Scand. J. Immunol. 26, 119-127 Autologous Red Blood Cells Potentiate Antibody Synthesis by Unfractionated Human Mononuclear Cell Cultures M. T. RUGELES. M. LA VIA. J.-M. GOUST, J. M. KILPATRICK, B. HYMAN & G. VIRELLA Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina. USA Rugcles. M.T.. La Via. M., Gousi. J.-M.. Kilpatriek. J.M.. Hyman. B. & Virella. G. Auto- logous Red Blood Cells Potentiate Antibody Synthesis by Unfractionated Human Mononuclear Cell Cultures. Scand. J. Immunol, ift, liy-127, 1987 We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ah) responses to tetanus toxoid (TI") and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear eells (PBMNC) were obtained from normal volunteers and stimulated with PWM. TT. KLH. and mixtures of PWM and antigens in the presence or absence of autologous red blood eells (RBC) (I:.MI ratio of PBMNC/RBC). The cultures were harvested on day II: immunogiobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for calibration. While ami-TV responses were easy to induce with PBMNC from recently hoosled individuals, ihe production of anii-IT from PBMNC obtained from non-rccently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in ihe presence of autologous RBC Similarly. anti-KLH responses were easier lo induce with PBMNC frt)m an immune donor; maximal response was observed after slimuiation wiih PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pre- treatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-y). However, addition of IFN-j'in different doses and at different limes to PWM-slimulaled PBMNC cultures was not as effeetive as addition of RBC in enhancing the production of immunogltibulin and antibody. Gabriel Virella, MD, PhD, Department of Microbiology and Immunology. Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2230, USA The development of simple and reproducible methods for the in vitro stimulation of immuno- globulin synthesis and the induction of specific antibody production in cultures of human lym- phocytes is essential for the study of human immunoregulator> circuits and for theobtention of adequately stimulated B cells to be used in immortalization protocols as a source of large amounts of human antibodies of defined specificities. However, considerable difficulties can be encountered in trying to set up protocols for in vitro stimulation of specific antibody responses [24\. Many different approaches have been tried in protocols for the induction of specific antibody production in human peri- pheral blood tnononuclear ceil (PBMNC) cul- tures, including the use of unfractionated lymphocytes from recently boosted individuals [6. 20. 38. 42. 4.-^. 45. 49]. the immobilization of antigens in microculture plates |6l or Sepharose beads [1(1. 17. 23], and the supplementation with interleukins I and 2 (IL-I and lL,-2) and inter- feron (IFN), alone or in combination |1, 4, 7, 9, 19. 27, 28. 30-32. 37. 39, 4I|. When PBMNC from recently immunized individuals are used, stimulation with nonspecific mitogcns alone has been shown to induce immunoglobulin and specific antibody release (17,29,38,45,461. 119