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Combine two color coded multimers for 25 epitopes and use for staining
QD655
QD705
Count QD800
CD8-FITC
CD8-FITC
TET QD800+
CD8+ / Dump- Cells
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QD605
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TET QD605+
Live / Dead Stain
CD8-FITC
CD8-FITC
CD8-FITC
CD8-FITC
PE
CD8-FITC
SSC-W TET APC+
TET PE+
TET QD655+
SSC-H
CD8-FITC
SSC-A
FSC-W FSC-H
FSC-A
Live Cells
SSC Single Cells
CD8-FITC
FSC Single Cells
Mononuclear Cells
TET PE-Cy7+
PE-Cy7
Supplementary Figure 1. Overview of combinatorial encoding approach. (A) MHC molecules charged with different peptides are each conjugated to a unique two-color fluorochrome code (exemplified by the red, green and blue fluorochromes). Sets of 25 pMHC multimers that each have a unique two-color code are combined for T cell staining. (B) Gating strategy applied for identification of pMHC-specific T cells by combinatorial coding. TILs were stained with a mixture of pMHC multimers plus the indicated antibodies. Single mononuclear cells were selected by FSC and SSC. Within the single cells, live cells were gated by a live/dead cell stain. Subsequently, anti-CD8-FITC positive and “dump-channel” (anti-CD4, -CD14, -CD19, -CD16-AF700) negative cells were selected (top right). (c) From the above gating, positive cells were selected in all eight fluorescent channels. Cells positive in two and only two different pMHC multimer channels are indicated in black, and cells negative in all channels are indicated in red. Cells positive in only one as well as cells positive in three or more pMHC multimer channels are gated out. The eight fluorochromes used are listed on the x-axis.
All antigens
10
20
D cumulative fraction of responses
0.05
0.10
0.15
0.20
0.25
0.30
measured responses random responses
5
10
15
20
25
number of patients
30
0.8 0
35
5
10
15
20
25
number of patients
30
35
30
35
OE antigens measured responses random responses
0.25
CT antigens
0
0.6
35
0.20
30
0.15
25
0.10
20
0.05
15
0.00
10
number of patients
0.00
cumulative fraction of responses
C
5
0.4
30
40
●
0.2
●
measured responses random responses
0.0
cumulative fraction of responses
50
60
experimental responses random responses
0
MDA antigens
B
0
cumulative number of responses
A
0
5
10
15
20
25
number of patients
Supplementary Figure 2. MD antigen- but not CT antigen-specific T cell responses are strongly focused towards specific epitopes. (A) The cumulative number of responses against epitopes in all antigen classes is shown as a function of the number of patients screened. The black line represents the mean +/- 95% confidence interval of 1000 randomized permutations (with replacement) of patient samples using the experimental data. The red line represents the predicted number of responses detected when 1000 randomized permutations are performed using randomly created patient-response tables (i.e., in which the probability of observing a T cell response is equal for each epitope, but in which the total number of responses is the same as in the experimental data). Note that there is a substantial saturation in the number of different responses observed in the experimental data. (B-D) The same analysis but now performed separately for the MD epitopes (B), CT epitopes (C), and OE epitopes (D), in each case expressed as a fraction of the total number of epitopes present within this antigen class. Note that the saturation observed in (a) can fully be attributed to recurrent recognition of MD epitopes.
Supplementary Figure 3. Random variability in TIL composition during in vitro culture. Contribution of indicated antigen-specific T cell populations (% of total CD8+ T cells) in parallel T cell cultures established from different tumor fragments of 2 patients (A, B). T cell cultures were initiated in separate wells, each containing one fragment, and cultured for approximately 2 weeks prior to analysis of T cell reactivity.
Supplementary Table 1. Set of HLA-A2-restricted melanoma-associated epitopes used for T cell profiling. Epitopes to which reactivity was detected in at least one patient are highlighted in grey.
