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Transferable anticancer innate immunity in spontaneous regression/complete resistance mice - PubMed

  • ️Sun Jan 01 2006

. 2006 May 16;103(20):7753-8.

doi: 10.1073/pnas.0602382103. Epub 2006 May 8.

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Transferable anticancer innate immunity in spontaneous regression/complete resistance mice

Amy M Hicks et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Spontaneous regression/complete resistance (SR/CR) mice resist very high doses of cancer cells that are lethal to WT mice even at low doses. In this study, we show that this resistance is mediated by rapid infiltration of leukocytes, mostly of innate immunity, in both primary and repeated challenges. Formation of rosettes with infiltrating natural killer cells, neutrophils, and macrophages was required for the subsequent destruction of cancer cells through rapid cytolysis. Highly purified natural killer cells, macrophages, and neutrophils from the SR/CR mice independently killed cancer cells in vitro. The independent killing activity by each subset of effector cells is consistent with the observation that the resistance was abolished by depleting total infiltrating leukocytes but not by depleting only one or two subsets of leukocytes. The resistance was completely transferable to WT recipient mice through SR/CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against subsequent cancer challenges or eradication of established malignancy at distant sites.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.

Leukocyte subset compositions of the peritoneal infiltrates during responses to primary and secondary challenges with cancer cells. TG-induced peritoneal leukocytes were isolated from unchallenged pups of SR/CR lineage and analyzed for the ability to kill S180 cancer cells in vitro as described in Materials and Methods. The pups displaying the SR/CR phenotype were then challenged with i.p. injection of 106 S180 cells for the first time. The responding leukocytes were washed from the peritoneal cavities, stained with the indicated cell surface markers, and profiled with flow cytometry (dark red bars indicate the first challenge). The populations of responding immune cells were also profiled for the second (green bars), third (brown bars), and fourth (orange bars) challenges with S180 cells. Subset percentages of total infiltrates are shown.

Fig. 2.
Fig. 2.

Formation of leukocyte rosettes during the killing of cancer cells in SR/CR mice. SR/CR mice were challenged i.p. with 107 S180 cells, and infiltrating peritoneal leukocytes were harvested after 24 h and analyzed to determine which leukocyte subsets were present in rosettes. (A) Composition of leukocyte subsets in the rosettes. Cytopreps were prepared and stained with cell-specific markers for each leukocyte subset. The percentage of each subset was quantified by direct counting. (B) Contact site morphology of rosettes. The contact sites between cancer cells (T) and PMN (arrowhead) and MΦ (arrow) were viewed by transmission electron microscopy. (C) Immunofluorescence microscopy of rosettes. SR/CR mice were injected i.p. with GFP-transfected S180 cells, and responding leukocytes were removed and analyzed by light microscopy. (a) Phase-contrast view of a rosette. (b) DAPI staining showing nuclear morphology. (c) GFP fluorescence of S180. (d) Rhodamine-conjugated anti-NK1.1 labeling of leukocytes in the rosette (arrows). (e) Composite picture of S180 and anti-NK1.1 staining in a rosette.

Fig. 3.
Fig. 3.

In vitro killing of cancer cells by purified leukocyte subsets from SR/CR mice. Individually purified leukocyte subsets from WT and SR/CR mice (described in Materials and Methods) were examined for their ability to kill S180 cells in a standard in vitro killing assay. The effector cells were incubated with S180 cells at a 10:1 ratio at 39°C for 24 h. Live S180 cells were identified by size, morphology, and fluorescence, and viability was determined by trypan blue exclusion. S, splenocytes; T, thioglycollate-induced peritoneal MΦ; M, MΦ; N/A, no leukocytes added. As a control, mixed populations of SR/CR leukocytes were incubated as described above with CHO or NIH 3T3 mouse fibroblast cells to determine whether S180 killing was specific to cancer cells or general to cultured cell lines.

Fig. 4.
Fig. 4.

Transfer of resistance from SR/CR mice to WT mice against established s.c. tumors. s.c. tumors were implanted in WT nude mice and allowed to grow for 4 days. Tumor-bearing mice received AT of SR/CR leukocytes, WT leukocytes, or no leukocytes. (A) Changes in tumor volume after tumor challenge and subsequent AT of leukocytes (n = 10 for SR and n = 8 for WT). The average tumor volume for control mice and mice receiving AT from SR/CR mice was calculated for each day. Control tumor volume was arbitrarily set at 100%, and tumor volume with AT from SR/CR mice was reported as a percentage compared with control. Experiments included five mice with AT of SR/CR leukocytes (■) and four control mice (♦). (B) Images of necrosis of tumor centers and subsequent regression in a single mouse. (C) Survival of the AT recipients and controls. Experiments included seven mice with AT of SR/CR leukocytes (▴) and five control mice (■).

Fig. 5.
Fig. 5.

Leukocyte infiltration in established S180 tumors. s.c. tumors were established in WT mice, which then received AT of SR/CR leukocytes. Regressing S180 tumors were sectioned, fixed, and stained with hematoxylin and eosin 24 days after receiving AT of SR/CR leukocytes. (A) Tumor cells are surrounded by MΦ and an outer layer of neutrophils, whereas a surprising number of plasma cells are observed in the outermost layer. (B and C) At higher power, small, scattered islands of surviving tumor cells are observed surrounded by MΦ and PMNs, with plasma cells seen in the periphery. Areas of scar tissue formation with fibroblast proliferation were also noticed. T, S180 tumor; M, MΦ; PMN, neutrophil; p, plasma cell; f, fibroblast. The brackets in C delineate the layers of infiltrating leukocytes.

Fig. 6.
Fig. 6.

Donor cells devoid of adaptive immune components were transferred into recipients deficient in adaptive immune responses. Rag1−/− WT mice received total tumor-induced leukocytes, innate immune cells (T/B depleted), or TG-induced leukocytes (>90% MΦ) from SR/CR donors. The recipient WT mice were then injected i.p. with S180 cells. Survival curves are shown for WT controls and for Rag1−/− recipients after initial S180 challenge.

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