseases who had received RICBT between March 2002 and June 2004 at Toranomon Hospital, Tokyo, Japan. Median age of the patients was 55 years (17–79). Primary diseases were advanced (n=88) or standard (n=19). Median follow-up was 14 months (0.5–27). Conditioning regimen was fludarabine 125 mg/m2, melphalan 80 mg/m2 and TBI 4 Gy. GVHD prophylaxis was cyclosporine (n=87) or tacrolimus (n=26). Median total nucleated cells (TNC): 3.2 x 10^7 cells (1.7–4.3); Median CD34+: 1.4 x 10^5 cells (0.21–3.28); HLA match: 6/6 (n=4), 5/6 (n=3), 4/6 (n=95), 3/6 (n−1). Time to event curves were plotted by using the actuarial method of Kaplan-Meier, and differences between curves were analyzed by log-rank tests. The following factors were considered potential predictors of outcomes about 1st and 2nd purpose: age, weight, sex, risk, HLA disparities, TNC, CD34+, GVHD, pre-engraftment reactions (PER) that we have reported (Clin Cancer Res.10:3586-92, 2004), and use of steroids for PER. All factors were tested for the proportional hazards assumption. Neutrophile (〉500/μL) and platelet recovery (〉20,000/μL) were observed in 77.9% at day 60 (median; 19.5 day), 48.7% at day 100 (45.5 day), respectively. Primary graft failure was occurred in 8% of all cases. Cumulative incidence of acute GVHD (II-IV) and chronic GVHD were 39.8% and 21.1%, respectively. Causes of NRM included infections (n=30), regimen related mortality (RRM) (n=19), and GVHD (n=6). Incidence of NRM at day 100 was 51.6% (95% CI: 42–61), 33 patients with use of steroids for PER had NRM at day 100 of 75% (95% CI: 67–94), as compared to 39% (95% CI: 25–48) in 77 patients without use of steroids (p