Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors - PubMed (original) (raw)

Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors

J P Medema et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The concept for cellular immunotherapy of solid tumors relies heavily on the capacity of class I MHC-restricted cytotoxic T lymphocytes (CTLs) to eliminate tumor cells. However, tumors often have managed to escape from the cytolytic machinery of these effector cells. Therefore, it is very important to chart the mechanisms through which this escape can occur. Target-cell killing by CTLs involves the induction of apoptosis by two major mechanisms: through death receptors and the perforin/granzyme B (GrB) pathway. Whereas tumors previously were shown to exhibit mechanisms for blocking the death receptor pathway, we now demonstrate that they also can resist CTL-mediated killing through interference with the perforin/GrB pathway. This escape mechanism involves expression of the serine protease inhibitor PI-9/SPI-6, which inactivates the apoptotic effector molecule GrB. Expression of PI-9 was observed in a variety of human and murine tumors. Moreover, we show that, indeed, expression results in the resistance of tumor cells to CTL-mediated killing both in vitro and in vivo. Our data reveal that PI-9/SPI-6 is an important parameter determining the success of T cell-based immunotherapeutic modalities against cancer.

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Figures

Figure 1

Figure 1

PI-9 is expressed in a subset of human tumors. RNA from human melanoma (A), breast and cervical carcinoma (B), primary colon carcinoma (C Left), and colon carcinoma cell lines (C Right) is isolated, and cDNA is generated. Subsequently, a control PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (A_–_C Lower) or PI-9 (A_–_C Upper) is performed.

Figure 2

Figure 2

SPI-6 binds GrB and is expressed in a subset of murine tumors. (A) In vitro translated [35S]methionine-labeled SPI-6 was incubated in the absence (−) or presence (+) of purified lysed granules (Left) or in the presence of neutrophil elastase (NE), cathepsin G (CatG), or GrB (Right). Complex formation was detected with the use of a mobility shift in an SDS/PAGE gel. (B) RNA isolated from different murine-tumor lines was used for reverse transcription–PCR. (Bottom) GAPDH PCR for 21, 23, and 25 cycles. (Upper) SPI-6 PCR for 24, 27, and 30 cycles. (C) Western blot analysis of lysates from 0.5 × 106 cells using a peptide-purified rabbit polyclonal antiserum against SPI-6. Please note that the SPI-6 in MFF/SPI-6 migrates at a different mobility because it is tagged with a short sequence derived from the vesicular stomatitis virus (VSV) glycoprotein at the N terminus.

Figure 3

Figure 3

SPI-6 expression is inversely correlated with lysis by CTLs. (A) Cells were incubated with the E1B-specific CTL clone in the presence of the relevant epitope; after 24 hr, the medium was analyzed for IFNγ secreted by the activated CTLs. (B) Cells were treated for 24 hr with crosslinked anti-CD95 and analyzed for DNA fragmentation with the Nicoletti assay (see Materials and Methods). (C and D) Cells were labeled with [3H]thymidine overnight and incubated with an E1B-specific CTL clone in the presence of the relevant epitope. After 6 hr, the remaining label was determined and served as a measure for CTL-induced DNA fragmentation (apoptosis). Note that_C_ and D represent one assay with the same CTL population. E:T ratio, effector-to-target ration.

Figure 4

Figure 4

SPI-6 prevents CTL-induced killing in vitro and_in vivo_. (A) Cells were labeled with [3H]thymidine overnight and incubated with the Moloney murine leukemia virus antigen-specific CTL clone. DNA fragmentation of MFF (▵) or MFF/SPI-6 (●) was determined 6 hr later. To determine the contribution of the perforin pathway, CTLs were preincubated with concanamycin A before the addition of the MFF cells (▴). (B) MFF cells were_in vitro_ labeled with CMTMR (orange fluorescence) and loaded with E1B peptide. MFF/SPI-6 cells, which enhanced green fluorescent protein, were loaded similarly with E1B peptide. MFF and MFF/SPI-6 cells were mixed subsequently at a 1:1 ratio and injected i.p. into nude mice. Mice were then injected i.p. with E1B-specific CTLs or were left untreated. At 24 or 48 hr later, tumor cells were isolated, and the MFF to MFF/SPI-6 ratio was determined from five mice per timepoint (standard deviation is given). The first bar is the ratio of cells that were injected (1:1). (C) One representative FACScan profile is shown per group.

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