Peptidspezifische zytotoxische T-Lymphozyten: Diskrepanz zwischen Antigenspezifität und Tumorerkennung

Zusammenfassung

Fragestellung

Es sollte untersucht werden, ob sich im T-Zellrepertoire gesunder Probanden prinzipiell T-Zellen mit Spezifität gegen tumorassoziierte Antigene nachweisen lassen, unter welchen Bedingungen sie in vitro expandierbar sind und ob sie ihre Effektorreaktionen gegen Mamma- bzw. Ovarialkarzinom-Zelllinien mit entsprechendem Antigenprofil entfalten können. Dazu wurden T-Zell-Reaktionen gegen die bereits definierten HLA-A*0201-restringierten Epitope Her-2/neu_{369 - 377} und MAGE-A1_{278 - 296} analysiert und dabei zwischen Peptidspezifität und Tumorerkennung differenziert.

Material und Methoden

Um antigenspezifische zytotoxische Killerzellen zu aktivieren und expandieren, wurden in autologen Systemen so genannte „In-vitro-Immunisierungen“ durchgeführt. Dabei dienten sowohl dendritische Zellen als auch aktivierte B-Zellen als antigenpräsentierenden Zellen in den T-Zell-Stimulationen. Sie wurden dafür zuvor mit definierten HLA-A*0201-bindenden Peptidantigenen beladen. Es sollte hier insbesondere der Einfluss verschiedener Antigenkonzentrationen auf die Spezifität und Funktionalität (Zytotoxizitätsprofil, Interferon-γ-Produktion) der generierten T-Zelllinien analysiert werden.

Ergebnisse

Es gelang, aus dem Blut gesunder Spender nach repetitiven In-vitro-Stimulationszyklen MAGE-A1_{278 - 296}- und Her-2/neu_{369 - 377}-spezifische T-Zelllinien zu etablieren. Dies wurde durch niedrigere Antigenkonzentrationen begünstigt. Dennoch konnten MAGE-A1_{278 - 296}-spezifische T-Zellen HLA-A*0201⁺/MAGE-A1⁺-Tumorzellen nicht lysieren, Her-2/neu_{369 - 377}-spezifische T-Zellen lysierten HLA-A*0201⁺/Her-2/neu⁺-Tumorzellen nur nach zusätzlicher exogener Peptidbeladung.

Schlussfolgerung

Von der Antigenspezifität zytotoxischer T-Zellen kann nicht auf deren lytische Kapazität gegenüber antigenexprimierenden Tumorzellen geschlossen werden. Die Avidität in vitro generierter T-Zellen hängt von der Konzentration des stimulierenden Peptides ab, wobei vermutlich suboptimale HLA/Peptid-Konzentrationen zur Anzucht höher affiner T-Zellen mit besserer lytischer Kapazität gegenüber Tumorzellen führen. Dies ist für die Optimierung von Kultivierungsprotokollen für den adoptiven T-Zelltransfer von Relevanz.

Abstract

Purpose

The majority of tumor-associated antigens are derived from nonmutated self proteins. Little is currently understood about the existence and nature of self-reactive T cells that recognize these antigens in healthy volunteers or cancer patients. Here we try to establish culture conditions to expand Her-2/neu- and MAGE-A1-specific T cells from the blood of healthy donors.

Material and Methods

Unseparated peripheral blood mononuclear cells of HLA-A*0201⁺ healthy donors were stimulated using the well characterized tumorantigens Her-2/neu_{369 - 377} and MAGE-A1_{278 - 296} for in vitro immunization. The influence of different peptide concentrations loaded on autologous antigen-presenting cells on the phenotype and function of the established T cell lines was elucidated. After repetitive stimulations T cells were assayed for peptide specificity as well as tumor recognition in cytotoxicity tests.

Results

Using in vitro immunization protocols it was possible to stimulate tumor antigen-specific cytotoxic T lymphocytes from the blood of healthy donors. This was supported by lower peptide concentrations. However, MAGE-A1_{278 - 296}-specific T cells failed to react with a panel of HLA-A*0201⁺/MAGE-A1⁺ tumor cells. Her-2/neu_{369 - 377}-specific T cells were able to lyse HLA-A*0201⁺/Her-2/neu⁺ tumor cell lines after additional exogeneous peptide-loading.

Conclusion

T cell responses toward peptides derived from tumor antigens can be easily generated in vitro. However, their antigen specificity did not necessarily reflect the lytic capacity towards antigen-expressing tumor cells. “Artificially” high concentrations of peptide presumably cause the expansion of low-avidity T cells which fail to respond to tumor cells with suboptimal antigen presentation. In contrast, cytotoxic T cells generated with low concentrations of peptide were demonstrated to be superior in peptide as well as in tumor recognition. Optimizing the conditions to expand self-reacting (tumor-specific) T cells in vitro is of relevance for the effectiveness of adoptive therapies.

Schlüsselwörter

Tumorimmunologie - zytotoxische T-Zellen - tumorassoziierte Antigene - MAGE-1 - Her-2/neu

Key words

Tumor immunology - cytotoxic T-cells - tumor-associated antigens - MAGE-1 - Her-2/neu

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