Donor-derived Cytotoxic T Lymphocytes Research Articles

Diagnosis of Intestinal Graft-versus-Host Disease and Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation

Severe diarrhea is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). Acute graft-versus-host disease (GVHD) has been one of the major causes of diarrhea after HSCT, which is triggered by donor-derived cytotoxic T-lymphocytes. On the other hand, intestinal thrombotic microangiopathy (TMA) sometimes coexists with acute GVHD, and intensified immunosuppression to treat acute GVHD could exacerbate intestinal TMA, presumably through the vascular endothelial cell damage. The differential diagnosis between intestinal TMA and acute GVHD of the gut has mainly relied on the pathological findings, as clinical diagnosis of intestinal TMA has not been established. Therefore, we aimed to assess the feasibility of our clinical diagnosis for the patients with diarrhea after HSCT. We made tentative clinical criteria for intestinal TMA and acute GVHD of the gut, based on the clinical manifestations, laboratory data and colonoscopic findings, and started treatment before pathological diagnosis were made. Subsequently, a pathologist retrospectively assessed the accuracy of clinical diagnosis in a blind manner. In this study, we enrolled 19 patients complicating watery diarrhea after HSCT, and diagnosed as having acute GVHD (n = 10), intestinal TMA (n = 3), or both (n = 6) according to our criteria. We demonstrated that our clinical diagnosis for intestinal TMA and acute GVHD of the gut was overall correct, in terms of the response to the therapy and the pathological diagnosis. The present study may provide a clue on making clinical diagnosis of patients with watery diarrhea after HSCT, which enables us to start a prompt therapy.

Clinical grade expansion of CD45RA, CD45RO, and CD62L-positive T-cell lines from HLA-compatible donors: High cytotoxic potential against AML and ALL cells

Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span, granulocyte macrophage colony-stimulating factor, interleukin-4, and calcium ionophore. All B-precursor ALL (N22) and AML (N13), but not T-cell ALL (N3), differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). Upon in vitro culture, donor-derived CTL acquired a memory T phenotype, showing concomitant high CD45RA, CD45RO, CD62L expression. CD8(+) cells, but not CD4(+) cells, were granzyme, perforine, and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%, 30:1 E:T ratio), but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%, 1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation.

In vivo expansion of LMP 1- and 2-specific T-cells in a patient who received donor-derived EBV-specific T-cells after allogeneic stem cell transplantation

Immunotherapy approaches with antigen-specific cytotoxic T lymphocytes (CTLs) have provided safe and effective prophylaxis and treatment of Epstein-Barr virus (EBV)-associated lymphomas arising after bone marrow transplantation. EBV is also associated with other malignancies including ∼40% of cases of Hodgkin's disease, making this tumor another potential target for EBV-targeted immunotherapy. This study describes a patient with multiple relapsed EBV positive Hodgkin's Disease who received both autologous and allogeneic EBV CTL lines. After multiple chemotherapeutic and radiotherapy regimens including two autologous stem cell transplants, he received two doses of gene-marked autologous EBV-specific CTL which resulted in disease stabilization for 6 months. The gene-marked EBV-CTL persisted for 12 months in the peripheral blood after which he proceeded to unrelated donor stem cell transplant followed by immunotherapy with donor-derived EBV-specific CTL. Despite low levels of donor chimerism, the patient remains in complete remission 5 years post-allogeneic SCT. Comparison of the autologous and the donor-derived CTL lines showed that the donor line had specificity for two tumor-associated EBV antigens, latent membrane protein (LMP)1 and 2 compared to the autologous line, which only had specificity for LMP2 epitopes. Following infusion of the donor-derived CTL, functional analyses showed that T-cells reactive with both LMP1 and LMP2 epitopes expanded in the peripheral blood, suggesting that strategies to increase their frequency may result in a broader cytotoxic response against EBV+ Hodgkin tumors.

Follow-Up of WT1-Specific Cytotoxic T Lymphocytes (CTL) with Tetramer-Based Analysis after Allogeneic Stem Cell Transplantation (SCT) in Patients with Renal Cell Carcinoma (RCC).

Background and Methods: Donor-derived CTL that respond to tumor antigens emerge after SCT, particularly in association with the status of immune recovery and disease remission. To analyze the frequency of CTL against WT1, PR1 and PRAME after SCT, tetramer-based analysis was performed in 69 samples taken from 34 patients with the HLA-A02 phenotype, including 2 with RCC (10 MDS, 8 AML, 6 NHL, 4 ALL, 2 CML, 2 RCC, 1 ATL, 1 PCL). Peripheral blood samples were stained with WT1 126, PR1 169, and PRAME 300 tetramers, and CMV pp65 tetramer was used as a positive control. Antigen -specific CTL was defined as CD3+, CD13−, CD19−, CD4−, CD8+, and tetramer + cells present in the lymphocyte gate, and the cut-off level was defined as 0.02% per CD8+ lymphocytes.Results: All samples from CMV-seropositive patients were positive for CMV-CTL with high titers (average 2.3%). Regarding PR1, only 1 sample showed the presence of tetramer-positive cells (0.04%). Similarly in PRAME, only 3 samples from 3 different patients were sporadically positive with low titers (<0.05%), but positive results were not found at different occasions. Regarding WT1-CTL, positive results were detected in 23 samples taken from 2 RCC, 2 NHL, 1 AML, 1 ALL, 1 ATL, and 1 PCL patient. A higher positive rate for WT1-CTL assay suggests that WT1 may be a potential candidate antigen for wider application compared to the other two antigens.Case monitoring: Based on these results, we performed serial analyses of WT1-CTL during the clinical course in 2 patients with RCC who underwent reduced-intensity stem cell transplantation (RIST). Before RIST, both RCC patients had undergone resection of the primary disease, but had developed multiple lung metastases, which were progressive despite interferon therapy. WT1-CTL in one of the RCC patients was detected at day 40 post-SCT after the development of skin GVHD, and the peak formation of WT1-CTL occurred on day 128 when oral chronic GVHD was noted. WT1-CTL became undetectable after day 268, but the disease has not progressed up to day 359. The other patient became positive for WT1-CTL on day 90, one week after the onset of liver acute GVHD. Cyclosporine was started at a dose of 400 mg/body and WT1-CTL disappeared. However, when cyclosporine was tapered to 100 mg/body, WT1-CTL became detectable again on day 146 following the development of skin GVHD. While the number of WT1-CTL was correlated with the presence of allo-reaction and cyclosporine treatment, the total lymphocyte count and CMV-CTL counts did not change significantly. This patient is currently showing stable disease beyond day 200. This is the first report of the precise monitoring of WT1-CTL after SCT in patients with RCC, and of the correlation of WT1-CTL with the clinical response. Further studies will be needed to evaluate the correlation between WT1-CTL and disease control. [Display omitted]

A human minor histocompatibility antigen specific for B cell acute lymphoblastic leukemia.

Human minor histocompatibility antigens (mHags) play an important role in the induction of cytotoxic T lymphocyte (CTL) reactivity against leukemia after human histocompatibility leukocyte antigen (HLA)-identical allogeneic bone marrow transplantation (BMT). As most mHags are not leukemia specific but are also expressed by normal tissues, antileukemia reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Here, we describe a novel mHag, HB-1, that elicits donor-derived CTL reactivity in a B cell acute lymphoblastic leukemia (B-ALL) patient treated by HLA-matched BMT. We identified the gene encoding the antigenic peptide recognized by HB-1-specific CTLs. Interestingly, expression of the HB-1 gene was only observed in B-ALL cells and Epstein-Barr virus-transformed B cells. The HB-1 gene-encoded peptide EEKRGSLHVW is recognized by the CTL in association with HLA-B44. Further analysis reveals that a polymorphism in the HB-1 gene generates a single amino acid exchange from His to Tyr at position 8 within this peptide. This amino acid substitution is critical for recognition by HB-1-specific CTLs. The restricted expression of the polymorphic HB-1 Ag by B-ALL cells and the ability to generate HB-1-specific CTLs in vitro using peptide-loaded dendritic cells offer novel opportunities to specifically target the immune system against B-ALL without the risk of evoking GVHD.

Generation of donor-derived antileukemic cytotoxic T-lymphocyte responses for treatment of relapsed leukemia after allogeneic HLA-identical bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) has been associated with an antileukemic effect, the graft-versus-leukemia (GVL) reactivity. Since T-cell depletion of the bone marrow graft performed to reduce the incidence and severity of graft-versus-host disease (GVHD) after BMT has been associated with an increase risk of relapse, the GVL reactivity has been attributed to the T lymphocytes from the graft. Previously, we demonstrated that leukemia-reactive cytotoxic T-lymphocyte (CTL) lines and clones could be generated from the peripheral blood of HLA-genotypically identical siblings of patients with leukemia by stimulation of the donor cells with irradiated leukemic cells from the patients. HLA class I as well as class II restricted CTL clones could be generated that recognized the leukemic cells. Some clones recognized the leukemic cells from the patient, but not the interleukin (IL)-2-stimulated lymphocytes from the same individual. To explore the possibility of clinically using donor-derived CTL lines directed against the leukemic cells from patients who relapsed after allogeneic BMT, a pilot study was performed using eight donor-recipient combinations. In seven of eight combinations donor-derived CTL lines could be generated that showed specific lysis of the leukemic cells from the patient. In five of these cases, the CTL lines showed reactivity with the leukemic cells, but not with the IL-2-stimulated lymphocytes from the same individual. In two cases, the CTL lines were cytotoxic for the IL-2-stimulated lymphoblasts as well as the leukemic cells.(ABSTRACT TRUNCATED AT 250 WORDS)