Activation signals leading to proliferation of normal and leukemic CD3+ large granular lymphocytes.

The activation signals leading to proliferation of normal and leukemic CD3+ large granular lymphocytes (LGL) were studied in vitro. Anti-CD3 monoclonal antibody (MoAb) alone (P less than .01) and recombinant interleukin-2 (IL-2) alone (P less than .01) caused significant stimulation of peripheral blood mononuclear cells (PBMC) from four CD3+ LGL leukemia patients, as measured in a 3H-thymidine incorporation assay. Recombinant interleukin-4 (IL-4) alone had no effect (P = .11). The combination signals of anti-CD3 MoAb and either IL-2 or IL-4 produced a proliferative response greater than anti-CD3 MoAb alone (P less than .01) or lymphokine alone (P less than .01). Leukemic LGL, purified by two-color sorting, were subsequently activated by anti-CD3 MoAb and IL-2 and assessed for DNA content by viable Hoechst No. 33342 (HO) staining. Results of these studies demonstrated that leukemic LGL were stimulated directly by anti-CD3 MoAb and IL-2, with the percentage of cells in cell cycle (S + G2/M) ranging from 16% to 72%. Normal CD3+ LGL were also stimulated to enter the cell cycle by anti-CD3 and IL-2. These results show that leukemic LGL proliferate in vitro after activation through the T-cell receptor and/or lymphokine.

Interleukin-2 receptor monoclonal antibodies have no effect on anti-CD3 monoclonal antibody-mediated cytotoxicity in CD3+ leukemic large granular lymphocytes.

We studied the role of Fc receptors and interleukin-2 (IL-2) receptor subunits in anti-CD3 monoclonal antibody (MAb)-mediated cytotoxicity of CD3+ leukemic large granular lymphocytes (LGL). Peripheral blood mononuclear cells from four patients with CD3+ LGL leukemia were cultured with 1 microgram/ml of anti-CD3 MAb. Anti-CD3 MAb-mediated cytotoxicity was not inhibited when K562 target cells were preincubated with heat-aggregated human IgG, suggesting that binding of the effector cell-bound anti-CD3 MAb to Fc receptors of target was not involved in cytotoxicity. Induction of cytotoxicity was not blocked by the addition of either anti-p55 or anti-p75 IL-2 receptor MAbs. These results show that the induction of cytotoxicity by anti-CD3 MAb is not mediated through IL-2 receptor subunits in CD3+ leukemic LGL.

Anti-CD3 monoclonal antibody-mediated cytotoxicity occurs through an interleukin-2-independent pathway in CD3+ large granular lymphocytes.

The mechanism of induction of cytotoxicity produced by anti-CD3 monoclonal antibody (MoAb) was studied in four patients with CD3+ large granular lymphocyte (LGL) leukemia. Anti-CD3 MoAb treatment resulted in increased target cell binding and increased granule formation. After activation, leukemic LGL remained Tac-, with the exception of a patient with CD4+ LGL leukemia. Radiolabeled interleukin-2 (IL-2) binding studies demonstrated that treatment with anti-CD3 MoAb resulted in upregulation of the number of p75 intermediate affinity IL-2 receptor sites per cell. Northern blot hybridization analysis showed expression of gamma-interferon gene transcripts 24 to 48 hours after activation. There was no evidence for expression of IL-2 messenger RNA or secretion of IL-2 after activation. Anti-CD3 MoAb and IL-2 provide different signals for activation of CD3+ LGL. Induction of cytotoxicity produced by anti-CD3 MoAb in leukemic CD3+ LGL is not associated with IL-2 production.

Rearrangement and expression of T-cell receptor genes in large granular lymphocyte leukemia.

Ten patients with large granular lymphocyte (LGL) leukemia were studied for rearrangement and expression of T cell receptor (TCR) genes. Eight patients with CD3+ LGL proliferation had unique TCR beta-gene rearrangements, supporting a clonal process. Each of five patients studied with CD3+ disease had evidence for expression of full-length TCR alpha-, beta-, and gamma-gene transcripts. In contrast, patients with CD3- LGL proliferation had no evidence for rearrangement or expression of TCR genes. These studies suggest that leukemic LGL arise from two different cell origins. Leukemic LGL may be a useful model for studying natural killer (NK) cell (CD3- LGL) and non-MHC-restricted cytotoxic T lymphocyte (CD3+ LGL) activation and differentiation.

Characterization of host cells involved in resistance to marrow grafts in dogs transplanted from unrelated DLA-nonidentical donors.

A canine model of marrow transplantation was used to further define the host cells mediating resistance to marrow engraftment. Recipient dogs were given 9.2 Gy of total body irradiation followed by marrow infusion from unrelated DLA-nonidentical donors. No postgrafting immunosuppression was given. At three and ten days posttransplantation recipient marrow and peripheral blood cells were obtained and characterized by the following in vitro studies: morphologic analysis; phenotypic analysis with monoclonal antibodies; assays for natural killer cell (NK) activity; and cocultures with donor marrow to study the effect on donor CFU-GM growth. Daily differential cell counts revealed a proliferation of peripheral blood mononuclear cells approximately eight days posttransplant. By day 10 surviving host cells were uniformly large granular lymphocytes which were phenotypically of T cell lineage, had NK activity, and were capable of suppressing donor marrow CFU-GM growth. Mononuclear cells from dogs given total body irradiation only and no marrow infusion (radiation control group), did not suppress CFU-GM growth when cocultured with marrow from unrelated DLA-mismatched dogs. These results suggest that radioresistant host cells with the morphology of large granular lymphocytes and NK activity and which proliferate in response to the infused donor marrow cells mediate resistance to DLA-nonidentical marrow grafts. It remains to be determined, however, whether in vitro functional studies reflect the mechanisms involved in vivo.

Purified canine monocytes fail to confer concanavalin-A responsiveness to accessory-cell-depleted lymphocytes.

Monocytes were purified from canine peripheral blood by subjecting Ficoll-Hypaque-separated mononuclear cells to either complement-dependent cytolytic treatment with a panlymphocyte antibody (DLy-6) or rosette formation with human red blood cells (RBCs) followed by adherence to fetal-calf-serum-coated plastic dishes, or sedimentation over discontinuous Percoll gradients. These techniques resulted in monocyte enrichment of 83 +/- 3%, 89 +/-6%, and 42 +/- 3%, respectively. Monocytes purified by either the monoclonal antibody method or rosette formation with human RBCs failed to confer concanavalin-A responsiveness to lymphocytes depleted of accessory cells by discontinuous Percoll gradients. The most potent accessory activity was present among cells from low-density fractions of Percoll gradients and populations that did not form rosettes with human RBCs (monocyte-depleted), indicating that cells other than monocytes are involved in accessory function.