Monoclonal antibody anti‐NC‐2 identifies a second receptor on cells mediating natural cytotoxicity in mice

Abstract

SUMMARYWe have previously reported the identification by the murine monoclonal antibody (mAb) 1C4 of the first leucocyte receptor which is involved in natural cytotoxicity (NC ) against WEHI-164; the NC-1.1 receptor. We report herein the identification and characterization of a second leucocyte receptor which is involved in NC, NC-2 (MW 50 000), identified by a rat anti-mouse mAb D9 (immunoglobulin G2a; IgG2a). Flow cytometric analysis showed that NC-2 was expressed on<6% of splenic leucocytes of different inbred mouse strains and 96% of the cells of a mast-cell line which has high NC activity. In vitro treatment of splenic leucocytes with the D9 mAb blocked effector cell-WEHI-164 target cell conjugation and NC by # 50% without affecting natural killing (NK ). Western blot analysis of affinity purified NC-2 and NC-1.1 using the D9 and 1C4 mAbs showed specific reactivity of the proteins with D9 and 1C4, respectively. Pretreatment of splenic leucocytes with both mAbs blocked NC 84%, a result which almost doubled that caused by either mAb alone. Flow cytometric screening of 16 different mouse cell lines showed that 19% of the cell lines expressed both receptors, 6% expressed only NC-2, 44% expressed mainly or only NC-1.1 and the remaining cells expressed neither receptor. These data indicate that D9 identifies a xenoantigen, NC-2, which is expressed on cells mediating NC and not NK, and that it is not the previously described NC-1.1 allo-antigen. We conclude that NC-2 is likely to be one of a number of receptor molecules on cells mediating NC against tumour cells.

Natural Cytotoxicity of NC‐2+ Cells Against the Growth and Metastasis of WEHI‐164 Fibrosarcoma

“…As reported previously [21], incubation of spleen cells from a number of inbred mouse strains, BALB/ c , C57BL/6, CBA and (CBA × C57BL/6) F1, with mAb D9 (anti‐NC‐2) reduced splenic NC activity against the NC‐sensitive WEHI‐164 target by approximately 50%, but NK activity against the NK‐sensitive YAC‐1 target in vitro was not affected [21]. BALB/ c and (CBA × C57BL/6) F1 mice were selected according to our previous finding about the cytotoxicity of splenic leucocytes from different inbred mouse strains.…”

“…BALB ⁄ c and (CBA · C57BL ⁄ 6) F1 mice of both sexes were selected according to our previous finding about the cytotoxicity of splenic leucocytes from different inbred mouse strains. The above-mentioned mice had higher NC activity against WEHI-164 tumour cells [21]. The animals were maintained in David Maddison Clinical Sciences Building Animal Facility under standard conditions and used between 10 and 12 weeks of age.…”

“…To our knowledge, only two receptors, NC‐1.1 [20] and NC‐2 [21], have been identified on murine leucocytes, which mediate NC against WEHI‐164 (NC‐sensitive target) whilst the activity of these cells against NK‐sensitive target (YAC‐1 tumour cells) was not affected. Studies showed that mice rendered deficient in NC by the administration of the anti‐NC‐1.1 mAb 1C4 have accelerated growth of a number of transplantable tumours [22] and exhibited increased susceptibility to chemical carcinogens [23] compared to control mice.…”

“…In vitro blocking studies showed that pretreatment of (CBA × C57BL/6) F1 mouse spleen cells with mAb D9 blocked NC against WEHI‐164, whereas NK activity against YAC‐1 (NK target) was unaffected. Furthermore, we have shown that mAbD9 blocked effector cells‐WEHI‐164 tumour cell conjugation approximately 50%, whilst conjugation to YAC‐1 tumour cells was not affected [21]. We predicted from the in vitro data that mAb D9, like mAb 1C4, would reduce splenic NC when administered into mice.…”

&lt;i&gt;Toxoplasma gondii&lt;/i&gt; But Not &lt;i&gt;Leishmania major&lt;/i&gt; or &lt;i&gt;Trichomonas vaginalis&lt;/i&gt; Decreases Cell Proliferation and Increases Cell Death on Fibrosarcoma Cancer Cells in Culture Medium

“…WEHI-164, Balb/C mouse fibrosarcoma cells provided by Pasture Institute, Tehran, Iran and cultured in Dulbecco's Modification of Eagles Medium (DMEM) purchased from Sigma company and was supplemented with 20 mm HEPES 0.2 mM L-glutamine, 50 µM 2-mercaptoethanol 0.15% sodium bicarbonate, 50 µg/ml gen-tamicin and 10% fetal calf serum (FCS) as reported previously [15]. Viability of tumor cells was checked by trypan blue staining.…”