0041-1337/98/6503-416$03.00/0 TRANSPLANTATION Vol. 65, 416 – 420, No. 3, February 15, 1998 Copyright © 1998 by Williams & Wilkins Printed in U.S.A. TREATMENT OF SEVERE COMBINED IMMUNODEFICIENCY MICE WITH ANTI-MURINE GRANULOCYTE MONOCLONAL ANTIBODY IMPROVES HUMAN LEUKOCYTE XENOTRANSPLANTATION 1 STEFANO M. SANTINI,MASSIMO SPADA,STEFANIA PARLATO,MARIANTONIA LOGOZZI, CATERINA LAPENTA,ENRICO PROIETTI,FILIPPO BELARDELLI, AND STEFANO FAIS 2 Laboratory of Virology, Istituto Superiore di Sanita ` , 00161 Rome, Italy Background. The residual resistance of severe com- bined immunodeficiency (SCID) mice to human graft is the main factor in conditioning both the extent of human cell reconstitution and the xenograft-to-xe- nograft variability. We have recently shown that an early and massive murine granulocyte recruitment is the main event in the SCID mouse reaction to the human graft. Methods. Here, we evaluate the importance of mouse granulocytes in the restriction of human cell engraft- ment in SCID mice. We injected SCID mice with a monoclonal antibody to murine granulocytes. Results. Injection of this antibody resulted in a marked depletion of polymorphonuclear cells in the hematopoietic organs of SCID mice. This depletion was associated with a significant increase in both the growth of human cell lines of different hematopoietic origin and the engraftment of human peripheral blood leukocytes. Moreover, the abolishment of the early granulocyte reaction markedly reduced the xenograft- to-xenograft variation, a major shortcoming of these xenochimeric models. Conclusions. These results provide new insights into the control of the natural immune response of SCID mice against human graft. Furthermore, treatments aimed at controlling the acute inflammatory reaction of SCID mouse-to-human cell transplantation can be considered useful experimental approaches for in- creasing the xenograft-to-xenograft reproducibility. Severe combined immunodeficient (SCID*) mice are un- able to reject either allogeneic or xenogeneic organ grafts and are now widely used to investigate in vivo engraftment, hom- ing, growth patterns, and responses of normal and neoplastic human hematopoietic cells (1). However, the SCID mutation does not affect natural immunity, and SCID mice retain fully functioning and highly efficient natural killer (NK) cell, mac- rophage, and granulocyte compartments (2). We previously described the reaction of SCID mice to intraperitoneal injec- tion of human peripheral blood leukocytes (hu-PBL) (3). This SCID reaction consists of an early (24 hr after hu-PBL trans- plantation) and massive neutrophil recruitment and an in- duced expression of a wide spectrum of murine cytokine mRNAs in the SCID mouse peritoneal cavity. This murine acute response is associated with a marked reduction in the number of human cells in the peritoneal cavity, occurring primarily within 24 hr after hu-PBL injection (3). Therefore, xenogenic transplantation in SCID mice seems to be limited not only by professional NK cell activity but also by an acute inflammatory reaction, which appears to play a prominent role. Previous studies aimed at suppressing the residual reac- tivity of SCID mice to human cell engraftment used anti– asialo-GM1 to inhibit murine NK cell activity (4–6). In this study, we investigated whether a monoclonal antibody (RB6 – 8C5) to mouse granulocytes could improve the engraft- ment of human cell lines of hematopoietic origin and PBLs in SCID mice. We report here that anti-granulocyte antibody treatment markedly improves both the growth of human hematopoietic cell lines and hu-PBL engraftment, thus en- hancing xenograft-to-xenograft reproducibility of human cell transplantation in SCID mice. MATERIALS AND METHODS Animals. C.B.-17 SCID/SCID female mice (Arlan Nossan, Italy) were used at 4 –5 weeks of age and were kept under specific patho- gen-free conditions. SCID mice were housed in microisolator cages, and all food, water, and bedding were autoclaved before use. Antibodies. Monoclonal anti-mouse granulocyte antibodies (RB6 – 8C5 hybridoma) were prepared as previously described (7). Purified rat serum was used as control. To deplete animals of neutrophils, SCID mice were given subcutaneous injections of 0.2 ml (100 mg/ mouse) of an anti-mouse granulocyte monoclonal antibody (mAb) as follows: for hematopoietic cell lines, intraperitoneal inoculation 3 days before subcutaneous cell injection and on day 0 and every 3 days for the first week; for hu-PBLs, subcutaneous inoculation 3 days and 1 day before hu-PBL intraperitoneal injection and every 3 days after reconstitution. Growth of U937 and CEM-cell tumors in SCID mice. Mice were given subcutaneous injections in the shoulder of 210 6 uninfected U937 or 210 7 CEM cells resuspended in 0.2 ml of saline. The two major diameters of each tumor nodule were measured by a caliper, and the mean tumor diameter for each mouse was calculated. Reconstitution with hu-PBLs. Hu-PBLs were obtained from the peripheral blood of healthy donors. All donors were screened for human immunodeficiency virus-1 and hepatitis before donation. The hu-PBLs were obtained by Ficoll-Paque density gradient centrifuga- tion. Twenty million cells were resuspended in 0.3 ml of RPMI 1640 and injected intraperitoneally into the SCID mice. Cell recovery from the peritoneal cavity and organs of the SCID mice. SCID mice were euthanized at different times after hu-PBL injection (24 hr and 1 week), and cells were collected from the peritoneal cavity and spleen. At each time, a peritoneal lavage was 1 This study was supported by grants from the Italian Ministry of Health (930/B, VIII Progetto di Ricerca sull’AIDS). 2 Address correspondence to: Dr. Stefano Fais, M.D., Ph.D., De- partment of Virology, Istituto Superiore di Sanita ` , V.le Regina Elena, 299, 00161 Rome, Italy. * Abbreviations: hu-PBL, human peripheral blood leukocyte; IFN, interferon; IL, interleukin; mAb, monoclonal antibody; NK, natural killer; PCR, polymerase chain reaction; PMN, granulocyte (polymor- phonuclear leukocyte); SCID, severe combined immunodeficiency. 416