Download Supplementary Data (doc 82K)

SUPPLEMENTARY DATA
Foglietta et al.
SUPPLEMENTARY METHODS
Vaccine formulation. The protocol was conducted under BB-IND 5779. Tumor Id
protein was isolated from plasma of MM patients by affinity chromatography (1) and conjugated
to carrier molecule KLH (Biomira, Edmonton, Canada) as previously described (2, 3). Briefly, Id
protein was isolated from plasma by clinical-grade Protein A chromatography (Repligen). Id
protein was conjugated to KLH using glutaraldehyde (Sigma) for 4 hours at a 1:1 ratio. The final
product was available as a solution containing 1.1 mL of conjugated protein diluted in sodium
chloride 0.9% (NS). The solution was contained in a 1.8 mL insert inside a sterile vial. The final
solution contained 0.5 mg/mL of KLH conjugated to 0.5 mg/mL of the patient-specific Id protein
and was stored at -20°C until ready for administration.
Product release criteria included
sterility, endotoxin (<350 EU/mg), and predominant loss of free Id and KLH based on SDSPAGE and Western blotting under reducing and non-reducing conditions. The Id-KLH (0.5 mg)
protein conjugate was administered with GM-CSF (250 g/m2) subcutaneously on day 1 of each
vaccination cycle. Additional doses of GM-CSF (250 g/m2) were administered subcutaneously
on days 2-4 of each cycle in close proximity to the vaccination site.
Antibody assays. To detect anti-KLH antibody responses, pre and postvaccine serum
samples were serially diluted over wells coated with KLH. Bound antibody was detected with
horse-radish peroxidase goat anti-human IgG or IgM antibodies (Caltag, Burlingame, CA). AntiId antibody responses were measured by ELISA as previously described (3, 4). A four-fold or
higher increase in antibody titer in the postvaccine sample as compared with negative controls
was considered to be a positive response. Negative controls included prevaccine serum reactivity
against Id/KLH, and postvaccine serum reactivity against irrelevant Id.
Cytokine induction assay. To assess KLH- and Id-specific cytokine production, fresh or
cryopreserved PBMC (1×106/ml) were cultured in a 48-well plate in complete medium in the
presence or absence of KLH, patient/donor-specific Id, or isotype-matched irrelevant Id proteins
(100 g/ml each). Culture medium was constituted with RPMI 1640 with 1X Glutamax
(Invitrogen, Carlsbad, CA), supplemented with 5% fetal bovine serum (Hyclone, Logan, UT), 1
mmol sodium pyruvate (Invitrogen), 20 mmol HEPES buffer (Invitrogen), 50 mol mercaptoethanol (Invitrogen), 100 units/ml penicillin and 100 g/ml streptomycin (Invitrogen),
and 10 g/ml gentamicin (Invitrogen). After 6 days, cytokine production was assessed in the
supernatants using a multiplex bead-based assay (Bio-Rad Laboratories, Hercules, CA) in a
Luminex-100 XYP System (Luminex Corporation, Austin, TX). A two-fold or higher increase in
cytokine production in the postvaccine sample as compared with negative controls was
considered to be a positive response. Negative controls included PBMC alone and prevaccine
PBMC reactivity against Id/KLH. The anti-Id response was considered specific if the response
was confined to the vaccinated but not irrelevant Id and polyspecific if present using vaccinated
Id and irrelevant Id.
Intracellular cytokine assay. Cryopreserved PBMC were cultured as above for 24 hours
with anti-CD28 antibody (5 g/ml; BD Pharmingen) in the presence or absence of KLH or
bovine serum albumin (BSA) (100 g/ml each). Brefeldin A (5 g/ml; Sigma-Aldrich) was
added during the last 14 hours of incubation. Intracellular cytokine staining and flow cytometric
analysis was performed as previously described (5). Antigen specific T cells were calculated by
subtracting percent of cytokine producing T cells in the absence of KLH from that in the
presence of KLH.
2
Regulatory T cell quantitation. For flow cytometric quantitation of Tregs, surface
(antibodies from BD Biosciences) and Foxp3 (clone PCH101; eBioscience) staining were
performed according to the manufacturer’s instructions. Samples were acquired using a BD
FACS Calibur system (BD Biosciences) and analyzed using FlowJo (Tree Star, Inc.) software.
The absolute number of Tregs at each time point was calculated as follows: (Percent of
CD4+CD127lowFOXP3+ cells in lymphocyte gate × Absolute lymphocyte count/l of peripheral
blood). Tregs were also quantitated by DNA methylation analysis of Foxp3 locus (Epitest assay,
Epiontis) as previously described (6).
3
SUPPLEMENTARY REFERENCES
1. Li Y, Bendandi M, Deng Y, et al. Tumor-specific recognition of human myeloma cells by
idiotype-induced CD8(+) T cells. Blood 2000;96:2828-33.
2. Kwak LW, Taub DD, Duffey PL, et al. Transfer of myeloma idiotype-specific immunity
from an actively immunised marrow donor. Lancet 1995;345:1016-20.
3. Kwak LW, Campbell MJ, Czerwinski DK, Hart S, Miller RA, Levy R. Induction of immune
responses in patients with B-cell lymphoma against the surface-immunoglobulin idiotype
expressed by their tumors. N Engl J Med 1992;327:1209-15.
4. Neelapu SS, Munshi NC, Jagannath S, et al. Tumor antigen immunization of sibling stem cell
transplant donors in multiple myeloma. Bone Marrow Transplant 2005;36:315-23.
5. Neelapu SS, Baskar S, Kwak LW. Detection of keyhole limpet hemocyanin (KLH)-specific
immune responses by intracellular cytokine assay in patients vaccinated with idiotype-KLH
vaccine. J Cancer Res Clin Oncol 2001;127 Suppl 2:R14-9.
6. Wieczorek G, Asemissen A, Model F, et al. Quantitative DNA methylation analysis of
FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.
Cancer Res 2009;69:599-608.
4
SUPPLEMENTARY TABLE 1. Donors’ characteristics and immune responses following
Id-KLH+GM-CSF vaccinations.
KLH-specific immunity
Id-specific immunity
Donor
Age/Sex
Ab
Th1
Th2
Ab
Th1
Th2
1
46/F
+
+
+
-
-
+
2
53/M
+
+
+
+
+
+
3
52/F
+
+
+
-
-
-
4
57/M
+
+
+
+
+
+
5
47/M
+
+
+
+
-
+
6
48/M
+
+
+
+
+
+
7
44/F
+
+
+
+
+
+
8
60/F
+
+
+
+
-
-
9
56/M
+
+
+
+
-
-
10
41/F
+
+
+
-
+
-
Ab, antibody response; Th1, T helper 1 immune response; Th2, T helper 2 immune response; M,
male; F, female.
5
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1. KLH and Id-specific antibody responses in individual donors and
recipients. Prevaccine (pre-V) or pre-hematopoietic stem cell transplant (pre-SCT) and
postvaccine (post-V) or post-SCT serum samples from the indicated time points in the donors
(A, B, E) and recipients (C, D, F) were tested in parallel for KLH (A-D) and Id- (E, F) specific
antibody responses by ELISA as described in the Materials and Methods. Post-SCT samples at 4
mo, 6 mo, 7 mo and 9 mo were obtained one month after the 1st, two months after the 2nd, and
one and three months after the 3rd post-SCT vaccination, respectively. Vaccination time points
are indicated by arrows as V1, V2, and V3 in the donors (A) and recipients (C). Fold-change in
optical density measurements at a serum dilution of 1:32 were calculated at each of the
postvaccine and post-SCT time points relative to prevaccine and pre-SCT time points,
respectively. Antibody responses against KLH were either IgM (A, C) or IgG (B, D) subtype.
Prevaccine serum samples were unavailable for testing in donors 6, 7, and 9. Therefore, the
average optical density of prevaccine samples from donors 1, 2, 3, 4, 5, 8, and 10 was used to
calculate fold-change in postvaccine samples for donors 6, 7, and 9. Serum samples from the
indicated time points were tested at various dilutions for reactivity against vaccinated Id or
isotype-matched irrelevant Id proteins (E, F). KLH- and Id-specific antibody responses are
presented as a heat map according to the scales shown. The anti-Id antibody response was
considered specific (indicated by colored box) if the response was confined to the vaccinated Id
but not irrelevant Id and polyspecific (indicated by colored box with ‘p’) if present using
vaccinated Id and irrelevant Id. n/a, not available.
Supplementary Figure 2. KLH-specific T cells were of both effector and central memory
phenotype. Cryopreserved (A, C) or fresh (B) PBMC samples from various time points in
6
donors and recipients were cultured in medium alone, KLH, or bovine serum albumin (BSA)
overnight (B) or for 24 h (A, C) with Brefeldin A added for the last 12-14 h. Production of TNF and IL-2 was assessed by intracellular cytokine staining as described in Materials and
Methods. (A) Representative data from prevaccine (pre-V) and postvaccine 3 (post-V3) PBMC
from Donor 5 (D5) and, 90 days post-SCT and 9 months post-SCT PBMC from Recipient 5 (R5)
are shown. Percentage of CD3+CD4+ T cells producing TNF-, IL-2, and/or both is shown in
each dot plot. (B) Representative data from postvaccine 3 (post-V3) PBMC from Donor 2 (D2)
and 90 days post-SCT PBMC from Recipient 2 (R2) are shown. Percentage of CD3+CD4+ and
CD3+CD8+ T cells producing TNF- (top panel) and IL-2 (bottom panel) is shown in each dot
plot. (C) Using multiparametric flow cytometry, the phenotype of KLH-specific CD4+ T cells in
donor-recipient pairs was determined. Representative data from Donor 6 (D6, apheresis product)
and Recipient 6 (R6, 6 mo post-SCT) PBMC are shown.
7