HLA-Independent Heterogeneity of CD8+ T Cell Responses to MAGE-3, Melan-A/MART-1, gp100, Tyrosinase, MC1R, and TRP-2 in Vaccine-Treated Melanoma Patients

Sandra R. Reynolds; Esteban Celis; Alessandro Sette; Ruth Oratz; Richard L. Shapiro; Dean Johnston; Marilena Fotino; Jean-Claude Bystryn

Article Figures & Data

Tables

View popup

Table I.

Pre-immunization responses to HLA-A2-restricted peptides

Protein	Peptide	IC₅₀ (nM)^a	Pre-Existing CD8⁺ T Cell Response
Protein	Peptide	IC₅₀ (nM)^a	% Patients with recognition^b (n = 22)	Response level^c
MAGE-3_271–279	FLWGPRALV	61.0	5	18.7
MAG-3_188–198	IMPKAGLLIIV	20.0	0
MAGE-3_159–169	QLVFGIELMEV	7.9	0
Melan-A/MART-1_31–39	GILTVILGV	4.6	5	8.3
Melan-A/MART-1_56–64	ALMDKSLHV	17.2	5	6.6
Melan-A/MART-1_27–35	AAGIGILTV	1063.8	5	8.5
gp100_178–186	MLGTHTMEV	14.3	0
gp100_13–21	AVIGALLAV	17.9	5	21.4
gp100_250–259	YLAEADLSYT	45.5	0
gp100_619–627	RLMKQDFSV	46.3	0
gp100_177–186	AMLGTHTMEV	51.9	5	5.5
gp100_585–593	IMPGQEAGL	64.9	0
gp100_11–19	HLAVIGALL	106.4	0
gp100_606–614	LMAVVLASL	111.1	0
gp100_232–241	FLRNQPLTFA	138.9	5	10.3
gp100_570–578	SLADTNSLA	454.6	0
Tyrosinase_9–17	LLWSFQTSA	15.2	0
Tyrosinase_8–17	BLLWSFQTSA	19.2	5	6.3
Tyrosinase_214–222	FLLRWEQEI	25.6	0
Tyrosinase_477–486	WLLGAAMVGA	32.1	0
Tyrosinase_2–10	LLAVLYCLL	33.3	0
Tyrosinase_490–499	ALLAGLVSLL	56.4	0
Tyrosinase_482–490	AMVGAVLTA	75.5	0
Tyrosinase_490–498	ALLAGLVSL	76.3	5	10.3
Tyrosinase_487–495	VLTALLAGL	82.0	5	15.6
Tyrosinase_491–499	LLAGLVSLL	95.2	0
Tyrosinase_473–481	RIWSWLLGA	100.0	0
Tyrosinase_207–215	FLPWHRLFL	112.3	0
Tyrosinase_207–216	FLPWHRLFLL	204.9	0
MC1R_99–197	LLLEAGALV	6.7	0
MC1R_251–259	FLCWGPFFL	38.5	5	12.5
MC1R_291–299^d	SVMDPLIYA	83.3	0
MC1R_9–17	RLLGSLNST	166.7	0
MC1R_291–299	AIIDPLIYA	238.1	0
MC1R_79–87	CLALSDLLV	357.1	0
MC1R_291–299^d	SIIDPLIYA	454.6	0
TRP-2_431–439	NMVPFFPPV	3.9	0
TRP-2_185–193	FVWLHYYSV	15.2	0
TRP-2_455–463	YAIDLPVSV	15.6	5	6.7
TRP-2_288–296	SLDDYNHLV	26.3	0
TRP-2_476–484	VMGTLVALV	27.8	0
TRP-2_180–188	SVYDFFVWL	35.7	5	6.4
TRP-2_475–483	VVMGTLVAL	66.7	0
TRP-2_156–164	YVITTQHWL	104.2	0
TRP-2_367–376	SLHNLVHSFL	263.2	5	19.0
CSP_334–342	YLKKIKNSL	NT^e	0

a IC₅₀ is defined as the concentration of peptide required to inhibit 50% of the binding of a test peptide to purified HLA-A*0201 molecules.
b The percent of patients out of those tested, who recognized the peptide.
c The actual CD8⁺ T cell responses, i.e. the number of IFN-γ-secreting DC8⁺ T cells/500,000 PBL. These were calculated by subtracting the number of IFN-γ-secreting cells in wells treated with anti-CD8 from wells without Abs, both in the presence of the same peptide. Mean standard deviation was ±0.95.
d Analog (three amino acids have been changed from the original sequence).
e NT, not tested.

View popup

Table II.

Post-immunization responses to HLA-A2-restricted peptides

Protein	Peptide	IC₅₀ (nM)^a	Vaccine-Induced CD8⁺ T Cell Response
Protein	Peptide	IC₅₀ (nM)^a	% Patients with response^b (n = 22)	Response level^c
MAGE-3_271–279	FLWGPRALV	61.0	14	10.6, 89.2, −17.6^d,e
MAGE-3_188–198	IMPKAGLLIIV	20.0	5	8.2
MAGE-3_159–169	QLVFGIELMEV	7.9	9	11.8, 5.8
Melan-A/MART-1_31–39	GILTVILGV	4.6	5	8.3
Melan-A/MART-1_56–64	ALMDKSLHV	17.2	9	45.5,^f 12.7
Melan-A/MART-1_27–35	AAGIGILTV	1063.8	5	11.7^f
gp100_178–186	MLGTHTMEV	14.3	5	13.0
gp100_13–21	AVIGALLAV	17.9	5	69.5^f
gp100_250–259	YLAEADLSYT	45.5	0
gp100_619–627	RLMKQDFSV	46.3	0
gp100_177–186	AMLGTHTMEV	51.9	5	−5.5^e
gp100_585–593	IMPGQEAGL	64.9	14	6.1, 8.4, 10.7
gp100_11–19	HLAVIGALL	106.4	5	7.7
gp100_606–614	LMAVVLASL	111.1	0
gp100_232–241	FLRNQPLTFA	138.9	5	−10.3^e
gp100_570–578	SLADTNSLA	454.6	5	14.8
Tyrosinase_9-17	LLWSFQTSA	15.2	0
Tyrosinase_8–17	BLLWSFQTSA	19.2	9	11.5, −6.3^e
Tyrosinase_214–222	FLLRWEQEI	25.6	5	22.4
Tyrosinase_477–486	WLLGAAMVGA	32.1	0
Tyrosinase_2–10	LLAVLYCLL	33.3	0
Tyrosinase_490–499	ALLAGLVSLL	56.4	0
Tyrosinase_482–490	AMVGAVLTA	75.5	9	6.8, 17.1
Tyrosinase_490–498	ALLAGLVSL	76.3	5	21.8
Tyrosinase_487–495	VLTALLAGL	82.0	9	9.3, 13.4
Tyrosinase_491–499	LLAGLVSLL	95.2	0
Tyrosinase_473–481	RIWSWLLGA	100.0	0
Tyrosinase_207–215	FLPWHRLFL	112.3	0
Tyrosinase_207–216	FLPWHRLFLL	204.9	0
MC1R_99–197	LLLEAGALV	6.7	0
MC1R_251–259	FLCWGPFFL	38.5	9	23.4^f
MC1R_291–299^g	SVMDPLIYA	83.3	0
MC1R_9–17	RLLGSLNST	166.7	0
MC1R_291–299	AIIDPLIYA	238.1	5	22.2
MC1R_79–87	CLALSDLLV	357.1	5	13.7
MC1R_291–299^g	SIIDPLIYA	454.6	0
TRP-2_431–439	NMVPFFPPV	3.9	0
TRP-2_185–193	FVWLHYYSV	15.2	0
TRP-2_455–463	YAIDLPVSV	15.6	9	37.1^f , 36.1
TRP-2_288–296	SLDDYNHLV	26.3	0
TRP-2_476–484	VMGTLVALV	27.8	0
TRP-2_180–188	SVYDFFVWL	35.7	9	16.6, −6.4^e
TRP-2_475–483	VVMGTLVAL	66.7	0
TRP-2_156–164	YVITTQHWL	104.2	0
TRP-2_367–376	SLHNLVHSFL	263.2	9	15.4, −19.8^e
CSP_334–342	YLKKIKNSL	NT^h	0

a IC₅₀ is defined as the concentration of peptide required to inhibit 50% of the binding of a test peptide to purified HLA-A*0201 molecules.
b The percent of patients out of those tested, who recognized the peptide.
c The actual CD8⁺ T cell responses, i.e. the number of IFN-γ-secreting CD8⁺ T cells/500,000 PBL. These were calculated by subtracting the number of IFN-γ-secreting cells in wells treated with anti-CD8 from wells without Abs, both in the presence of the same peptide. Mean standard deviation was ±0.95.
d More than one value indicates multiple patient responses.
e −, Response decreased after vaccine immunization.
f Response increased further by vaccine immunization.
g Analog (three amino acids have been changed from the original sequence).
h NT, Not tested.

View popup

Table III.

Vaccine-induced CD8⁺ T cell responses to melanoma-associated Ags

Patient ID	gp100	Tyrosinase	Melan-A/MART-1	MAGE-3	TRP-2	MC1R	Any Peptide
191	6.1^a	0^b	0	0	0	0	+
375	0	0	45.5	89.2	0	0	+
418	0	0	8.3	10.6	0	0	+
422	0	0	0	11.8	0	0	+
436	10.7, −10.3^c	22.4, −6.3	0	0	36.1, 16.6^d	23.4, 13.7	+
442	0	0	0	8.2, 5.8	0	0	+
443	−5.5	0	0	0	0	0	−
445	0	0	0	0	0	0	−
458	7.7	0	11.7	0	0	22.2	+
460	13.0	11.5	0	0	0	0	+
462	0	0	0	0	0	0	−
465	0	0	0	0	0	0	−
467	0	21.8, 6.8	0	0	0	0	+
468	69.5, 8.4	0	0	0	37.1, −19.0	0	+
471	0	0	0	0	0	0	−
475	0	0	0	−17.6	0	0	−
477	0	0	0	0	0	0	−
478	0	17.1	12.7	0	−6.4	0	+
479	0	13.4	0	0	15.4	0	+
482	14.8	0	0	0	0	0	+
483	0	0	0	0	0	0	−
484	0	0	0	0	0	0	−

%	27%	23%	18%	18%	13%	9%	59%

a Number of vaccine-induced peptide-specific CD8⁺ T cells/500,000 PBL. These were calculated by subtracting the number of peptide-specific CD8⁺ T cells present prior to immunization from that after four immunizations in the same patient.
b 0, Values that were between −4.9 and 4.9 after subtraction of the preimmunization response. This range on either side of 0 is approximately twice the maximum standard deviation of our assay.
c A value with a − indicates that the pre-immunization response was greater than the postimmunization response.
d Multiple values indicate stimulation of CD8⁺ T cells to more than one peptide on the same Ag.

View popup

Table IV.

Heterogeneity in CD8⁺ T cell responses to gp100 peptides

gp100 Peptide	Patient ID
gp100 Peptide	191	436	458	460	468	482
MLGTHTMEV	0	0	0	13.0^a	0^b	0
AVIGALLAV	0	0	0	0	69.5	0
YLAEADLSYT	0	0	0	0	0	0
RLMKQDFSV	0	0	0	0	0	0
AMLGTHTMEV	0	0	0	0	0	0
IMPGQEAGL	6.1	10.7	0	0	8.4	0
HLAVIGALL	0	0	7.7	0	0	0
LMAVVLASL	0	0	0	0	0	0
FLRNQPLTFA	0	−10.3	0	0	0	0
QLVFGIELMEV	0	0	0	0	0	14.8

a Number of vaccine-induced peptide-specific CD8⁺ IFN-γ-secreting T cells/500,000 PBL. These were calculated by subtracting the number of peptide-specific CD8⁺ T cells present prior to immunization from that after four immunizations in the same patient.
b 0, Values that were between −4.9 and 4.9 after subtraction of the pre-immunization response. This range on either side of 0 is approximately twice the maximum standard deviation of our assay.

View popup

Table V.

Clinical data

Patient ID	HLA Type	Stage^a	Adjuvant	Vaccine- Induced CD8⁺ T Cell Response^b	Progression- Free at 1 Year
445	A*0201	II	IL-2 lip^c	−	Yes
475	A*0201	IV	Alum^d	−	Yes
443	A*0201	III	Alum	−	No
471	A*0201	IV	IL-2 lip	−	No
462	A*0201	III	IL-2 lip	−	No
477	A*0201	II	IL-2 lip	−	No
483	A*0201	III	IL-2 lip	−	No
484	A*0205	II	IL-2 lip	−	No
465	A*0201	IV	Alum	−	No
436	A*0201	III	IL-2 lip	+	Yes
191	A*0201	III	IL-2 lip	+	Yes
375	A*0201	III	IL-2 lip	+	Yes
422	A*0201	III	IL-2 lip	+	Yes
442	A*0201	IV	IL-2 lip	+	Yes
460	A*0201	II	IL-2 lip	+	Yes
468	A*0201	II	IL-2 lip	+	Yes
458	A*0201	III	Alum	+	No
418	A*0201	IV	IL-2 lip	+	No
482	A*0201	IV	IL-2 lip	+	No
467	A*0201	III	IL-2 lip	+	No
478	A*0201	II	IL-2 lip	+	No
479	A*0201	II	IL-2 lip	+	No