Vox Sang. 34: 1-7 (1978)

Human Granulocyte Antigens Detected on Leukemia Cells and a Chronic Myelogenous Cell Line’ S . I . Drew, R . Billing, 0 .J . Bergh and P . I . Terasaki Department of Surgery, School of Medicine, University of California, Los Angeles, Calif.

Abstract. Normal human granulocyte alloantigens were found on chronic myelogenous, acute myeloblastic leukemia cells, and a cell line of chronic myeloblastic origin (K562). Antigens were detected by human antisera positive for normal peripheral blood granulocytes but devoid of HLA activity. Very few acute lymphoblastic leukemia cells reacted positively, and none of the chronic lymphocytic leukemia cells seemed to bear granulocyte surface antigens. The recognition of these normal tissue isoantigens on myeloblastic leukemia cells is a necessary prerequisite for the identification of “ leukemia-specific” or “leukemia-associated” antigens.

The rapid increase in knowledge of cell surface antigens and receptors identifying different subpopulations of lymphocytes [17] has enabled a greater insight into the cellular origin and differentiation pathways of leukemic and lymphoid neoplasms. Whereas certain cell markers disclose similarities between differing leukemic classes, others have demonstrated cellular heterogeneity in morphologically indistinguishable leukemic proliferations. As examples, the majority of chronic lymphocytic leukemia (CLL) cells seem to possess cell surface 1 This work was supported in part by contract USPHS AM 02375 and A1 12366-02 from the National Institutes of Health.

characteristics similar to those of normal B (bursal-equivalent processed) lymphocytes [l,171; conversely, acute lymphoblastic (ALL) cells may be categorized by the presence or absence of T-cell (thymus-derived) surface receptors [5]. Furthermore, recent studies demonstrating the occurrence of a B-lymphocyte antigen on a large percentage of acute myelogenous leukemia (AML), CLL, and ALL cells, have provided a more complete understanding into the ontogenetic derivation of these malignant cells [2,3,8, 211 * The presence of a non-HLA polymorphic system of antigens on normal human granulocytes [13,141 led us to investigate the distribution of these alloantigens on

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Drew/Billing/Bergh/Terasaki

human leukemia cells and their relative occurrence in the different morphological leukemia classes.

these antibodies. 15 (21%) of the sera were found to contain B-cell antibodies. This enabled a division of the sera panel into two major groups: group A sera which were granulocytotoxic alone; and group B sera which were cytotoxic to both granulocytes and B lymphocytes.

Materials and Methods Cell Preparation Heparinized blood was drawn from leukemia patients in relapse and purified by Ficoll-Hypaque density sedimentation. Leukemia cells recovered from the interface were stored frozen at -196OC in a mixture comprising 20% dimethylsulfoxide and McCoy’s medium containing 0.5% fetal calf serum. Where possible, fresh cells were tested. The panel of 64 leukemia cells was comprised of 7 chronic myelogenous leukemia (CML), 14 ALL, 7 CLL, and 36 AML cells. In addition, one cell line, K562, of chronic myelogenous blast cell origin [6] (kindly supplied by Dr. 1.Minowada) was tested. The viability of all blast cells was satisfactory. Normal granulocytes were obtained from heparinized blood as previously described [ll] and tested in a microgranulocytotoxicity assay. Antisera Selection 72 human alloantisera, known to be granulocytotoxic positive by previous testing against a panel of 70 normal random granulocyte donors, were selected for testing. The sera were obtained from donors alloimmunized by previous pregnancies or blood transfusions. All sera were screened for the presence of HLA antibodies against a panel of lymphocytes representing the majority of first and second locus HLA antigens (HLA-1, 2, 3, 9, 10, 11, 5, 7, 8, 12, 13, W28, W29, W23, W24, W25, W26, W30, W32, W14, W18, W27, W15, W16, W17, W21, W22, W5) and found to be negative on an extended 3-hour incubation period performed at 23 O C . The presence of B-lymphocyte alloantibodies [22] in the granulocytotoxic sera was detected under similar assay conditions by reacting the sera against Ibenriched lymphocyte [2] populations from 10 unrelated healthy individuals. Furthermore, the AML, CLL, and ALL cells, that are known to carry B-lymphocyte alloantigens, served as additional controls for the presence of

Microcytotoxicity Testing The leukemia cells were tested against the selected sera in a 2-stage microcytotoxicity assay [16] with a 1.5-hour incubation period at 23 OC. Undiluted rabbit complement, absorbed at 0 OC for 1 h with human red blood cells at a 1O:l ratio, was used. A positive score was given to reactions showing greater than 50% cytotoxicity. Each cell was retested on two independent occasions, and excellent reproducibility was observed. Absorption of Antiserum Pooled normal granulocytes from 13 donors were used to absorb a granulocyte positive group A serum (E7689), at a 1:2 packed cell to serum ratio. The serum was absorbed at 37 OC for 1.5 h on a rocking platform, and retested in dilutions against the leukemia cells, K562, and normal granulocytes known to react positively with the same unabsorbed serum. Granulocyte Antigenic Specificities In a separate study, 499 alloimmune granulocytotoxic sera were tested against a panel of 70 normal random granulocyte donors in an improved microgranulocytotoxic assay [unpublished], and a total of six allelic antigen groups were defined. Where possible, sera of the present study were assigned one appropriate granulocyte antigen group, by reacting them in parallel with sera of known granulocyte antigen specificities against a panel of 70 random granulocyte donors. Sera having granulocyte antigen specificities similar to those previously determined were identified with the aid of Mickey’s Boolean regression computer analysis [lo]. In addition, the frequency of reactions for each of the 72 sera was determined against the same panel of 70 granulocyte donors. Statistical Analyses Unless otherwise indicated, differences in the frequency of serological reactions among the different leukemia classes were analyzed serum for serum using the sign test.

Granulocyte and 8-lymphocyte posltive sera (Group 8 )

Granulocyte positive sera (Group A)

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T Fig. 2. Percent positive reactions occurring in serum panel for each leukemia class and K562.

Fig. 3. Differences in percent cytotoxicity of granulocytotoxic group A sera for CML ( 0 )or normal granulocytes (0).

ure 1 is reduced to graph form in figure 2 where the percent reactivity (expressed as the percent positive reactions occurring in the serum panel for each of the leukemia in a loss of cytotoxicity for both the CML types and KS62) is plotted for both groups and AML cells, as well as the K562 (table I) of sera. In the group A granulocyte positive cell line. Of the 57 group A sera, 18 (32%) sera, the percent reactivity of the CML cells could be assigned one of the six granulocyte was significantly higher than that of all antigen specificities. No clear-cut pattern other types of leukemia cells (p < 0.001). reflecting either an increase or decrease of Similarly, Ah4L cells also reacted more any individual granulocyte antigen specificommonly than ALL and CLL cells (p < city was discernible on the leukemia cells. Also, where more than one serum defined 0.001). Although the difference in reactivity a single granulocyte antigen group, leukeamong the various leukemia type cells on mia cells did not react uniformly with all exposure to group B antisera was not signif- the sera of that group. icant, the CML cells were lysed more frequently than AML cells (p < 0.06) (fig. Discussion 2). A comparison of the frequency of reactiLeukemia cells exhibit a variety of cell vity of group A sera for normal granulocytes or CML cells showed that 3/57 (5.3%) of surface antigens, of which normal tissue the sera reacted more frequently with granu- antigens, differentiation, embryonic and leulocytes (p

Human granulocyte antigens detected on leukemia cells and a chronic myelogenous cell line.

Vox Sang. 34: 1-7 (1978) Human Granulocyte Antigens Detected on Leukemia Cells and a Chronic Myelogenous Cell Line’ S . I . Drew, R . Billing, 0 .J ...
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