Activated Allogeneic NK Cells as Suppressors of Alloreactive Responses

H2^d ALAK cells from BALB/c mice significantly suppress the (A) proliferation and (B) killing capacity of H2^b splenocytes from C57BL/6 mice in allogeneic MLR stimulated with irradiated H2^d splenocytes from BALB/C mice. Allogeneic MLR was set up with 5 × 10⁵ splenocytes from both C57BL/6 and BALB/c mice. The ALAK cell numbers were as indicated in the figure. The same number of BALB/c splenocytes was also added as controls. ^∗Indicates P < .01.The data shown are the representative of four experiments.

Only the ALAK cells of stimulator origin can effectively suppress the allo-responses in the allogeneic MLR. The same number of H2^d ALAK cells and H2^b ALAK cells were added to the H2^b anti-H2^d allogeneic MLR same as in Figure 1. The (A) proliferation and (B) killing capacity of H2^b splenocytes from C57BL/6 mice were measured at the end of the 3-day culture. (C) 1.25 × 10⁵ H2^d or H2^k ALAK cells were added to H2^b anti-H2^d MLR. The same number of H2^k ALAK cells was also added to H2^b anti-H2^k MLR. The proliferation of H2^b splenocytes was measured at the end of the 3-day culture. (D) The killing capacity of H2^b splenocytes was also measured at the end of the 3-day culture. ^∗Indicates P < .01. The data shown are the representative of 3 experiments.

The suppressive effect of allogeneic ALAK cells was not dependent on TGF-β secretion. Anti-TGF-β and the isotype control antibodies (35 μg/mL) were added to the allogeneic MLR described in Figure 1. (A) The levels of TGF-beta secreted in the MLR reaction with or without anti-TGF-beta antibody were measured by ELISA. ND indicates not detected. (B) The proliferation of H2^b splenocytes from C57BL/6 mice stimulated with irradiated H2^d splenocytes were measured at the end of the 3-day culture by ³H-thymidine incorporation assay. (C) The killing capacity of H2^b splenocytes killing H2^d tumor cells (P815) were measured at the end of the 3-day culture by cytotoxicity assay. ^∗Indicates P < .01. #Indicates P < .05. The data shown are the representative of 3 experiments.

The increased number of H2^d ALAK cells could cause the diminishing of the H2^b CTLs in the allogeneic MLR. The CFSE-labeled or unlabeled H2^d ALAK cells were added to the allogeneic MLR described in Figure 1. (A) The capacity of H2^d ALAK cells to inhibit H2^b splenocytes killing H2^d tumor target was not affected by CFSE labeling. (B) The number of live H2^b CTLs left in the culture was significantly decreased by adding H2^d ALAK cells. ^∗Indicates P < .01. The data shown are the representative of 3 experiments.

The suppressive effect of allogeneic ALAK cells was partially dependent on perforin-mediated killing mechanism. The ALAK cells from wild type C57BL/6 mice, gld mice, and pfp mice (both on C57BL/6 background) were used in the MLR assay. The (A) proliferation and (B) killing capacity of H2^d splenocytes from BALB/c mice were measured at the end of the 3-day culture. ^∗Indicates P < .01. The data shown are the representative of 2 experiments.

Donor ALAK cells promoted donor engraftment in nonmyeloblative allogeneic BMT. The ALAK cells from BALB/c mice (H2^d NK) were infused into host C57BL/6 mice during nonmyeloblative allogeneic BMT. The ALAK cells from syngeneic C57BL/6 mice (H2^b NK) were also infused as control. (A) Flow analysis of the percent of donor engraftment 2 months after BMT. (B) Summary and statistical analysis of all the experiments.

Donor ALAK cells could suppress host alloreactive responses in nonmyeloablative allogeneic BMT. The ALAK cells from BALB/c mice (H2^d NK) were infused into host C57BL/6 mice during nonmyeloablative allogeneic BMT. The ALAK cells from syngeneic C57BL/6 mice (H2^b NK) were also infused as control. (A) Percent of host derived CD4⁺ and CD8⁺ T cells (H2^b T cells) out of total T cells 2 months after BMT. The host (B) proliferative responses and IL-2 production responding to donor type antigens (irradiated BALB/c splenocytes) and (C) the killing capacity to donor type tumor targets (P815 cells) were measured 2 months after BMT. Base control was the readout without alloantigen stimulation. ^∗Indicates P < .01. The data shown are the representative of 3 experiments.