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. 2008 Oct;31(8):742-51.
doi: 10.1097/CJI.0b013e31818403d5.

Minimally cultured tumor-infiltrating lymphocytes display optimal characteristics for adoptive cell therapy

Khoi Q Tran  1 , Juhua Zhou, Katherine H Durflinger, Michelle M Langhan, Thomas E Shelton, John R Wunderlich, Paul F Robbins, Steven A Rosenberg, Mark E Dudley
Affiliations

Minimally cultured tumor-infiltrating lymphocytes display optimal characteristics for adoptive cell therapy

Khoi Q Tran et al. J Immunother. 2008 Oct.

Abstract

Adoptive cell therapy (ACT) with tumor-reactive lymphocytes in patients with refractory melanoma can result in tumor regression and prolonged survival. Generating tumor-reactive lymphocyte cultures is technically difficult and resource intensive; these limitations have restricted the widespread application of ACT. Tumor-infiltrating lymphocytes (TIL) from melanoma contain tumor antigen-reactive cells. The "standard" method for producing TIL cultures for clinical administration requires extended in vitro expansion in interleukin-2, then identification of tumor-reactive cells by immunologic assays. We show here that limitations in reagents and methods during screening underrepresent the actual reactivity of TIL cultures. Furthermore, the extended culture times necessitated by the screening assays resulted in telomere shortening and reduced expression of CD27 and CD28 in the TIL cultures, properties that our prior studies showed are correlated with in vivo persistence and clinical response. We have thus developed an alternative "young" TIL method that demonstrated superior in vitro attributes compared with standard TIL. This approach uses the entire resected tumor to rapidly expand TIL for administration without in vitro testing for tumor recognition. Our observations suggest that younger TIL can have an undetermined but high level of antigen reactivity, and other advantageous attributes such as long telomeres and high levels of CD27 and CD28. We suggest that minimally cultured, unselected lymphocytes represent an alternative strategy for generating TIL cultures suitable for use in ACT that, if effective in vivo, may facilitate the widespread application of this approach to a broader population of patients with melanoma.

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Figures

Figure 1
Figure 1
In vitro assessment of TIL 2427 initially failed to demonstrate recognition of shared tumor antigens, but eventually revealed significant recognition of autologous tumor targets. A) IFNγ ELISA results (pg/ml) after overnight coculture of TIL and tumor targets. Each melanoma cell line is shown above its HLA-A loci. AK1700-3 and JKF6 are “control” TIL with know specificities. F1, F2, F3, F4, and F7 are independent TIL derived from tumor 2427. Values that are twice background and >200pg/ml are indicated in bold font. B) Inoperable scalp lesion of patient 2427 prior to ACT treatment. C) Scalp area of patient 2427 after two courses of ACT treatment consisting of non-myeloablative chemotherapy, TIL 2427 F7 and high dose IL-2 therapy.
Figure 2
Figure 2
Composition of TIL cultures changed significantly over time. Each line connects the frequency of marker expression for young TIL (left) with the frequency of marker expression for standard TIL (right) generated from the same tumor. Filled circles represent TIL generated from enzymatic digests of tumor and open squares represent TIL from tumor fragments. Mean age was 15 days for young TIL and 31 days for standard TIL. A. Young TIL contained significantly more CD4+ cells than standard TIL. B. There were no significant differences in CD8+ cells between young and standard cells. C. Young TIL contained significantly more CD3-CD56+ cells than standard TIL.
Figure 3
Figure 3
The phenotype of CD8+ lymphocytes changed over time as TIL were maintained in culture. Each line connects the frequency of marker expression for young TIL (left) with the frequency of marker expression for standard TIL (right). Filled circles represent TIL generated from enzymatic digests of tumor and open squares represent TIL from tumor fragments. Young TIL were in culture for a mean of 15 days while standard TIL were in culture for a mean of 31 days. A. CD8+ young TIL expressed significantly more CD27 than standard TIL. B. CD8+ young TIL expressed significantly more CD28 than standard TIL. C. CD8+ young TIL expressed significantly more CD27 and CD28 than standard TIL.
Figure 4
Figure 4
Telomere length decreased over time as TIL were maintained in culture. Telomere lengths of 495 TIL from 48 consecutive patients were evaluated by quantitative fluorescent in-situ hybridization. Mean telomere lengths calculated from 10 or more independent samples are indicated by large circles, and from less than 10 samples are indicated by small circles. Error bars indicate standard errors. The trend line was calculated from all 495 individual data points.
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