Christine Cameron

Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin 2.

The immunologic consequences of prolonged infusions of rIL-2 in doses that produce physiologic serum concentrations of this cytokine were investigated. rIL-2 in doses of 0.5-6.0 x 10(6) U/m2 per d (3.3-40 micrograms/m2 per d) was administered by continuous intravenous infusion for 90 consecutive days to patients with advanced cancer. IL-2 concentrations (25 +/- 25 and 77 +/- 64 pM, respectively) that selectively saturate high-affinity IL-2 receptors (IL-2R) were achieved in the serum of patients receiving rIL-2 infusions of 10 micrograms/m2 per d and 30 micrograms/m2 per d. A gradual, progressive expansion of natural killer (NK) cells was seen in the peripheral blood of these patients with no evidence of a plateau effect during the 3 mo of therapy. A preferential expansion of CD56bright NK cells was consistently evident. NK cytotoxicity against tumor targets was only slightly enhanced at these dose levels. However, brief incubation of these expanded NK cells with IL-2 in vitro induced potent lysis of NK-sensitive, NK-resistant, and antibody-coated targets. Infusions of rIL-2 at 40 micrograms/m2 per d produced serum IL-2 levels (345 +/- 381 pM) sufficient to engage intermediate affinity IL-2R p75, which is constitutively expressed by human NK cells. This did not result in greater NK cell expansion compared to the lower dose levels, but did produce in vivo activation of NK cytotoxicity, as evidenced by lysis of NK-resistant targets. There was no consistent change in the numbers of CD56- CD3+ T cells, CD56+ CD3+ MHC-unrestricted T cells, or B cells during infusions of rIL-2 at any of the dosages used. This study demonstrates that prolonged infusions of rIL-2 in doses that saturate only high affinity IL-2R can selectively expand human NK cells for an extended period of time with only minimal toxicity. Further activation of NK cytolytic activity can also be achieved in vivo, but it requires concentrations of IL-2 that bind intermediate affinity IL-2R p75. Clinical trials are underway attempting to exploit the differing effects of various concentrations of IL-2 on human NK cells in vivo.

Extended continuous infusion low-dose recombinant interleukin-2 in advanced cancer: prolonged immunomodulation without significant toxicity.

In previous clinical trials, recombinant interleukin-2 (rIL-2) has been infused at high doses over short periods of time to generate lymphokine-activated killer (LAK) cells in vivo. These trials have been limited by severe toxicities, and the immunologic effects of rIL-2 have been transient. The present study was designed to assess the toxicity and immunologic effects of prolonged administration of low doses of rIL-2. In this phase I study, patients with advanced cancer were scheduled to receive intravenous (IV) infusion of rIL-2 without interruption for 3 months in an outpatient setting. Twenty-one patients received rIL-2 at doses ranging from 0.5 x 10(5) to 6.0 x 10(5) U/m2/d. Treatment was extremely well tolerated, and no patient experienced grade 3 or grade 4 toxicity. The lowest dose level (0.5 x 10(5) U/m2/d) did not have demonstrable immunologic activity. At doses of 1.5 x 10(5) and 4.5 x 10(5) U/m2/d, rIL-2 infusion resulted in the specific expansion of natural-killer (NK) cells (sixfold and ninefold increases, respectively, at these two dose levels) without any changes in B cells, T cells, neutrophils, or monocytes. Grade 2 toxicity was observed at the dose of 6.0 x 10(5) U/m2/d, as three patients required interruption of therapy and two patients who completed therapy developed transient hypothyroidism. In patients with increased NK cells, enhancement of non-major histocompatibility complex (MHC)-restricted cytotoxicity and increased generation of LAK cells in vitro were also demonstrated. Therapy with low-dose rIL-2 can be given safely in an uninterrupted fashion for prolonged periods of time in an outpatient setting. This results in selective expansion of NK cells in vivo with minimal toxicity. Further investigation of this schedule for immunomodulation in vivo should be pursued in phase II studies of both malignant and immunodeficient disease states.

Functional Consequences of APO-1/Fas (CD95) Antigen Expression by Normal and Neoplastic Hematopoietic Cells

Murine monoclonal antibody (mAb) 7C11 binds to the same cell surface epitope as anti-APO-1 and anti-Fas and reacts specifically with cells transfected with a cDNA encoding the human Fas antigen. Furthermore, incubation with 7C11 causes death of hematopoietic cell lines that express APO-1/Fas but not APO-1/Fas-negative cell lines. 7C11 therefore recognizes the human APO-1/Fas (CD95) antigen, a 40 to 50 kDa cell surface glycoprotein that can trigger apoptosis or programmed cell death. Expression of APO-1/Fas antigen by normal and neoplastic hematopoietic cells was determined by flow cytometry using 7C11. APO-1/Fas is expressed by approximately 30 to 40% of resting peripheral blood T cells, B cells, and monocytes and by approximately 5% of resting NK cells and thymocytes. It was not detected on granulocytes, erythrocytes, or platelets. Approximately 80 to 90% of activated T cells, B cells, and thymocytes express APO-1/Fas, as do the majority of activated NK cells. Perturbation of APO-1/Fas by 7C11 does not affect the viability of resting lymphocytes or monocytes. In contrast, activated T cells and NK cells undergo apoptosis within 3 hours of exposure to 7C11. Other mAb that stimulate T cells or NK cells do not cause rapid induction of programmed cell death. APO-1/Fas antigen is expressed by many cell lines of lymphoid and myeloid lineage. However, this antigen was detected on neoplastic cells from only one of 69 patients with acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, or multiple myeloma. Only 3 out of 25 tumor samples from patients with non-Hodgkins lymphoma were found to express APO-1/Fas. All three of these lymphomas harbored the bcl-2-Ig fusion gene associated with the chromosomal translocation t (14;18). Conversely, only 27% of lymphomas that possessed the bcl-2-Ig gene were found to express the APO-1/Fas antigen. Like normal activated lymphocytes, leukemia and lymphoma cells that expressed APO-1/Fas antigen were found to undergo apoptosis in vitro after incubation with 7C11. The APO-1/Fas antigen appears to regulate the growth of normal hematopoietic cells, and the marked upregulation of this antigen on activated normal lymphocytes contrasts sharply with the absence of APO-1/Fas on neoplastic cells of hematopoietic lineage. Defects in the apoptotic signal delivered through this antigen might contribute to the pathogenesis of hematopoietic neoplasms. Thus, the gene encoding APO-1/Fas can be considered a novel type of tumor suppressor gene, just as bcl-2 can be considered a cellular proto-oncogene.

Defects in HLA class II antigen presentation in B-cell lymphomas

Burkitt Lymphoma (BL) is a high grade B-cell malignancy occurring most frequently in children in areas with holoand hyper-endemic malaria and with lesser frequency in all other parts of the world [1,2]. This neoplasm is one of the fastest growing malignancies in humans and is one of the nonHodgkin lymphomas (NHL) [1 – 3]. Non-Hodgkin Follicular Lymphoma (FL) is also a common type of B-cell lymphoma, comprising approximately 30% of all cases. Transformation of low grade non-Hodgkin lymphomas to high-grade diffuse large B-cell lymphomas are also found in a significant number of patients with B-cell malignancies [4]. Studies have long focused on eliciting tumor-specific CD8þ cytotoxic T-cells (CTL) because of their potent antitumor activity [5]. The resulting treatments, however, have been surprisingly poor at inducing complete tumor rejection. The existence of direct antitumor, effector cytotoxic CD4þ T-cells also remains elusive, although a few studies have shown that CD4þ CTL can directly kill tumor cells [6,7]. By contrast, it is widely believed that helper, HLA class II-restricted non-cytotoxic CD4þ T-cells are important for the induction and maintenance of antitumor immunity exerted by cytotoxic CD8þ CTL. Because the majority of B-cell tumors express HLA class II molecules, these tumors could be potential targets for CD4þ T-cells. Our current study suggests that there is a severe defect in HLA class II antigen (Ag) presentation in B-cell lymphomas. In this letter, we will also show that BL-as well as FL-associated molecules alter the HLA class II pathway for Ag presentation diminishing CD4þ T cell recognition. To examine whether B-cell lymphomas functionally present antigenic peptides to CD4þ T-cells, we analyzed four primary B-cell tumors from B-lymphoma patients. Briefly, lymph node cells and blood samples were obtained from one BL (TB2952) and three FL (TB2759, TB2767 and TB2769) patients through our Hollings Cancer Center Tissue Bank (Medical University of South Carolina, Charleston, USA). TB2952 cells are Burkitt-like lymphoma/ leukemia (EBV status unknown) and positive for CD33, CD10, CD19, CD20 and CD38 as determined by flow cytometric analysis. TB2759 cells are follicular lymphoma/leukemia and positive for CD10, CD19, CD20 and CD52. TB2767 cells are grade 2 FL that expressed CD10, CD19, CD20, CD38, CD45 and CD52. TB2769 cells are grade 3 FL and positive for CD10, CD19, CD20, CD23, CD25, CD38, CD45 and CD52. Blood samples were also obtained from healthy individuals with written consent. Healthy control B-cells and malignant primary tumors (TB2759, TB2767, TB2769 and TB2952) were subjected to western blotting to determine HLA class II protein levels. Western blot analysis showed that these primary B-cells as well as B-cell tumors expressed HLA-DR molecules. To study class II-restricted CD4þ T cell recognition, we transfected these cells with a distinct class II allele HLA-DR4 as described previously [8]. TB2759, TB2767, TB2769 and TB2952 cells expressed measurable surface DR4 molecules as determined