In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity - PubMed (original) (raw)

In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity

Ryosei Nishimura et al. Blood. 2008.

Abstract

Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. CIK cells are cytotoxic against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD). However, the precise mechanism of reduced GVHD is not fully understood. Therefore, we evaluated the trafficking and survival of luciferase-expressing CIK cells in an allogeneic bone marrow transplant model. The initial trafficking patterns of CIK cells were similar to conventional T cells that induced GVHD; however, CIK cells infiltrated GVHD target tissues much less and transiently. CIK cells accumulated and persisted in tumor sites, resulting in tumor eradication. We evaluated different properties of CIK cells compared with conventional T cells, demonstrating a slower division rate of CIK cells, higher susceptibility to apoptosis, persistent increased expression of interferon gamma (IFN-gamma), and reduced acquisition of homing molecules required for entry of cells into inflamed GVHD target organs that lack expression of NKG2D ligands recognized by CIK cells. Due to these properties, allogeneic CIK cells had reduced expansion and caused less tissue damage. We conclude that CIK cells have the potential to separate graft-versus-tumor effects from GVHD.

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Figures

Figure 1

Figure 1

CIK cells induce much less GVHD in a major-mismatched BMT model. Lethally irradiated Balb/c (H-2d) hosts received BM with either CIK cells or fresh splenocytes at the indicated dose from wild-type FVB (H-2q) mice. Percent survival (A) and percent body weight change (B) are presented. □ indicates BM (n = 5); ▵, CIK cells 5 × 106 (n = 5); ×, CIK cells 10 × 106 (n = 5); ◇, CIK cells 20 × 106 (n = 5); ▿, fresh splenocytes 2.5 × 106 (n = 5); ■, radiation control (n = 5). Results from one representative experiment are shown.

Figure 2

Figure 2

Trafficking and survival of CIK cells upon adoptive transfer. Lethally irradiated host allogeneic (Balb/c, H-2d, n = 5) and syngeneic (FVB/N, H-2q, n = 5) recipients were injected with 5 × 106 FVB wild-type BM cells with 10 × 106 CIK cells generated from luc+ FVB mice (L2G85). To compare GVHD development, 10 × 106 L2G85 splenocytes were transplanted with BM cells (n = 5). (A) BLI of mice after transplantation. Donor-derived CIK cells in the allogeneic mice homed to and proliferated in secondary lymphoid organs, followed by the infiltration of gut and skin (top row), which was similar to GVHD progression (bottom row). However, in CIK cell–receiving mice, the speed of spread to peripheral tissue was slower and maximum peak signal from gut was lower than that in animals receiving fresh splenocytes. In syngeneic mice (middle row), there was no significant CIK cell proliferation and only a transient signal was observed on day 14 in cervical lymph nodes and abdomen. (B) Quantitation of photon emission of BLI. (C) Ex vivo imaging demonstrated compared with naive splenocytes, CIK cell homing to and proliferating in gastrointestinal tissues over time. (D) Histopathology of selected tissues on day 7 after transplantation. Magnification was ×200 or ×400 as indicated. Goblet cells (dashed arrow); crypt abscesses (solid arrow); lymphocytic infiltration surrounding the bile ducts (red arrow). (E) Mice receiving splenocytes (n = 3) or CIK cells (n = 3) were humanely killed on day 7 after BMT. GVHD histopathologic score was calculated on liver, small intestine, and large intestine damage on day 7 after BMT (CIK vs splenocytes, P = .002). Error bars represent SD.

Figure 3

Figure 3

Comparison of CIK cells trafficking between major- and minor-mismatched recipients. Balb/c (H-2d, major mismatch, n = 5) and Balb/b (H-2b, minor mismatch, n = 5) recipients were lethally irradiated, and then injected with 5 × 106 C57BL/6 (H-2b) wild-type BM with 10 × 106 CIK cells generated from B6-L2G85 (H-2b) luc+ mice. (A) CIK subpopulations. (B) BLI images and (C) quantitative analysis of BLI over time. (D) Percent body weight change over time. Error bars represent SD.

Figure 4

Figure 4

Cell division rate of CIK cells in vivo compared with naive splenocytes. Lethally irradiated Balb/c recipient mice were injected with 5 × 106 C57BL/6 (H-2b, Thy1.2) wild-type BM cells with either CFSE-labeled 10 × 106 CIK cells or splenocytes generated from congenic C57BL/6 (H-2b, Thy1.1). (A) On day 3, the cell division rate from splenocytes (top row) and CIK cells (bottom row) in the spleen was analyzed by a CFSE-based cell proliferation assay. Similar results were obtained in 3 allogeneic and 3 syngeneic receipient mice. (B) Proliferation index of CD4 and CD8 T cells of splenocytes and CIK cells. Error bars represent SD. *P < .05; **P < .01.

Figure 5

Figure 5

Rate of apoptosis of transferred T cells. Lethally irradiated host mice (Balb/c, H-2d) were injected with 5 × 106 C57BL/6 (H-2b, Thy1.2) wild-type BM cells with either 10 × 106 CIK cells generated from congeneic C57BL/6 (H-2b, Thy1.1) or 10 × 106 splenocytes. (A) Early apoptotic cells from CIK cells (n = 3) and GVHD effector cells (n = 3) from the spleen were analyzed on day 7 after BMT separately in CD8+ and CD4+ lymphocytes by FACS using annexin V and PI staining. Error bars represent SD. **P < .01. (B) Comparison of Fas and FasL expression between GVHD controls and CIK cell–receiving mice on day 7 after BMT. One representative result of 3 similar experiments is shown.

Figure 6

Figure 6

Expression of chemokine receptors and homing molecules between CIK cells and splenocytes. Lethally irradiated host mice (Balb/c, H-2d) were injected with C57BL/6 (H-2b, Thy1.2) wild-type 5 × 106 BM cells with either 10 × 106 CIK cells or splenocytes generated from congenic C57BL/6 (H-2b, Thy1.1). Expression of chemokine receptors, homing molecules, and IFN-γ expressed on donor-derived cells were analyzed at indicated days and tissues after BMT by FACS. (A) α4β7 and CCR9. (B) E-selectin ligand and P-selectin ligand. (C) CCR5 and CXCR3. (D) IFN-γ. ND indicates not done. Samples were not obtained because all GVHD control mice died by day 9. LN indicates lymph node. Error bars represent SD. *P < .05; **P < .01.

Figure 7

Figure 7

CIK cells retain tumor killing activity and home to tumor site in vivo. (A) NKG2D ligands were expressed on A20 tumor cells, (B) but not on GVHD target tissues such as small (SB) and large bowel (LB), skin, and liver on day 3 after irradiation. (C) Cytolysis against A20 cells. Effectors (allogeneic CIK cells) were combined with targets at indicated ratio either alone or in the presence of isotype control antibodies or NKG2D antibodies. *P < .05. (D) A20 luc+ leukemia/lymphoma cells (106; H-2d) were injected into Balb/c (H-2d) hosts in the right flank subcutaneously after lethal irradiation, followed by injection of 5 × 106 FVB wild-type (H-2q) TCD-BM with or without 10 × 106 CIK cells generated from FVB wild-type animals. Tumor regression was observed in CIK cell–receiving mice. (E) Emitted photons from luc+ tumor cells over time. (F) A20 cells (106) were injected into the right flank of Balb/c hosts subcutaneously after lethal irradiation, followed by injection of 5 × 106 FVB wild-type TCD-BM with 10 × 106 CIK cells generated from luc+ L2G85 animals. Accumulation of luc+ CIK cells was observed in the tumor site. (G) CIK cells were collected on day 7 after BMT and compared with expanded CIK cells prior to injection. Lethally irradiated Balb/c (H-2d) mice were given 5 × 106 C57BL/6 BM plus 10 × 106 CIK cells generated from GFP+C57BL/6 splenocytes. On day 7, GFP+ cells were recovered from the host spleen by FACS sorting and then used in a 51Cr release cytotoxicity assay against A20 cells. Error bars represent SD. (H) Activation status of allogeneic CIK cells from the host spleen in vivo was analyzed on day 7 after BMT.

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