0022-1767/87/13810-3385802.00/0 THE JOURNAL OF hUUNOLOCY apyr@ht @ 1987 by The Amerlcan Association of Immunologists Vol. 138.3385-3391. No. 10. May 15.1987 Prlnted In U.S.A. A COMPLEMENT-RESISTANT HeLa CELL LINE (T638) IS BLOCKED AT THE STEP OF C3 DEPOSITION1 A complement-resistant line of HeLa cells (T638) was derived by serial passage of complement-sus- ceptible HeLa cells in anti-&microglobulin (b2m) antiserum and complement. The T638 line main- tained stable complement resistance when passed for an additional 1500 generations in the absence of antiserum and complement. T638 cells expressed equivalent levels of cell-associated b2m as did the parent HeLa cell line. Furthermore, T638 cells were resistant to killing by complement and anti-HeLa antiserum with specificity for molecules other than b2m. These results indicate that the resistance of T638 cells does not simply reflect loss of anti-b2m binding antigens. We next investigated the mecha- nism of resistance of T638 cells to complement- mediated killing. Antibody-sensitized HeLa and T638 cells both consumed CHso activity completely from normal human sew cytotoxicity was not mediated via the alternative complement pathway. HeLa and T638 cells caused equivalent utilization of C4 from normal human serum in the presence of antibody. Consumption of C2, greater with T638 than with HeLa cells during incubation in serum, was complete when cells bearing purified C1 and limited C4 were incubated with C2. T638 cellsbound more 'H-C4 than HeLa cells during incubation in serum, but binding of 3H-C3 by T638 cells was four- fold to fivefold less than by HeLa cells. Finally, we investigated the rate of decay in the capacity of C142 on HeLa and T638 to cleave and deposit 'H- C3. The TV2 for decay of Cl42-mediated binding of 3H-C3 onHeLa was 3.9 min. whereas minimal C3 deposition was detected on T638 cells at all time points. These results show that T638 cells evade complement-mediated lysis despite activating early components of the classical complement pathway. The mechanism of resistance is a failure to form an effective C3convertase. Resistance to complement attack is an attribute of selected procaryotic and eukaryotic cells. Conceptually, Received for publicatlon August 22. 1986. Accepted for publication January 30. 1987. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement In accordance with 18 U.S.C. Section 1734 solely to Indi- cate this fact. slglio Nazlonale delle Rlcerche. ' This work was supported by Progetto Finalizzato Oncologla del Con- Cllnical Investigation. National Institute of Allegr and Infectious Ms- a To whom correspondence should be addressed at the Laboratory of eases. Natlonal Institutes of Health. Ekthesda, MD 20892. complement resistance can be due to factors affecting the formation of lytic channels, or to factors affecting the efficiency of the channels to lyse the relevant target. In fact, experimentalevidence has accumulated support- ing each of these mechanisms. Cells may activate com- plement inefficiently, in both the presence and the ab- sence of antibody. For example, alterations in the density of membrane antigensmay drastically alter the capacity of bound IgG to activate C1 (1,2).Once activated, control may be exerted on completion of the activation sequence by membrane factors acting at the step of C3 and C5 convertase formation (3-6) or at the steps of C8 and C9 binding (7-9). Both of these mechanisms function most efficiently to control complement activation and lysis in homologous systems, Le., those circumstances in which the species of the complement proteins and the target cells are matched. Finally, factors affecting membrane lipid composition and hormonal regulation of lipid syn- thesis can alter the capacity of a formed C5b-9 complex to effectively insert into the membraneto create a lytic channel (10, 11). Once the latter step has occurred, nu- cleated cells have devised several mechanisms to circum- vent the otherwise lethal effects of the complement chan- nel. C5b-9 may be endocytosed by a calcium-dependent process (1 2,13). The complement channel can be actively extruded from neutrophils on membrane blebs (14). Fi- nally, platelets bearing C5b-9 demonstrate increased ac- tivity of the Na+-K+ pump, a process that repolarizes the membrane during sublethal complement attack (15). We studied the phenomenon of complement resistance in a line of HeLa cells that were rendered resistant to antibody-dependent complement lysis by serial passage in the presence of normally cytotoxic levels of anti-bz- microglobulin (b2m)3 and complement. Our results show that these cells are resistant to complement attack de- spite the capacity to bind normally cytotoxic antibodies to either b2m, HLA, or other antigens. These studies differ from those previously reported (1 0- 13, 16, 17) in that the resistant cell line was derived from the sensitive cell line by serial passage with immune antibody and complement: no enzyme or mutagen was used for deri- vation of the resistant line. Interestingly, in comparison to previous studies, a block in complement activation at the step of C3 convertase activity was noted in the re- sistant cell line. Abbreviations used in this paper: b2m. @a-micrcglobulin: NHS. normal human serum: ETA, ethyleneglycol-bis-(@-amlnoethylether)-N,N'-tetra- acetic acid. 3385