Regulation of B cell lymphomagenesis by a malignant Qal+ inducer T cell clone.

A series of Thy-1.2+ Ly-1+ Qa-1+ malignant T cell clones have been isolated from murine sarcoma virus-murine leukemia-Moloney (MSV-MuLV-M)-induced B cell lymphomas or from MSV-MuLV-M-infected B6 mice. These T cell clones enhance both antigen-independent and -dependent lymphocyte differentiation and function. They also induce the differentiation of granulocytes and erythrocytes in the stem cell compartment, a function that parallels the immunopathology of the disease in vivo. The malignant T cell appears to sustain B lymphoma growth in vivo by releasing a factor (BCGF) that promotes B cell proliferation.

Amplification of granulopoiesis by T cell subpopulations.

Regulation of granulopoiesis by activated splenic T lymphocyte subpopulations was investigated. The addition of syngeneic T cells activated by the mitogen concanavalin A or by mixed lymphocyte response (MLR) to normal bone marrow cultures stimulated granulopoiesis, while, in contrast, resting T cells did not. Functional analyses of Fluorescence Activated Cell Sorter (FACS) purified subpopulations of MLR activated T cells showed that both subsets enhanced granulocyte colony forming cells (CFUc) differentiation. Further studies using Ly 23+ T cells stimulated in vitro to suppress the primary generation of plaque forming cells (PFC) showed that these "suppressor cells" simultaneously enhanced CFUc differentiation. These results show that T cell "helper" function in hematopoietic regulation is not restricted to the inducer subset of T cells.

T-cell regulation of erythropoiesis during acute lymphoblastic leukemia.

How and where erythropoiesis is maintained during advanced leukemic disease is an important and, as yet, unresolved question in hematology. To address the potential role of T-lymphocytes as cells that regulate CFU-E differentiation during leukemogenesis, an experimental model of disease has been developed in inbred Balb/c mice. Specifically, three-week-old Balb/c By mice were injected with murine sarcoma virus-murine leukemia virus-Moloney (MSV-MuLV-M), which resulted 6-8 months later in the development of immunoblastic T-cell sarcomas with a leukemic phase. Splenic T cells from either normal or tumor-bearing mice were assessed for their relative ability to modulate erythroid differentiation. Quantitatively, T cells, Ly1 or Ly 2,3 T-cell subsets isolated from tumor-bearing animals significantly enhanced erythropoiesis when compared with comparable normal T-cell subsets. These data suggest that the compensatory shift of erythropoiesis from the bone marrow to the spleen observed during leukemogenesis was facilitated by splenic T cells. In this circumstance, the enhanced erythropoietic function may be mediated by splenic T cells, which are selectively activated by virus.

CD34+ selection of hematopoietic blood cell collections and autotransplantation in lymphoma: overnight storage of cells at 4 degrees C does not affect outcome.

The purpose of this study was to investigate whether storing mobilized peripheral blood progenitor cell (PBPC) collections overnight before CD34+ selection may delay platelet count recovery after high-dose chemotherapy and CD34+-enriched PBPC re-infusion. Lymphoma patients underwent PBPC mobilization with cyclophosphamide 4 g/m2 i.v. and G-CSF 10 microg/kg/day subcutaneously. Patients were prospectively randomized to have each PBPC collection enriched for CD34+ cells with the CellPro CEPRATE SC System either immediately or after overnight storage at 4 degrees C. Thirty-four patients were randomized to overnight storage and 34 to immediate processing of PBPC; 15 were excluded from analysis due to tumor progression or inadequate CD34+ cell mobilization. PBPC from 23 patients were stored overnight, while 30 subjects underwent immediate CD34+ selection and cryopreservation. Median yield of CD34+ enrichment was 43.6% in the immediate processing group compared to 39.1% in the overnight storage group (P = 0.339). Neutrophil recovery >500 x 10(9)/l occurred a median of 11 days (range 9-16 days) in the overnight storage group compared to 10.5 days (range 9-21 days) in the immediate processing group (P = 0.421). Median day to platelet transfusion independence was 13 (range 7-43) days in the overnight storage group vs 13.5 (range 8-35) days in those assigned to immediate processing (P = 0.933). We conclude that storage of PBPC overnight at 4 degrees C allows pooling of consecutive-day collections resulting in decreased costs and processing time without compromising neutrophil and platelet engraftment after infusion of CD34+-selected progenitor cells. Bone Marrow Transplantation(2000) 25, 559-566.

The isolation and functional characterization of autoimmune clones expressing inappropriate Ia.

The MRL/lpr mouse is an inbred strain widely accepted as a model for autoimmune disease both in murine and human systems. Developed from a series of crosses involving four strains of mice, the MRL/lpr (H-2k) genome is a composite estimated to contain approximately 75% of its parental LG/J (H-2d) genome. To explore the cellular mechanism underlying lymphoproliferation in the MRL/lpr mouse, we have isolated a series of clones from the lymph nodes of MRL/lpr mice with autoimmune disease. Extensive immunofluorescent analyses of these clones, designated the PAC series, reveal expression of IAk and IEk (beta-chain) cell surface antigens, as well as inappropriate expression of IAd, IEd (beta-chain), and H-2d. PAC cells also express MAC-1, MAC-2, RA3-2C2, and RA3-6B2 and contain esterase-positive cytoplasmic granules. The capacity of PAC cells to present antigen was investigated by co-culturing PAC with IA-restricted, antigen-specific T cell hybridomas +/- antigen. These assays demonstrated the PAC inability to present antigen to IAk-restricted T cell hybridomas, as well as their capacity to present antigen to IAd-restricted T cell hybridomas. In addition, activation of MRL/lpr peritoneal macrophages using gamma-interferon resulted in increased fluorescent staining for IAd and IEd concomitant with decreased fluorescent staining for IAk. Based on these findings, we propose a model of lymphoproliferation in which Ly-1+, H-2K+ T cells proliferate to inappropriate d haplotype antigens expressed by a small subset of monocytes in the MRL/lpr lymph node. The major genomic contribution of the LG/J (H-2d) mouse may be in part responsible for inappropriate antigen expression either by age-dependent expansion of d haplotype cells or by age-regulated expression of Iad and H-2d genes.

Isolation of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes that lyse Reed-Sternberg cells: implications for immune-mediated therapy of EBV+ Hodgkin's disease.

A subset of Hodgkin's disease (HD) patients have detectable Epstein-Barr virus (EBV) genomes in the malignant Reed-Sternberg (R-S) cells. R-S cells express only a limited set of latent EBV proteins, but only LMP1 and LMP2 can potentially elicit a CD8+ cytotoxic T-lymphocyte (CTL) response. We have evaluated if either of these proteins could be used as targets for specific adoptive T-cell therapy for EBV-positive (EBV+) HD. The success of this strategy requires that R-S cells are susceptible to lysis by CD8+ CTL, and that CTL specific for LMP1 and LMP2 can be detected and potentially amplified in HD patients. Antigen presentation and CTL sensitivity was evaluated with an in vitro maintained, phenotypically representative R-S cell line, HDLM-2. The R-S cells were able to process and present viral proteins, and to be efficiently lysed by specific CTL in a Class I-restricted manner. Since CTL responses to LMP1 and LMP2 do not represent the dominant responses to EBV, we examined if CTL clones specific for these proteins could be isolated despite the presence of weak or nondetectable responses in polyclonal T-cell lines. LMP-specific clones were generated from individuals either by cloning from the polyclonal EBV-reactive T-cell lines or by direct stimulation of peripheral blood mononuclear cells (PBMC) with cells expressing LMP1 or LMP2 as the only EBV protein. Our ability to isolate CTL specific for LMP proteins from individuals with HD and the sensitivity of R-S cells for CTL-mediated lysis suggest that the pursuit of specific adoptive immunotherapy represents a viable strategy for the subset of HD patients with EBV+ tumors.