Howard M. Gebel

Multidrug resistance activity in human lymphocytes

The multidrug resistance gene (mdr1) is a member of the recently described ATP binding cassette (ABC) superfamily of transporters. Family members include: (1) the cystic fibrosis transmembrane conductance regulator gene; (2) the hlyB gene of bacteria, and (3) the histocompatibility antigen modifier (HAM) gene. The level of expression of mdr1 correlates with multidrug resistance (MDR), the ability of cells to efflux otherwise toxic doses of several chemotherapeutic agents. MDR activity is also associated with the efflux of cationic lipophilic compounds such as the fluorescent dye rhodamine 123. Recently it was reported that normal lymphocytes efflux rhodamine 123, suggesting that these cells possess MDR-like activity due to the expression of mdr1. In this study, using two-color flow cytometric analysis, we observed that the ability to efflux rhodamine 123 was heterogeneous among human lymphocyte subsets in the order of CD8 greater than CD4 greater than CD2O. Rhodamine 123 efflux and accumulation in lymphocytes was sensitive to the known MDR reversing agents, verapamil and Solutol HS 15. Collectively, these data suggest that an MDR-like transport system is present in normal lymphocytes and may be important for trafficking of molecules involved in lymphocyte function.

Diverse multidrug-resistance-modification agents inhibit cytolytic activity of natural killer cells

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells selected for resistance to one chemotherapeutic agent simultaneously acquire resistance to several apparently unrelated drugs. MDR in tumor cells is associated with the over-expression of P-glycoprotein, an ATP-dependent cell-membrane transport molecule. P-glycoprotein is also expressed in several normal tissues but its physiological role(s) is unknown. We recently observed that a hierarchy of MDR-like activity exists among human peripheral blood lymphocytes in the order CD8>CD4>CD20 (cytoxic/suppressor T cells, helper T cells and B cells respectively). In this study, we report that natural killer (NK) cells also express MDR-like activity. This activity could be inhibited with verapamil or solutol HS-15, two agents that reverse MDR in tumor cells. These, and four additional reversing agents, were used to investigate the possible role of P-glycoprotein in NK cells. We observed that at 10% of their IC50, five of six reversing agents inhibited NK-cell-mediated cytotoxicity; at higher (but non-toxic) doses, all six agents were inhibitory. These data suggest that NK-cell-mediated cytotoxicity may require the functional expression of an efflux molecule similar or identical to P-glycoprotein.

T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor α, β, or γ genes

To test the hypothesis that the T-cell receptor (Tcr) λ gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16+ lymphocytosis were analyzed for rearrangements and expression of the human Tcr α, β, and λ genes. Two of the clones displayed distinct rearrangements of their Tcr β and λ genes and expressed mature Tcr α, β, and αl RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr λ gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr α and β gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.

IL-4 inhibits P-glycoprotein in normal and malignant NK cells

Patients presenting with a natural killer (NK) cell leukemia generally have a poor prognosis. NK cell tumors are generally resistant to numerous chemotherapeutic drugs and even combination chemotherapy usually results in only short term remissions. The drug resistance of NK cell leukemias may be at least partially explained by their expression of the multidrug resistant transporter, P-glycoprotein (Pgp). In this study, we demonstrate that the expression and function of Pgp activity on NK cells (leukemic and normal) can be reversed with IL-4.

Differential surface marker expression in patients with Cd-16+ lymphoproliferative disorders: In vivo model for NK differentiation

In this study, we report on three patients, each with a CD-16+ lymphoproliferative disorder. Peripheral blood lymphocyte from all three patients were evaluated for lymphocyte morphology, natural killer (NK) function, and surface marker expression. In addition, two-color flow cytometric analysis was performed to determine the phenotype of the CD-16+ cells. Our findings indicate that the presence of increased numbers of CD-16+ cells alone is not a good predictor of NK activity. However, we observed a differential expression of the HLA class II molecules DR and DQ on the CD-16+ cells obtained from these patients that was associated with NK function. Hence, a CD-16+, Leu-7-, Leu-19+ (NKH-1A) and HLA class II+ phenotype did correlate with NK function in contrast to a CD-16+, Leu-7+, Leu-19- (NKH-1A) and HLA class II- phenotype. Of importance was the fact that the CD-16+, HLA class II+ cells did not express CD-25 or TFR, nor did they mediate cytotoxicity against solid tumor targets, suggesting that these CD-16+ cells are not activated. Thus, in contrast to previous studies of NK ontogeny that utilized in vitro activated NK cells, studies of patients with CD-16+ lymphoproliferative disorders may provide an alternative approach for examining NK differentiation.

Differential sensitivity of resting and IL-2 activated NK cells to R-verapamil

Natural killer (NK) cells are the first lymphoid population to reconstitute the peripheral blood compartment of immunologically compromised bone marrow transplant (BMT) recipients. Recent data suggest that, among patients transplanted for leukemia, NK cells can prevent or delay disease relapse by mediating a cytotoxic graft vs leukemia (GvL) response. Although the major mechanism by which NK cells mediate target cell lysis involves degranulation and release of cytolytic effector molecules (granzymes, proteoglycans, perforin), accumulating evidence suggests that NK cells possess additional pathways to mediate target cell killing. In fact, it is well recognized that recombinant cytokines such as IL-2 enhance the in vitro cytolytic activity of NK cells. In this study, we observed that the lytic activity mediated by resting and IL-2 activated NK cells against the same target cell appears to occur via two distinct pathways, as distinguished by their differential response to R-verapamil. Specifically, we observed that 25 microM R-verapamil inhibited the lytic activity of resting NK cells against K562 targets by approximately 50%. However, the lytic activity of IL-2 activated NK cells was unaffected by this concentration of R-verapamil. Additional studies suggested that the inhibitory effect of R-verapamil on NK cytotoxic activity was associated with its ability to prevent degranulation of cytotoxic granules. Specifically, R-verapamil inhibited BLT esterase release from resting but not IL-2 activated NK cells. These data suggest that IL-2 activated NK cells can promote target cell lysis by a pathway (possibly degranulation independent) distinct from that used by resting NK cells. We speculate that the target of R-verapamil on resting NK cells is P-glycoprotein (Pgp), an ABC transporter that we recently reported was expressed on NK cells and whose functional activity is known to be inhibited by R-verapamil.

Ultraviolet-B irradiation of leukapheresis products: Dose-response relationship with the mixed lymphocyte reaction

Ultraviolet‐B (UVB) irradiation of blood constituents intensifies their anti‐rejection effect in pretransplant donor‐specific transfusions. UVB‐induced inhibition of the mixed lymphocyte reaction (MLR) between UVB‐irradiated donor cells and prospective recipient cells is a predicator of this anti‐rejection effect. In order to define the dose‐response relationship between the incident UVB irradiation on leukocyte concentrates and subsequent inhibition of their MLR responses, we collected 4 ± 2 × 109 leukocytes (93 ± 7% lymphocytes) in 200 ml plasma from each of three volunteers by leukapheresis and exposed them to rapid, serial doses of UVB irradiation which was delivered by a blood product irradiator (4R4440 UVB Irradiator, Baxter, Inc) with aliquots removed between doses. Lymphocytes from each aliquot were placed in MLR with panel donors and studied in three groups: 1) the panel donor cells were γ‐irradiated (1,500 rads) (i.e., only the UVB‐irradiated cells could proliferate), 2) the UVB‐irradiated cells were γ‐irradiated (i.e., only the panel lymphocytes could proliferate), and 3) no γ‐irradiation (i.e., both cell populations could proliferate). Each group had a similar UVB dose‐related diminution in the MLR (p = .79, ANOVA). A single dose of 6 J/cm2 extinguished the MLR to baseline in all groups. This dose should theoretically prevent transfused cells from producing either graft‐versus‐host disease or allosensitization, and might heighten their tolerogenic effect. This dose will be employed in our study of donor‐specific leukocyte transfusion in clinical renal transplantation.

Association of Allograft Rejection and Inducible Interleukin-2 Receptor Expression on CD8 Lymphocytes

Acute rejection of the transplanted kidney is a leading cause of graft failure in renal transplant recipients. Early clinical diagnosis of rejection in these patients is difficult because other processes such as drug toxicity and infection can diminish graft function, elevate serum creatinine, and mimic rejection. Recently, we had begun testing a new peripheral blood assay for the diagnosis of rejection among solid organ allograft recipients. A significant increase in inducible interleukin-2 receptor (IL2R) expression on CD8 lymphocytes from hepatic allograft recipients was observed during periods of rejection versus quiescence (37±5 vs 9±2; p<0.001) documented by serial liver biopsies (1). In this study, we obtained peripheral blood from ten renal allograft recipients at least bi-weekly post-transplant to determine the predictive value of monitoring inducible IL2R on CD8 lymphocytes.