Development and characterization of a canine-specific anti-CD94 (KLRD-1) monoclonal antibody

Abstract

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94 + cells were CD3 +/-, CD8 +/-, CD4-, CD21-, CD5 low , NKp46 + , and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on

CD94 Ex Vivo Cultures in a Bone Marrow Transplantation Setting

“…Mononuclear cells were washed at 180 rcf for 10 min then resuspended in Hank’s Balanced Salt Solution (Invitrogen) containing 2% horse serum (2% HH; Gibco) at 20 million cells/mL. Anti-canine CD94 clone 8H10 (mouse IgG 1 ) 14 was added at 10 μg/mL, then incubated on ice for 20 min and washed with 2% HH at 400 rcf for 10 min. CD94-bound cells were labeled for magnetic separation in Miltenyi Running Buffer (Miltenyi Biotech) using either anti-mouse whole-Ig or -IgG 1 coated Miltenyi microbeads according to the manufacturer’s instructions, then selected on a CliniMACS (n = 2) or over LS columns (n = 3).…”

“…Cell culture conditions were based on those previously described for obtaining large granular lymphocyte with NK cell characteristics. 14 , 15 Cells were cultured in Iscove’s Modified Dulbecco’s Medium containing 10% FCS, GlutaMAX, and 15 ng/mL recombinant human IL-15 (Shenandoah) plus 100 ng/mL recombinant canine IL-2 (R&D Systems) in upright T-75 tissue culture flasks (Corning, NY) with lethally irradiated (Elite 3000; Atomic Energy of Canada) K562mbIL21 16 at a ratio of 1:2-1:10 (CD94+ cell: K562mbIL21) and at up to 1 million total cells per ml. Cultures were counted daily for live and Trypan positive K562mbIL21 cells and for live, non-K562mbIL21 cells (cultured CD94-selected cells containing NK) and fed with additional medium to maintain total cell density of no more than 2 million total cells per milliliter.…”

“…NK (CD3-CD94+), NKT (CD3+CD94+), and T-cell (CD3+CD94−) percentages were found using a combination of anti-canine CD3 and CD94 mAb as previously described. 14 Cells per microliter were calculated by multiplying the percentage of immunophenotyped cells found in a lymphocyte (FSC lo SSC lo ) gate by the absolute lymphocyte count obtained from a respective CBC.…”

Developments and translational relevance for the canine haematopoietic cell transplantation preclinical model

“…38 Recently, we produced an anti-canine CD94 mAb that is expressed on NK and NKT cells. 39 The mAb proved effective in enriching NK/NKT cells from dog PBMC. Flow cytometry indicated anti-CD94 bound to cultured and in vitro expanded cells that retained potent cytolytic activity when tested against the canine cell line CTAC.…”

A Role for Dogs in Advancing Cancer Immunotherapy Research

“…Numbers and percentages of neutrophils and monocytes in blood of both species are also very similar. However, recent reagent development for canine NK cells may improve our ability to quantitate dog NK cell responses (28,29). Dogs also have circulating gamma-delta T cells, though little is known regarding how their numbers may change in disease states (30).…”