Highly sensitive in vitro cytokine release assay incorporating high-density preculture

Figures & data

Table 1. Cytokine release from human PBMC induced by various immobilized antibodies.

	TNFα (pg/ml)		IFNγ (pg/ml)		IL-2 (pg/ml)		IL-6 (pg/ml)
Ab	HDC (−)	HDC (+)	HDC (−)	HDC (+)	HDC (−)	HDC (+)	HDC (−)	HDC (+)
Panitumumab	447 ± 243	872 ± 667	10 ± 7	18 ± 17	11 ± 6	9 ± 6	315 ± 361	258 ± 400
Trastuzumab	1701 ± 639^††	2260 ± 939^†	167 ± 259	71 ± 45^§	7 ± 5	8 ± 7	2006 ± 2115	80 ± 135
Rituximab	1475 ± 824^§	1753 ± 889	147 ± 312	52 ± 27^†	6 ± 6	9 ± 6	2410 ± 3203	27 ± 31
Alemtuzumab	1430 ± 780^§	5074 ± 2812^#,§	119 ± 113	299 ± 365	6 ± 7	11 ± 8	2180 ± 1720^§	1171 ± 1606
Anti-human CD3 mAb	4159 ± 1362^§§	10768 ± 3839^##,§§	12736 ± 12549	26551 ± 24321^§	31 ± 14^††	146 ± 153	1075 ± 1083	838 ± 758
Anti-human CD28 SA mAb	13689 ± 9120	32740 ± 12166^*,§§	167691 ± 185393	184273 ± 124381^§§	11681 ± 8146^§	21679 ± 13991^§	3736 ± 2492^§	3065 ± 1526^§§
PBS	35 ± 34	36 ± 21	3 ± 3	6 ± 9	7 ± 5	8 ± 4	77 ± 106	14 ± 23
Human IgG1 Ab	2393 ± 1078	3449 ± 2183	166 ± 292	85 ± 66	7 ± 6	12 ± 10	4159 ± 3017	553 ± 1217*
Human IgG2 Ab	1243 ± 844	932 ± 455	168 ± 273	51 ± 58	13 ± 7	10 ± 7	2260 ± 3046	35 ± 48
Mouse IgG1 Ab	890 ± 724	658 ± 384	99 ± 125	7 ± 9	11 ± 5	9 ± 6	2267 ± 2712	74 ± 151
Mouse IgG2a Ab	1134 ± 734	1513 ± 987	197 ± 299	48 ± 56	13 ± 7	9 ± 6	2651 ± 3324	85 ± 152
LPS	2255 ± 694	9773 ± 4485^##	1004 ± 925	4079 ± 4968	6 ± 3	10 ± 7	6743 ± 2136	18680 ± 4448**
PHA	4302 ± 1531	8520 ± 3081*	14751 ± 11711	46155 ± 32634	522 ± 174	2923 ± 1560^#	6337 ± 2048	8920 ± 3704
	IL-8 (pg/ml)		IL-10 (pg/ml)		MIP-1α (pg/ml)
Ab	HDC (−)	HDC (+)	HDC (−)	HDC (+)	HDC (−)	HDC (+)
Panitumumab	29752 ± 17498	30670 ± 15700	10 ± 7	16 ± 15	4172 ± 2613	7133 ± 4275
Trastuzumab	80787 ± 13971^††	72653 ± 30762^†	21 ± 11	18 ± 11	9609 ± 5471	16884 ± 10123
Rituximab	69528 ± 22765^††	59461 ± 27416^†	22 ± 17	17 ± 11	9521 ± 4693^†	15478 ± 10415
Alemtuzumab	75555 ± 20952^††	132635 ± 54324^*,§§	20 ± 14	49 ± 28^*,†	7458 ± 5163	25748 ± 20217
Anti-human CD3 mAb	52335 ± 23611	77460 ± 36608^§	2689 ± 1123^§§	5675 ± 2824^*,§§	11398 ± 6015^†	24675 ± 11504^*,§
Anti-human CD28 SA mAb	73767 ± 35004^†	94046 ± 30799^††	735 ± 328^§§	1521 ± 610^*,§§	16869 ± 12021^§	24686 ± 11495^§
PBS	2708 ± 1680	2134 ± 839	7 ± 6	4 ± 2	86 ± 67	64 ± 43
Human IgG1 Ab	86839 ± 18289	102142 ± 41129	34 ± 19	21 ± 15	9595 ± 5915	17216 ± 11048
Human IgG2 Ab	54804 ± 31507	41241 ± 12546	22 ± 17	17 ± 11	7350 ± 4724	7672 ± 4127
Mouse IgG1 Ab	51888 ± 38017	29983 ± 14752	25 ± 19	12 ± 6	5066 ± 4007	4760 ± 3753
Mouse IgG2a Ab	54219 ± 32924	51568 ± 19417	25 ± 17	15 ± 10	6006 ± 3519	9108 ± 5302
LPS	35620 ± 13987	152723 ± 22079**	662 ± 364	1272 ± 669	7441 ± 4184	14476 ± 5866*
PHA	30283 ± 15867	114862 ± 32006**	1524 ± 597	2255 ± 1275	10710 ± 5130	19517 ± 10637

Ab: antibody, mAb: monoclonal antibody, HDC: high-density preculture, LPS: lipopolysaccharide, PHA: phytohemagglutinin, SA: super-agonist. A 0.2 ml suspension of human PBMC (2 × 105 cells/well) was incubated with immobilized Ab (0.1, 0.3, 1, and 3 µg/well) for 48 hr exposure. Cytokine levels in well supernatants were measured after the 48 hr exposure. Maximum responses to mAb are shown. Values shown are the means ± SD of six donors from six independent experiments. *p < 0.05 (Student's t-test), **p < 0.01 (Student's t-test), ^#p < 0.05 (Welch's test), ##p < 0.01 (Welch's test): compared with the response in HDC (-). ^†p < 0.05 (Student's t-test), ^††p < 0.01 (Student's t-test), ^§p < 0.05 (Welch's test), ^§§p < 0.01 (Welch's test) compared with the response of panitumumab in the corresponding conditions.

Table 2. Cytokine release response to various immobilized antibodies by donor PBMC.

	TNFα (pg/ml)				IFNγ (pg/ml)
	HDC (−)		HDC (+)		HDC (−)		HDC (+)
Ab	Fold-change range	# responding donors	Fold-change range	# responding donors	Fold-change range	# responding donors	Fold-change range	# responding donors
Trastuzumab	2.3–6.6	5	1.1–7.7	4	2.3–35.7	5	1.8–8.9	5
Rituximab	2.0–4.7	3	0.7–4.5	3	1.4–40.7	4	1.7–10.4	2
Alemtuzumab	1.3–5.1	4	1.7–11.2	5	1.5–51.6	4	3.8–27.4	6
Anti-human CD3 mAb	5.6–18.0	6	5.0–36.0	6	282.0–3833.1	6	664.4–3304.4	6
Anti-human CD28 SA mAb	16.3–55.6	6	16.9–101.9	6	3472.7–60752.6	6	3511.2–26743.7	6
	IL-2 (pg/ml)				IL-6 (pg/ml)
	HDC (−)		HDC (+)		HDC (−)		HDC (+)
Ab	Fold-change range	# responding donors	Fold-change range	# responding donors	Fold-change range	# responding donors	Fold-change range	# responding donors
Trastuzumab	0.2–1.1	0	0.0–4.3	1	3.0–11.4	5	0.0–12.2	1
Rituximab	0.0–1.3	0	0.0–4.6	1	2.1–20.6	3	0.0–4.9	1
Alemtuzumab	0.0–1.1	0	0.0–5.0	1	3.7–14.8	6	0.1–483.6	5
Anti-human CD3 mAb	1.5–8.2	2	1.9–48.9	5	2.8–6.1	5	0.1–475.1	5
Anti-human CD28 SA mAb	424.8–1399.0	6	843.8–4737.0	6	6.9–37.3	6	2.3–1208.9	5
	IL-8 (pg/ml)				IL-10 (pg/ml)
	HDC (−)		HDC (+)		HDC (−)		HDC (+)
Ab	Fold-change range	# responding donors	Fold-change range	# responding donors	Fold-change range	# responding donors	Fold-change range	# responding donors
Trastuzumab	1.8–5.0	3	1.4–7.0	2	0.8–9.8	3	0.0–6.4	1
Rituximab	1.7–4.7	2	1.0–3.9	1	0.4–12.9	1	0.0–8.5	1
Alemtuzumab	1.4–4.8	3	2.2–8.8	4	0.3–6.4	2	0.4–11.4	4
Anti-human CD3 mAb	1.3–2.6	0	1.4–6.6	2	145.4–689.3	6	103.1–1181.4	6
Anti-human CD28 SA mAb	1.7–4.1	1	1.9–5.8	3	30.5–272.6	6	33.4–415.6	6
	MIP-1α (pg/ml)
	HDC (−)		HDC (+)
Ab	Fold-change range	# responding donors	Fold-change range	# responding donors
Trastuzumab	0.9–4.0	2	1.0–3.9	3
Rituximab	1.6–4.2	1	0.8–3.5	1
Alemtuzumab	0.9–2.2	0	1.6–7.0	3
Anti-human CD3 mAb	1.9–4.4	2	1.9–9.1	4
Anti-human CD28 SA mAb	2.3–4.6	5	2.4–8.0	6

Ab: antibody, mAb: monoclonal antibody, HDC: high-density preculture, SA: super-agonist.

Fold-change in cytokine level associated with each mAb relative to corresponding levels induced by panitumumab. Fold-change range shows minimum-maximum fold-change of cytokines for all six donors. Number of responding donors is number of positive responding donors showing a fold-change of ≥3. Results in which more than half of the donors showed a positive response are shown in bold.

Table 3. Cell proliferation induced in donor PBMC by various immobilized antibodies.

	Cell proliferation (OD 450)
Ab	HDC (−)	HDC (+)
Panitumumab	0.366 ± 0.027	0.364 ± 0.052
Trastuzumab	0.344 ± 0.036	0.363 ± 0.032
Rituximab	0.371 ± 0.056	0.382 ± 0.050
Alemtuzumab	0.368 ± 0.066	0.399 ± 0.047
Anti-human CD3 mAb	0.870 ± 0.276	1.406 ± 0.475*
Anti-human CD28 SA mAb	1.046 ± 0.347	1.264 ± 0.407
PBS	0.387 ± 0.046	0.414 ± 0.036
Human IgG1 Ab	0.344 ± 0.036	0.375 ± 0.039
Human IgG2 Ab	0.372 ± 0.043	0.353 ± 0.030
Mouse IgG1 Ab	0.370 ± 0.050	0.370 ± 0.036
Mouse IgG2a Ab	0.379 ± 0.063	0.359 ± 0.025
LPS	0.323 ± 0.028	0.501 ± 0.057**
PHA	0.746 ± 0.155	1.151 ± 0.339*

Ab: antibody, mAb: monoclonal antibody, HDC: high-density pre-culture, LPS: lipopolysaccharide, PHA: phytohemagglutinin, SA: super-agonist.

A 0.2 ml suspension of human PBMC (2 × 10⁵ cells/well) was incubated with immobilized antibodies (0.1, 0.3, 1, and 3 μg/well) for 48 hr. Cell proliferation was determined using a WST-8 assay over the final 3 hr of incubation. Maximum responses to mAb/Ab are shown. Values shown are means ± SD of six donors of six independent experiments. *p < 0.05 (Student’s t-test), **p < 0.01 (Student’s t-test) compared with that in HDC (−).

Figure 1. TNFα release and cell proliferation induced by various immobilized antibodies from human peripheral blood mononuclear cells for 24 or 48 hr. mAb: monoclonal antibody; SA: super-agonist. A 0.2 ml suspension of human PBMC (2 × 10⁵ cells/well) following high-density pre-culture (10⁷ cells/ml) was incubated with immobilized Ab (0.1, 0.3, 1, and 3 μg/well) for 24 or 48 hr. (A) TNFα levels in the supernatants after 24 or 48 hr of exposure. (B) Cell proliferation determined by WST-8 assay over last 3 hr of 24 or 48 hr incubations. Values shown are means ± SD of 5–6 donors from six independent experiments. *p < 0.05, **p < 0.01 (Student’s t-test), ^#p < 0.05, ^##p < 0.01 (Welch’s test): compared with values at 24 hr.

Table 4. Cytokine release induced by various immobilized antibodies from F(ab’)₂ fragment pretreated human PBMC.

mAb/Ab	F(ab')2 fragment	TNFα		IFNγ		IL-2		IL-6		IL-8		IL-10		MIP-1α
		(pg/ml)	(%)^a	(pg/ml)	(%)	(pg/ml)	(%)	(pg/ml)	(%)	(pg/ml)	(%)	(pg/ml)	(%)	(pg/ml)	(%)
Panitumumab	Mouse IgG1	834 ± 358	–	3 ± 2	–	6 ± 4	–	13 ± 9	–	33126 ± 10150	–	2 ± 1	–	12146 ± 6803	–
	Anti-FcγRI	627 ± 176	75	2 ± 1	91	8 ± 5	119	46 ± 72	338	26905 ± 6841	81	2 ± 1	109	10104 ± 6909	83
	Anti-FcγRII	949 ± 650	114	14 ± 28	540	8 ± 4	121	138 ± 301	1021	34710 ± 11441	105	4 ± 5	201	10618 ± 5179	87
	Anti-FcγRIII	496 ± 262	59	2 ± 2	78	5 ± 3	79	9 ± 4	63^#	22262 ± 10439	67	2 ± 1	89	7673 ± 6006	63
Trastuzumab	Mouse IgG1	1585 ± 569	–	80 ± 81	–	6 ± 4	–	65 ± 59	–	54744 ± 24372	–	3 ± 3	–	14369 ± 10801	–
	Anti-FcγRI	1756 ± 559	111	89 ± 86	112	5 ± 3	80	191 ± 195	295	51732 ± 11369	94	3 ± 2	93	13240 ± 8643	92
	Anti-FcγRII	1541 ± 348	97	100 ± 111	125	7 ± 6	123	79 ± 110	123	52918 ± 20553	97	2 ± 1	67	12329 ± 7693	86
	Anti-FcγRIII	1012 ± 591	64^#	28 ± 35	35^##	4 ± 2	68	22 ± 14	34^#	33792 ± 17113	62	1 ± 2	36	7552 ± 3771	53
Rituximab	Mouse IgG1	1299 ± 717	–	38 ± 50	–	4 ± 3	–	24 ± 15	–	46230 ± 23286	–	2 ± 1	–	11188 ± 7998	–
	Anti-FcγRI	1386 ± 527	107	39 ± 49	103	3 ± 3	75	52 ± 54	211	46840 ± 15503	101	1 ± 1	57	12657 ± 7732	113
	Anti-FcγRII	1117 ± 396	86	33 ± 35	87	4 ± 2	88	32 ± 20	132	43291 ± 11688	94	1 ± 1	62^#	9599 ± 4653	86
	Anti-FcγRIII	756 ± 525	58^#	9 ± 8	24^##	3 ± 3	69^#	9 ± 3	37^#	27798 ± 15283	60^#	1 ± 1	54	5820 ± 3266	52
Alemtuzumab	Mouse IgG1	4090 ± 2258	–	298 ± 389	–	3 ± 2	–	1116 ± 1550	–	124309 ± 45291	–	42 ± 34	–	20157 ± 13475	–
	Anti-FcγRI	2198 ± 801	54^#	200 ± 205	67	3 ± 2	86	311 ± 243	28	68553 ± 19565*	55^#	24 ± 25	58	14255 ± 6362	71
	Anti-FcγRII	2427 ± 910	59^#	269 ± 316	90	3 ± 2	91	364 ± 211	33	90795 ± 28831	73	34 ± 35	81	18938 ± 14971	94
	Anti-FcγRIII	1239 ± 692^#	30^##	69 ± 54	23^##	2 ± 1	67^#	109 ± 84	10^##	40699 ± 19697**	33^##	16 ± 17	39^#	8828 ± 3334	44
Human IgG1 Ab	Mouse IgG1	3257 ± 2367	–	83 ± 156	–	6 ± 5	–	367 ± 427	–	76234 ± 22876	–	12 ± 16	–	17838 ± 11449	–
	Anti-FcγRI	1744 ± 756	54^#	88 ± 129	105	5 ± 6	86	171 ± 181	47	51125 ± 14977*	67^#	4 ± 3	35	11787 ± 7302	66^#
	Anti-FcγRII	2446 ± 1122	75^#	90 ± 129	108	15 ± 8*	227	312 ± 345	85	75232 ± 20096	99	6 ± 4	50	15874 ± 10765	89
	Anti-FcγRIII	1826 ± 1168	56^##	18 ± 13	21	12 ± 9	181	204 ± 326	55^#	50841 ± 22035	67^##	5 ± 5	40^#	11682 ± 7503	65^##
Human IgG2 Ab	Mouse IgG1	1142 ± 622	–	6 ± 3	–	7 ± 2	–	84 ± 163	–	42019 ± 15412	–	3 ± 1	–	12545 ± 10775	–
	Anti-FcγRI	946 ± 574	83	8 ± 8	131	7 ± 6	108	32 ± 25	38	34973 ± 14853	83	2 ± 2	85	8336 ± 4727	66
	Anti-FcγRII	969 ± 401	85	7 ± 7	124	6 ± 4	94	25 ± 13	29	40061 ± 12630	95	2 ± 2	77	10534 ± 6447	84
	Anti-FcγRIII	647 ± 413	57^##	4 ± 4	73	5 ± 5	82	10 ± 7	12^##	27174 ± 11740	65^##	2 ± 1	60^#	6635 ± 3442	53^#

Ab: antibody, LPS: lipopolysaccharide, PHA: phytohemagglutinin.

High-density precultured human PBMC (2 × 10⁵ cells/well) were incubated with F(ab’)₂ fragments of neutralizing antibodies to F_cγRI (CD64), F_cγRII (CD32), or F_cγRIII (CD16) for 45 min on ice. Then, a 0.2 ml suspension was incubated with immobilized antibodies (3 μg/well) for 48 hr, after which cytokine levels in well supernatants were measured. Values shown are means ± SD of six donors from two independent experiments.

^aPercentage cytokine release induced by various immobilized antibodies from F(ab’)₂ fragment pretreated PBMC compared with that due to mouse IgG₁ F(ab’)₂ fragment treatment is shown. Percentage = 100 × [absolute average value of six donors in each F(ab’)₂ treatment/absolute average value of six donors with mouse IgG₁ F(ab’)₂ treatment]. *p < 0.05 (Student’s t-test), **p < 0.01 (Student’s t-test), ^#p < 0.05 (Welch’s test), ^##p < 0.01 (Welch’s test) compared with response with mouse IgG₁ F(ab’)₂ fragment treatment.