A case of membranous nephropathy associated with chronic GVHD successfully treated with rituximab

Abstract Abstract 4480 Introduction: Adult T-cell leukemia (ATL) endemic in southwestern Japan is an HTLV-I-induced mature T-cell neoplasm. It has a dismal prognosis when treated with conventional chemotherapy, mainly because ATL cells overexpress multidrug-resistance genes. Allogeneic hematopoietic stem cell transplantation (allo-SCT) has been reported to improve the prognosis. However, it is also known that allo-SCT shows high treatment-related mortality (TRM) because of disease characteristics of severely deteriorated cellular immunity. Therefore, we evaluated the outcome of reduced-intensity stem cell transplantation (RIST), compared to conventional stem cell transplantation (CST) for ATL patients (pts). Patients and methods: We retrospectively analyzed consecutive 20 pts with aggressive ATL (acute type, 16; lymphoma type, 4) who underwent allo-SCT between 2000 and 2010 in our institute. The age of pts who received RIST was significantly older than that of CST (P = <.001). The other parameters were balanced between the two groups (Table 1). All pts received induction chemotherapy with Japan Clinical Oncology Group (VCAP-AMP-VECP) regimen or biweekly CHOP prior to SCT. Nine pts received a conventional myeloablative conditioning with CY/TBI-based (n=8) or BU/CY-based (n=1) regimens and 11 pts received a reduced-intensity regimen with fludarabine(Flu)/melphalan(Mel)±TBI (Flu 25 mg/m2 on days -6 to -2 + Mel 40 mg/m2 on days -4 and -3 ± TBI 4 Gy on day -1). Graft-versus-host-disease (GVHD) prophylaxis consisted of cyclosporine plus short-term methotrexate (sMTX) for HLA-matched sibling donor recipients and tacrolimus plus sMTX for the others. Cyclosporine or tacrolimus was promptly tapered when complete chimerism was achieved. Results and discussion: The estimated 3-year overall survival (OS) and 3-year progression-free survival (PFS) rates for all pts were 59% and 55%, respectively (Fig. 1). At the median follow-up duration of 25 months (1–67), 12 pts were alive and 8 pts dead. Causes of death were disease progression (n=4) and treatment-related complications including acute GVHD and infection (n=4). The cumulative incidences of disease-associated mortality and TRM at 3 years were 21% and 20%, respectively. Importantly, after 1 year post-transplantation, the curve of PFS reached plateau phase. Therefore, allo-SCT could be a curable therapeutic option for aggressive ATL. It is notable that RIST showed excellent 3-year OS. Although not significant, 3-year OS for RIST and CST were 70% and 44% (P =.25), respectively (Fig. 2). TRM of RIST trended to be low compared with that of CST (18% vs 22%, P =.87). In addition, chronic GVHD was observed in 91% of the evaluable 11 pts (limited type, 6; extensive type, 5). It is suggested that both improvement of TRM and the induction of graft-versus-leukemia effects may contribute to the favorable outcome in RIST for ATL. Furthermore, it should be stressed that older pts who preferentially received RIST rather than CST achieved favorable survival comparable to younger pts (≥ 50 years: 58% vs < 50 years: 60%, P =.94). We would suggest that RIST should be applied to all ATL pts eligible for allo-SCT, regardless of the age. Univariate analysis for all pts revealed that only male sexuality was a significant poor prognostic factor for OS (male: 25% vs female: 82%, P =.006). Regarding disease status at SCT, CR or non-CR did not significantly affect OS (CR: 67% vs non-CR: 58%, P =.98). However, PD was an adverse impact on 3-year OS (PD: 33% vs non-PD: 64%, P =.11). It is recommended that ATL pts receive allo-SCT in early phase before the disease becomes chemo-resistant, even if they do not achieve CR. Conclusion: Allo-SCT for ATL remarkably improved the poor prognosis. Especially, RIST is a promising procedure for excellent outcome. Disclosures: No relevant conflicts of interest to declare.

Twelve patients with T cell large granular lymphocyte leukemia and associated hematocytopenia were treated in a phase I dose-escalation trial with the murine monoclonal antibody Mikbeta1. Mikbeta1 identifies CD122, the beta-subunit shared by the IL-2 and IL-15 receptors. At the doses administered in this study the antibody inhibited the actions of IL-15 on both natural killer and T cells and that of IL-2 when the intermediate-affinity IL-2 receptor was expressed. Mikbeta1 treatment was not associated with significant toxicity or with the development of an immune response to the infused monoclonal antibody. At these doses of Mikbeta1, >95% saturation of the IL-2/IL-15beta receptor (CD122) on the surfaces of the leukemic cells was achieved. Furthermore, in seven patients this led to the down-modulation of the receptor from the surfaces of the leukemic cells. Nevertheless, no patients manifested a reduction in peripheral leukemic cell count or an amelioration of their hematocytopenia. This latter observation may reflect the fact that the monoclonal T cell large granular lymphocyte leukemia leukemic cells of the patients did not produce IL-2 or IL-15 or require their actions for cell survival. In light of the lack of toxicity and lack of immunogenicity of the antibody observed in the present study and the role for IL-15 in the pathogenesis of autoimmune diseases, clinical trials should be performed using the humanized version of Mikbeta1 in groups of patients with human T cell lymphotropic virus I-associated myelopathy/tropical spastic paraparesis, rheumatoid arthritis, multiple sclerosis and refractory celiac disease.

High-dose therapy with peripheral blood stem cell transplantation (HDT/PBSCT) was performed as one aspect of front-line therapy in patients with poor-risk aggressive non-Hodgkin's lymphoma (high-intermediate/high risk) according to the age-adjusted international prognostic index (aaIPI). Twenty-nine patients were enrolled in this study between November 1994 and March 1999. CHOP + etoposide (CHOP-E) was used as an initial chemotherapy and as a chemotherapy agent for the purpose of cell harvesting. Peripheral blood stem cells were harvested from 17 patients, and HDT with CEC (carboplatin, etoposide, cyclophosphamide)/PBSCT was performed in 11 patients. Eighteen patients dropped out, including five for whom CHOP-E therapy was ineffective and 5 who did not give consent for cell harvesting or HDT/PBSCT. CHOP-E therapy produced complete remission (CR) in 15 out of 26 patients (58%) after discounting the 3 who were ineligible among the 29 who were initially enrolled. The median observation period after PBSCT in the 11 patients who underwent HDT/PBSCT was 25 months (3 to 50 months), and the 3-year disease-free survival rate was 73%. No serious complications associated with the transplantation were observed. We were able to confirm the feasibility and safety of HDT/PBSCT as one form of front-line therapy for aggressive non-Hodgkin's lymphoma in patients under 60 years of age.

We investigated the efficacy and safety of two methods of granisetron injection to treat nausea and vomiting induced by chemotherapy for tumors in hematopoietic organs. The methods of administration were intravenous drip infusion over 30 minutes, which is the conventional method, and intravenous bolus injection. In this study, 89.5% of patients in both groups (17/19 for each) were free from vomiting. No serious adverse events were observed in either administration group. Abnormal laboratory test values suspected to be related to granisetron were observed in 3 cases in the bolus injection group and in 2 cases in the drip infusion group. but did not pose any clinical problem. These results demonstrated the safety of both methods of administration. In conclusion, it is considered that granisetron intravenous bolus injection can be considered as the method of choice for the prevention of nausea and vomiting induced by chemotherapy for tumors in hematopoietic organs.

Evans syndrome is a rare disease defined as autoimmune hemolytic anemia plus immune thrombocytopenia. We describe a 57-year-old man with Evans syndrome whose thrombocytopenia was refractory to conventional therapy, including prednisolone, danazol, azathiopurine, cyclophosphamide, vincristine, gamma-globulin and splenectomy. The patient was then treated with three cycles of pulsed high-dose dexamethasone (40 mg/day for 4 sequential days every 4 weeks) followed by cyclosporine A therapy (300 mg/day). The platelet counts dramatically increased from 2,000/microliter to 200,000/microliter. Although thrombocytopenia appeared again with the tapering of cyclosporine A, the platelet counts recovered by re-increase of cyclosporine A, and have remained above 100,000/microliter for more than 14 months. Treatment with cyclosporine A can provide an effective therapeutic choice for refractory Evans syndrome.

An 18-year-old man was admitted to our hospital because of 39 degrees C fever for over one month, marked hepatosplenomegaly, and pancytopenia. Malignant histiocytosis, malignant lymphoma, or hemophagocytic syndrome were ruled out by bone marrow aspiration and liver biopsy. A diagnosis of chronic EB virus infection was made according to his characteristic clinical features, abnormally high titiers of anti-EBV antibodies (VCA-IgG x 2560, EA-IgG x 1280), and the detection of EBV genome in the peripheral blood mononuclear cells by polymerase chain reaction. He also manifested granular lymphocyte proliferative disorder (GLPD). The phenotype of the proliferating granular lymphocytes was CD2 (+), CD3 (-), CD56 (+), and IL-2R beta (+), showing the NK lineage of these cells. Chromosomal abnormality of the cells cultured for a short time with IL-2 and a monoclonal junctional DNA structure of EB virus terminal repeat analyzed by the Southern blotting provided definitive evidence for the monoclonal expansion of the granular lymphocytes. These findings indicate a causative role of EV virus in NK-GLPD or NK-leukemia.

We examined nine cases of adult non-T lymphoid leukemia to investigate the cell surface inducibility of interleukin 2 receptor alpha chain (IL-2R alpha) and beta chain (IL-2R beta) after in vitro culture with and without recombinant human interleukin-1 beta (rhIL-1 beta). Induction of IL-2R alpha was observed in four of six cases with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) and in all of three cases with B-cell mature lymphoid neoplasm (two chronic lymphocytic leukemia and one leukemic phase of non-Hodgkin's lymphoma). All of the IL-2R alpha-inducible cases could express this spontaneously even without rhIL-1 beta, while IL-2R beta did not appear on leukemic cells from any of the cases tested. IL-2R alpha-inducible pre-B ALL cases displayed stem cell antigen CD34 and induced myeloid-associated antigen CD13 simultaneously. These results suggest that IL-2R alpha but not IL-2R beta is easily inducible in certain cases of mature B-cell lymphoid neoplasm and pre-B ALL with immature characteristics.

The interleukin 2 (IL-2) receptor (IL-2R) is a multisubunit receptor that includes three major IL-2 binding subunits, the IL-2R alpha, beta, and gamma chains. We have detected and analyzed cooperative interactions between the IL-2R alpha and beta chains (IL-2R alpha and IL-2R beta, respectively) in COS cells transfected with cDNAs encoding the IL-2R alpha, the IL-2R beta, or both cDNAs. We demonstrated that IL-2 F42A, an analog that fails to bind to the isolated IL-2R alpha subunit and would be predicted by the hierarchical affinity-conversion model to have impaired binding to cells expressing both chains, instead readily binds to the IL-2R alpha/beta heterodimer in COS cells. Furthermore, this binding is abolished by the antibody HIEI that separates the two IL-2R subunits. The monoclonal antibodies anti-Tac and Mik-beta 1 directed at the IL-2-binding sites on IL-2R alpha and IL-2R beta, respectively, block ligand binding to the heterodimer. This binding pattern is inconsistent with the strict hierarchical affinity-conversion model that mandates an initial binding of IL-2 to IL-2R alpha followed by binding of the IL-2/IL-2R alpha complex to IL-2R beta. Instead, our results support an alternative model of preformed complexes of IL-2R beta with other IL-2R subunits. In this alternative model, IL-2R alpha and -beta exist in part as preformed complexes in which the affinity of IL-2R beta for IL-2 is altered by the proximity of IL-2R alpha, through mechanisms that do not require the prior binding of IL-2 to IL-2R alpha.

Leukemic cells from 27 adult patients with acute myelocytic leukemia (AML) were investigated to determine the cell surface inducibility of interleukin-2 receptor (IL-2R) in in vitro culture with and without interleukin-1 beta (IL-1 beta). IL-2R alpha chain (IL-2R alpha) was induced on leukemic cells in 11 of 27 cases. Most of the cases could induce IL-2R alpha spontaneously without IL-1 beta, while the IL-2R beta chain (IL-2R beta) did not appear on leukemic cells from any of the cases tested. AML cases expressing CD7 or HLA-DR antigen could induce IL-2R alpha more frequently than any other type of AML. Among interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF) and G-CSF, IL-3 showed a more prominent effect on DNA synthesis in IL-2R alpha inducible cases than in its uninducible cases. These results suggest that IL-2R alpha but not IL-2R beta was easily inducible on AML cells with immature characteristics.

IL-2Rs are expressed by T cells activated in response to foreign histocompatibility Ags but not by normal cells. This difference in IL-2R expression is exploited by blockade of IL-2Rs to achieve immunosuppression. High affinity IL-2Rs involve three subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma. Murine Mik beta 1, a mAb that blocks IL-2 binding to IL-2R beta, was developed as an immunosuppressive agent. There was modest prolongation of cynomolgus cardiac allograft survival in animals treated with murine Mik beta 1 (mean survival 11.8 +/- 1.6 days compared with 8.2 +/- 0.4 days in untreated animals; p = 0.06). However, murine Mik beta 1 is ineffective in recruiting primate effector cells and is neutralized by monkey Abs directed toward the infused Ab. To circumvent these limitations, a humanized form of Mik beta 1, which is a largely human IgG1k Ab, except that murine hypervariable regions are retained, was developed. In vivo plasma survival of humanized Mik beta 1 was threefold longer than simultaneously administered murine Mik beta 1 (terminal t1/2, 104 +/- 10 h vs 37 +/- 2 h). Furthermore, humanized Mik beta 1 manifests Ab-dependent cellular cytotoxicity, an activity that is absent with the parental murine Mik beta 1. Graft survival was significantly prolonged by humanized Mik beta 1 treatment with survivals of 22, 22, 24, 27, 44, and > 300 days (p vs control < 0.01; p vs murine Mik beta 1 < 0.01). Survival was not prolonged further (p > 0.3) by the addition of humanized anti-Tac, which blocks interaction of IL-2 with IL-2R alpha subunits. There was no toxicity attributable to the use of Mik beta 1 Abs. Thus, humanized Mik beta 1 prolonged cardiac allograft survival in primates without toxicity and may be effective as an adjunct to standard immunosuppressive therapy.

Cytokine is a generic term of biologically active molecules which are mainly produced by the immune-competent cells and regulate the immune response, inflammation and hematopoiesis. This includes interleukins (IL), colony-stimulating factors (CSF), interferons (IFN), tumor necrosis factors (TNF) and so on. These cytokines are glycoproteins with a molecular weight of 20,000-40,000 kD and work at very low concentrations of pM order. ILs and CSFs transduce their signal via specific cell-membrane receptors which usually consist of at least two subunits and belong to a newly identified superfamily of cytokine receptors. Characterization of cytokine/receptor system has had a considerable impact on many clinical fields including pathophysiology of diseases and therapy. For example, IL-4 and IL-5 has been revealed to play essential roles in IgE production in allergic diseases and eosinophilia in a hypereosinophilic syndrome, respectively. Receptor abnormality has also been proven to cause diseases; patients for X-linked severe combined immunodeficiency (X-SCID) have a specific defect in the gamma chain of the IL-2 receptor which is critical for thymic maturation of T cells. EPO, G-CSF, M-CSF, IFN, and IL-2 are already commercially available for therapeutic use. IL-1, IL-3, IL-6, and TNF may also be useful for mycosis fungoides, aplastic anemia, thrombocytopenia, and malignant melanoma, respectively. On the other hand, it is possible to modulate the immune response by using the monoclonal antibody directed to the cytokine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the expression of interleukin‐2 receptors (IL‐2R) in 60 adult patients with mature lymphoid neoplasms by flow cytometric analysis, using two monoclonal antibodies, anti‐Tac for IL‐2R α‐chain (IL‐2Rα) and Mik‐β for IL‐2R β‐chain (IL‐2Rβ). Among B‐cell malignancies, IL‐2Rα was found in 13/25 (52%) cases of chronic lymphocytic leukemia (CLL) and its variants, 3/14 (21%) of a heterogenous group of non‐Hodgkin's lymphoma (NHL) and none of the plasma cell diseases. IL‐2Rβ was not observed in any of B‐cell neoplasms. IL‐2Rα was more frequently expressed in CD11 b(+) B‐cell neoplasms than in CD11b(‐) ( P < 0.05). In T‐cell disorders, all three cases of adult T‐cell leukemia/lymphoma expressed IL‐2Rα but not IL‐2Rβ. IL‐2Rβ was detected in 3/8 cases of CLL and 2/3 of NHL and none of these cases expressed IL‐2Rα. CD8(+) malignant T‐cells commonly displayed IL‐2Rβ. These data indicate that the IL‐2Rα and IL‐2Rβ in mature lymphoid neoplasms was expressed independently each other and was associated with the particular phenotypical characteristics of neoplastic cells, respectively. © 1994 Wiley‐Liss, Inc.

Mikbeta1 is a mouse mAb directed at the beta-subunit of the human IL-2R (Tac) that inhibits IL-2 binding and inhibits IL-2 induction of large granular lymphocytes (LGL). Mikbeta1 alone does not inhibit IL-2-induced T-cell proliferation, but acts synergistically with anti-Tac to inhibit IL-2-induced proliferation of activated T cells. To evaluate these effects for possible therapy in humans, we constructed two humanized Mikbeta1 antibodies by combining the complementarity-determining regions of the murine antibody with human framework and constant regions. Compared with murine Mikbeta1, the two humanized Mikbeta1 antibodies, which differ in their degree of humanization, had similar affinities for IL-2Rbeta. The murine Mikbeta1 and one of the humanized Mikbeta1 antibodies were equivalent in competing for IL-2 binding to IL-2Rbeta and inhibiting IL-2 induction of LGL cytotoxicity. The activity of the second humanized antibody was significantly reduced. The three Mikbeta1 antibodies act synergistically to varying degrees with humanized anti-Tac to prevent IL-2-induced proliferation of activated T cells. This capacity to synergize paralleled their abilities to inhibit IL-2 binding. In addition, both humanized antibodies directed antibody-dependent cell-mediated cytotoxicity. We hope that humanized Mikbeta1 in combination with humanized anti-Tac will provide a new immunosuppressive therapy for the treatment of autoimmune diseases, graft-versus-host disease, and prevention of allograft rejection.

Rabbits can be infected with human retroviruses such as human T-cell leukemia virus-1 (HTLV-1) and human immunodeficiency virus (HIV), and provide useful animal models to study retroviral diseases such as adult T-cell leukemia and HIV. Previously we have succeeded in generating monoclonal antibodies (mAbs) against rabbit CD4, CD5 and CD11a antigens. To make this animal species more amenable to cellular and molecular studies, we have attempted to extend the panel of mAbs against rabbit CD antigens. Here we report on the generation of three neutralizing mAbs against interleukin-2 receptor alpha chain (IL-2R alpha) (CD25), Kei-alpha 1 (IgG2b), Kei-alpha 2 (IgG2a) and Kei-alpha 3 (IgG1). They specifically recognize the rabbit Mr 55,000 IL-2 binding protein, IL-2R alpha, and completely inhibit both high- and low-affinity IL-2 binding to F648b cells that express IL-2R alpha as well as IL-2R beta. The use of mAb Kei-alpha 1 confirmed that the rabbit IL-2R alpha is not only a low-affinity IL-2R on its own but also an essential component of high-affinity IL-2R as found in other animal species, and that rabbit activated T cells including HTLV-1-transformed cell lines express high levels of the IL-2R alpha. Together with mAbs against various rabbit CD antigens that we reported previously, these neutralizing mAbs to IL-2R alpha will be valuable for studies of human retrovirus infections, such as those induced by HTLV-1 or HIV, in rabbits.

Functional studies of the interleukin 2 receptor (IL-2R) of two (ED515- D and Kit225) IL-2-dependent and three (ED515-I, 3T3-alpha beta 11, and Hut102) IL-2-independent cell lines were done. All of these cell lines appeared to express high as well as low affinity IL-2R. However, ED515- I and 3T3-alpha beta 11, which expressed the IL-2R beta chain, did not bind IL-2 at all when IL-2 binding to their IL-2R alpha chain was blocked with anti-Tac monoclonal antibody, whereas the intermediate affinity binding in ED515-D, Kit225, and Hut102 cells remained. We tentatively called the high affinity IL-2R of the former cells pseudo- high affinity IL-2R. The dissociation constant of pseudo-high affinity IL-2R was higher than that of ordinary high affinity IL-2R. Internalization of cell-bound 125I-IL-2 into ED515-I and 3T3-alpha beta 11 cells was less efficient than that into ED515-D cells. The addition of IL-2 neither promoted cell growth nor upregulated IL-2R alpha chain expression in ED515-I and 3T3-alpha beta 11 cells. Furthermore, tyrosine phosphorylation of the cellular proteins (p120, p98, p96, p54, and p38) was induced or enhanced in response to the addition of IL-2 in ED515-D and Kit225 cells, but not in the cell lines expressing pseudo- high affinity IL-2R. Finally, 125I-IL-2 crosslinking followed by SDS- PAGE analysis showed an 80-kD band corresponding to p65 + IL-2, in addition to bands corresponding to IL-2R alpha and beta chain + IL-2 in cells bearing ordinary high affinity IL-2R but not in cells with pseudo- high affinity IL-2R. Taken together, we consider that another protein whose molecular mass is approximately 65 kD is functionally important in IL-2 binding and subsequent signal transduction and may be the third component of IL-2R.