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. 2013 Feb 12:11:37.
doi: 10.1186/1479-5876-11-37.

IL-21 promotes the expansion of CD27+ CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells

Saskia J A M Santegoets  1 , Annelies W Turksma, Megan M Suhoski, Anita G M Stam, Steve M Albelda, Erik Hooijberg, Rik J Scheper, Alfons J M van den Eertwegh, Winald R Gerritsen, Daniel J Powell Jr, Carl H June, Tanja D de Gruijl
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VSports - IL-21 promotes the expansion of CD27+ CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells

Saskia J A M Santegoets et al. J Transl Med. .

"V体育ios版" Abstract

Background: Adoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency VSports手机版. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and -21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma. .

Methods: We applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-γ receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine V体育安卓版. .

Results: Although IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+ CD28+ "young" phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs V体育ios版. .

Conclusion: Our data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of "young" effector TIL for ACT. VSports最新版本.

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Figures

Figure 1
Figure 1
Efficiency of tumor infiltrating lymphocytes (TIL) expansion via IL-2, IL-15, IL-21 or no cytokine aAPC. NSCLC, OvCa and melanoma (mel)-derived TIL were expanded by stimulation with IL-2, IL-15, IL-21 or no cytokine-producing aAPC and used for further phenotypic analysis by flow cytometry after two rounds of stimulation. A) Fold expansion of total number of TIL cells in the IL-2, IL-15, IL-21 or no cytokine aAPC-expanded cultures is depicted for 16 NSCLC/OvCa/mel patients. B) Percentage of CD3+ T cells within the total cell population and C) percentage of CD3-CD56+ NK cells is shown for 12 patients. D) Percentage of CD8+ T cells within the CD3+ population is shown for 14 patients. Differences were compared using repeated measures ANOVA’s with a Tukey post test when data followed Gaussian distribution and by using a Friedman test with a Dunn’s post test when data did not pass the normality test. Differences were considered significant when p < 0.05, as indicated with an asterisk (* p < 0.05, ** p < 0.01), *** p < 0.001).
Figure 2
Figure 2
Frequencies of CD27+CD28+ and CD4+FoxP3+ T cells in IL-2, IL-15, IL-21 or no cytokine aAPC-expanded TIL. NSCLC/OvCa/mel-derived TIL were expanded by stimulation with IL-2, IL-15, IL-21 or no cytokine producing aAPC and used for further phenotypic and functional analysis. Percentage of CD27+CD28+ T cells was determined after two stimulations, and percentage of CD4+FoxP3+ T cells was determined after one or two stimulations by flow cytometry. Percentages of CD27+CD28+ is given for 13 NSCLC/OvCa/mel patients within A) the CD3+CD8+ T cell population and B) the CD3+/CD4+ T cell population. Percentage of suppressive FoxP3+ T cells is given for 13 patients C) as percentage of FoxP3 within CD4+ T cell population or D) as percentage of CD4+FoxP3+ cells within the total TIL population. Differences were compared using repeated measures ANOVA’s with a Tukey post test when data followed Gaussian distribution and by using a Friedman test with a Dunn’s post test when data did not pass the normality test. Differences were considered significant when p < 0.05, as indicated with an asterisk (* p < 0.05, ** p < 0.01), *** p < 0.001).
Figure 3
Figure 3
Cytotoxic capacity of TIL expanded with IL-15, IL-21 or no cytokine producing aAPC. Percentage of granzyme B- or perforin-expressing CD8+ T cells was determined by flow cytometry. A) Dot plots with percentages of granzyme B+ cells (top panel) and perforin+ cells (bottom panel) within the CD8+ T cell population are shown for a representative patient. B) Percentage of granzyme B+ cells (left) and perforin+ cells (right) within the CD8+ T cell population are shown for 12 patients. Differences were compared using repeated measures ANOVA’s with a Tukey post test when data followed Gaussian distribution and by using a Friedman test with a Dunn’s post test when data did not pass the normality test. Differences were considered significant when p < 0.05, as indicated with an asterisk (* p < 0.05, ** p < 0.01), *** p < 0.001).
Figure 4
Figure 4
Cytotoxic and tumor-recognizing capacity of TIL expanded with IL-15, IL-21 or no cytokine producing aAPC. Cytotoxic capacity of the IL-2, IL-15 and IL-21 aAPC-expanded TIL was determined by flow cytometry-based re-directed cytotoxicity assay. To this end, CFSE- and anti-CD3-pulsed no cytokine aAPC cells were cultured in a 1:2 ratio in triplicate for 20 hours with CD8+ T cells derived from IL-2, IL-15 or IL-21 aAPC-expanded total TIL, after which percentage of killing was determined by 7-AAD staining. A) Data is presented as percentage of 7-AAD+ target cells for three patients. B) MHC-I restricted autologous tumor recognition of expanded TIL from one donor was determined by IFN-γ secretion assay. To this end, autologous/CD45-depleted tumor cells were cultured in a 1:2 ratio in triplicate for 20 hours with CD8+ T cells derived from IL-2, IL-15 or IL-21 aAPC-expanded total TIL in the presence of either irrelevant IgG2a antibody (left) or anti-MHC class I blocking antibody (w6/32; right), after which supernatants were harvested and tested by IFN-γ ELISA. Differences were compared using repeated measures ANOVA’s with a Tukey post test, and considered significant when p < 0.05, as indicated with an asterisk (* p < 0.05, ** p < 0.01), *** p < 0.001).
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