Article Figures & Data
Figures
- FIGURE 1.
Frequency and diversity of anti-vaccine and anti-tumor T cells in the blood of patient LB2586. A, Clinical evolution of the patient. NED, No evidence of disease. B, Frequencies of anti-vaccine and anti-tumor T cells. Circles refer to frequencies based on in vitro cultures (MLPC), squares refer to frequencies based on clonotypic PCR. All evaluations were conducted under limiting dilution conditions and details are given in Tables I and II. The asterisk (∗) indicates that the value is based on a single positive group in the limiting dilution analysis. The frequencies of anti-MAGE-3.A1 CTL and anti-tumor CTL are referred to total blood CD8 T cells, while the frequencies of anti-MAGE-3.DP4 T cells are referred to total blood CD4 T cells. C, Diversity of anti-vaccine (top panel) and anti-tumor (bottom panel) T cells. T cell clones derived from individual microcultures are represented with different numbers (anti-vaccine) or letters (anti-tumor) corresponding to different TCR sequences. The occurrence of repeated clones is also indicated. The target Ag is mentioned; for some clones, it was not determined (ND).
- FIGURE 2.
Development of the mixed tumor response in patient LB2586. Regressing lesions on the posterior face of the right leg are encircled. The squares frame lesions that progressed over the same period of time.
- FIGURE 3.
Frequency and diversity of anti-vaccine and anti-tumor T cells in various skin metastases of patient LB2586. A, Diversity of anti-tumor T cells obtained from freshly prepared single cell suspensions from tumor fragments. TIL clones emerging from individual microcultures restimulated with tumor cells are referred to by different capital letters corresponding to different TCR sequences. The occurrence of repeated clones is indicated. The target Ag is mentioned; for some clones, it was not determined (ND). B, Frequency of anti-vaccine and anti-tumor TCR β-chains. Total RNA was extracted from fragments of resected frozen metastases and the total number of TCR β-chain transcripts was quantified. The frequencies of TCR β-chains of anti-vaccine clonotypes 1 and 2 and of anti-tumor clonotype A were evaluated among total TCR β-chains under limiting dilution conditions either by β-chain clonotypic PCR or by TCR-β screening. Values are given in Table II. The asterisk (∗) indicates that the value is based on a single positive group in the limiting dilution analysis.
Tables
- Table I.
Frequencies of anti-MAGE-3.A1 and anti-MAGE-C2.B44 CD8 T cells and anti-MAGE-3.DP4 CD4 T cells measured in the blood (PBMC)a
2002 2003 2004 October December January April June October November December February June August CD8 anti-MAGE-3.A1 (clonotype 1) MLPC <3.4 × 10−7 1.1 × 10−6 5.2 × 10−6 5.6 × 10−6 5.5 × 10−6 2.8 × 10−5 (2.9 × 106) (8.8 × 105) (9.7 × 105) (1.4 × 106) (9.2 × 105) (5.8 × 105) α- and β-chains clonotypic PCR <8.3 × 10−7 7.5 × 10−6 5.1 × 10−5 (1.2 × 106) (2.8 × 105) (2.8 × 105) CD8 anti-MAGE-C2.B44 (clonotype A) α- and β-chain clonotypic PCR <8.3 × 10−7 1.5 × 10−5 4.2 × 10−5 2.8 × 10−5 (1.2 × 106) (7 × 104) (2.8 × 105) (2.8 × 105) CD4 anti-MAGE-3.DP4 (clonotype 2) Ex vivo tetramer <3 × 10−7 8 × 10−6 5.4 × 10−5 (3.5 × 106) (1.2 × 106) (5.5 × 105) β-chain clonotypic PCR 2.5 × 10−5 6.6 × 10−5 (1.2 × 106) (1.2 × 105) a Values correspond to frequency among CD8 or CD4 T cells, respectively. The number of CD8 or CD4 T cells analyzed to establish the frequencies are represented in parentheses.
- Table II.
Frequencies of anti-MAGE-3.A1 and anti-MAGE-C2.B44 CD8 T cells and anti-MAGE-3.DP4 CD4 T cells measured in tumor samples (cutaneous in transit metastases)a
2002 2003 2004 October December January April June October November December February June August CD8 anti-MAGE-3.A1 (clonotype 1) β-chain clonotypic PCR 3.3 × 10−6 <1.4 × 10−5 (7 × 104) 1.5 × 10−5 (7 × 104) 6.7 × 10−5 (7 × 104) (3 × 105) CD8 anti-MAGE-C2.B44 (clonotype A) β-chain clonotypic PCR(i) or TCRβ screening(ii) 6.2 × 10−6 (i) 1.5% (ii) (400) 0.7% (ii) (151) 2.2% (ii) (387) (3 × 105) CD4 anti-MAGE-3.DP4 (clonotype 2) β-chain clonotypic PCR <1.2 × 10−5 <1.2 × 10−5 (8 × 104) 1.4 × 10−5 (7 × 104) (8 × 104) a Values correspond to frequency among TCR β-chains. The number of TCR β-chains analyzed to establish the frequencies is represented in parentheses.
- Table III.
Expression of cancer-germline and melanocytic differentiation genes in tumor samples from patient LB2586
Before Vaccination After Vaccination Cutaneous Metastases Cutaneous Metastases Cell Cultures A B 2586-MEL.B LB2586-MEL.G MAGE-A1 − − − (+) +++ MAGE-A3 + ++ + +++ +++ MAGE-A4 − (+) − ++ + MAGE-A10 − − (+) (+) ++ MAGE-C2 + + + ++ +++ LAGE-1 − − − − − NY-ESO1LAGE-2 − − − − − NA17-A ++ ++ ++ +++ +++ Tyrosinase +++ +++ + +++ +++ Melan-AMART-1 +++ +++ ++ +++ +++ gp100 + ++ − + − Cutaneous metastases were resected in August 2002 (before vaccination, A and B) and in October 2003 (after vaccination). Cell cultures were derived from cutaneous metastases resected in June 2003 (LB2586-MEL.B) and October 2004 (LB2586-MEL.G). Gene expression levels were evaluated semiquantitatively using the intensity of the bands obtained by separating PCR products in agarose gel. The results are expressed as +++, ++, +, or (+) if the amount of the PCR-amplified product is equal or greater than that obtained with 1/1, 1/3, 1/9, and 1/27 dilutions of the reference RNA used for each gene. Lower levels of expression are scored negative. Reference RNA is extracted from tumor cell lines expressing the relevant genes.
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