Murine anti-third-party central-memory CD8(+) T cells promote hematopoietic chimerism under mild conditioning: lymph-node sequestration and deletion of anti-donor T cells

Abstract

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donorderived central memory CD8(+) T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti-third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

Figure 1

Anti–third-party Tcms without GVH reactivity support engraftment of TDBM allografts in sublethally irradiated mice. Sublethally irradiated (5.5Gy) Balb/c (H-2^d) mice were transplanted with 20 × 10⁶ C57BL/6-nude (H-2^b) BM cells with or without: (A) Five × 10⁶ allogeneic C57BL/6 Tcm (H-2^b) or (B) 5 × 10⁶ CB6 F1 Tcms (H-2^bd). Percentage of donor cells in peripheral blood was analyzed 90 days after transplant by FACS using anti-donor (H-2K^b) antibodies. Data represent results of at least 3 independent experiments (***P < .001). (C) Average weight change during 100 days after transplant of nude-BM or nude-BM and allogeneic Tcms.

Figure 2

Anti–third-party Tcms can be used with decreased irradiation dose when coadministered with low-dose rapamycin. Sublethally irradiated (4.5Gy TBI) Balb/c (H-2^d) mice were transplanted with 20 × 10⁶ C57BL/6-nude (H-2^b) BM cells with or without: 5 × 10⁶ allogeneic C57BL/6 Tcm (H-2^b; ▵ and ) or 5 × 10⁶ CB6 F1 Tcm (H-2^bd; ●). The indicated groups received subcutaneous injections of 0.5 mg/kg bw rapamycin during 5 days (days −1 to +4). Percentage of donor cells in peripheral blood, analyzed 90 days after transplant by FACS using anti-donor (H-2K^b) antibodies is presented. Data represents results of at least 2 independent experiments (*P < .05; ***P < .001).

Figure 3

Anti–third-party Tcms mediated chimerism specifically protects donor skin graft from rejection. Sublethally irradiated (5.5Gy/4.5Gy TBI) Balb/c (H-2^d) mice were transplanted with 20 × 10⁶ C57BL/6-nude (H-2^b) BM cells with or without administration of 5 × 10⁶ C57BL/6 Tcm. The indicated groups received subcutaneous injections of 0.5 mg/kg rapamycin during 5 days (days −1 to +4). On day 30 after BM transplantation skin grafting of donor-type (C57BL/6, H-2^b) as well as third-party (C3H, H-2^k) was carried out (each recipient was transplanted with both grafts). Recipients of skin grafts were either BM control mice (“5.5Gy+BM” and “4.5+BM” Groups) which had rejected the donor-BM or those Tcm-treated mice which had achieved donor-chimerism. (A) Representative images of skin grafts across experimental groups taken 4 months after skin grafting. (B) Bars show percentage of chimeras that accepted donor skin grafts across experimental groups during a > 4-month follow-up period after skin grafting. Data were pooled from 6 independent experiments (***P < .001 by X²).

Figure 4

Tcms systemically eliminate host anti-donor T cells. Seven-hundred TCR transgenic 2c-Luc⁺ CD8⁺ cells were injected on day −1 into supralethally irradiated (4Gy on day −5 and 10Gy on day −2) C57BL/6 mice. One day after 2c administration, the mice received 3 × 10⁶ Balb/c-nude BM cells (“HTC only”) or BM+5 × 10⁶ (CB6)F1 Tcm (“HTC+Tcm”). (A) Images were taken at indicated days after transplantation in live anesthetized animals using IVIS. Color intensity is normalized for all images. (B) On day 9 after transplantation, organs (LN, spleen, BM, lung, and liver) were harvested from 5 animals from each group and numbers of live (7aad⁻) CD8⁺ 2c cells were assessed by FACS analysis. Total numbers of 2c cells harvested from all organs tested in the absence (black bar) or presence (gray bar) of Tcm treatment are presented. Data represent mean ± SD of total numbers of 2c cells from 5 animals in each group (***P < .001). (C) Survival curves of mice from the different treatment groups.

Figure 5

Anti–third-party Tcm administration induces LN accumulation of host anti-donor HTCs. Seven-hundred TCR transgenic 2c-Luc⁺ CD8⁺ cells were injected on day −1 into supralethally irradiated (4Gy on day − 5, and 10Gy on day − 2) C57BL/6 mice. One day after 2c administration, the mice received 3 × 10⁶ Balb/c-nude BM cells (“HTC only”) or BM+5 × 10⁶ (CB6)F1 Tcm (“HTC+Tcm”). (A-C) Representative IVIS images depicting the difference in 2c-Luc⁺ cell distribution within the body of untreated (A, “HTC only”) versus treated (C, “HTC and Tcm”) mice on day 10 after transplantation. Color intensities were adjusted according to signal for each individual image. (B-D) On day 9 after transplantation, organs (LN, spleen, BM, lung, and liver) were harvested from 5 animals from each group and numbers of live (7aad negative, viability staining solution) CD8⁺ 2c cells were assessed by FACS analysis. (B) Distribution of 2c cells in the recipients' organs in the absence of Tcm treatment. (D) Distribution of 2c cells in the recipients' organs in the presence of Tcm treatment. Data represent mean ± SD of total number of 2c cells of 5 animals in each group (*P < .05; **P < .01; ***P < .001).

Figure 6

Tcm conjugation with antigen-specific HTC leads to decelerated and confined HTC movement in the LNs. Lethally (10Gy) and sublethally (6.5Gy) irradiated C57BL/6 mice received 0.5 to 2 × 10⁶ 2c CD8⁺ cells (dyed with SNARF; red for lethal irradiation and magenta for sublethal irradiation [RIC]), and 1.5 to 10 × 10⁶ CB6 (“specific”) or C57BL/6 (“nonspecific”) derived Tcms (green, each dyed with CMTMR). Videos, using 2-photon microscopy were recorded at the indicated time points (see supplemental Methods). (A-B) 2c-CD8⁺-Tcm conjugates detected 4 to 16 hours after injection of 2 × 10⁶ 2c CD8⁺ and 10 × 10⁶ CB6 cells (arrows mark conjugates). (Picture presented is a single frame from supplemental Videos 1 and 2). A video was recorded 16 hours after injection of 0.5 × 10⁶ 2c CD8⁺, and 1.5 × 10⁶ CB6 or C57BL/6 cells. (C-D) Dot plot depicting velocity of individual cells at the LN (2c HTCs in the presence of “nonspecific” or “specific” Tcms). (E-F) Dot plot depicting displacement rate of individual 2c HTCs at the LN (in the presence of “nonspecific” or “specific” Tcms). Red horizontal bars indicate mean. (G-J) 3-D paths of tracked cells normalized to their starting coordinates in the presence of “nonspecific” (G-I), or “specific” (H-J) Tcms. Axes represent a distance of ± 100 μm for lethal irradiation and ± 30 μm for sublethal irradiation (RIC). Results presented represent 1 of 3 independent experiments (***P < .001).