Table 1.

Effect of LAG-3 mutations on LAG-3/class II MHC interaction

Mutation	Localization of residue	Anti-LAG-3 mAb reactivity			Level of LAG-3 expression	Relative adhesion
		17B4	11E3	15A9
Wt	—	+	+	+	1.0	1
GAL	Neg. control	−	−	−	0	0.09  ±  0.06
Q13A	AB	+	+	+	1.0	0.92  ±  0.06
D30A	BC	+	+	+	0.81	0.41  ±  0.17
H56A	Extra loop	−	+	+	0.7	0.45  ±  0.28
H56F	Extra loop	−	+	+	0.76	0.89  ±  0.36
H63A	Extra loop	+	+	+	0.89	0.83  ±  0.10
H63F	Extra loop	+	+	+	0.95	0.91  ±  0.18
R73E	Extra loop	+	−	+	1.1	3.25  ±  1.00
R75A	Extra loop	+	+	+	0.67	4.20  ±  0.66
R75E	Extra loop	+	+	+	1.4	4.10  ±  0.90
R76E	Extra loop	+	+	+	0.85	2.95  ±  1.00
Y77F	C"	+	+	+	0.75	0.05  ±  0.06
R88A	C"D	+	+	+	0.82	0.18  ±  0.07
R103A	DE	+	+	+	0.93	0.49  ±  0.03
R107K	E	+	+	+	1.1	1.02  ±  0.19
D109E	E	+	+	+	0.81	0.18  ±  0.19
R115A	EF	+	+	+	0.74	0.23  ±  0.05
D133A/R134A	FG	+	+	+	0.99	0.78  ±  0.24
D225L	FG (D2)	+	+	+	0.85	0.89  ±  0.13
(D2)−	D2 deletion	+	+	+	0.6	0.11  ±  0.09
(54/66)−	Extra-loop deletion	−	±	+	0.6	0.08  ±  0.12

The table shows the binding of ⁵¹Cr-labeled Daudi B cells to COS-7 cells expressing LAG-3 mutants. All mutations were in Ig-like domain 1 except for D225L. Nonspecific binding to control-transfected cells was not subtracted from experimental mean cpm. The results expressed relative to wt value should be compared with the background level as defined by the GAL (the LAG-3 insert cloned in reverse orientation in pCDM8)-transfected control cells. Relative expression level of mutant LAG-3 versus wtLAG-3 is also shown. The level of mutant LAG-3 expression was studied with 15A9 reactivity, and relative mAb reactivity was measured as follows: +, 1–0.6; +/−, 0.2–0.6; −, 0–0.2.