ALBERT

Description: Allografting as a curative approach for many hematological malignancies is hampered by the occurrence of acute graft-versus-host disease (aGvHD). Interleukin (IL)-18 stimulates T helper 1 (Th1) and Th2-mediated immune responses and has been shown to modulate aGvHD. It is still unknown whether increased IL-18 levels during aGvHD are of host or donor origin and how the absence of IL-18 impacts migration and expansion of conventional CD4+CD25− (Tconv) and CD4+CD25+ regulatory (Treg) T cells in vivo. By utilizing IL-18 gene deficient donor versus recipient animals we found that the major cytokine production during the early phase of aGVHD induction was recipient derived, while donor hematopoietic cells contributed significantly less. By generating IL-18−/ − luciferase transgenic mice we were able to investigate the impact of IL-18 on Tconv and Treg expansion and trafficking with in vivo bioluminescence imaging. While migration to secondary lymphoid organs was not significantly impacted by the absence of host IL-18, Tconv but not Treg expansion increased significantly. Absence of host IL-18 production translated into lower IFN-γ levels in the early phase after transplantation. We conclude that host derived IL-18 is a major factor for IFN-γ production that may have a protective effect on CD4+ mediated aGvHD, but is non-essential for Treg expansion in an allogeneic environment.

Description: NKT cells, which are CD1d reactive and express an invariant TCR, are thought to play an immunoregulatory role in suppressing dysfunctional immune reactions including graft vs. host disease (GVHD). Whether non-manipulated donor-type NKT can suppress GVHD following adoptive transfer has not been addressed, nor has the trafficking pattern of NKT in a hematopoetic transplantation (HCT) setting. To determine how effectively NKT proliferate and traffic in a HCT setting, 5.5x105 highly purified (〉95%) NKT (DX5+TCRβ+CD4+) from luciferase positive (luc+) C57BL/6 (H-2b) mice were transferred into lethally irradiated Balb/c (H-2d) recipients along with 5x106 T-cell depleted bone marrow (TCD-BM) from wild-type C57BL/6 mice. Proliferation and migration of luc+NKT was monitored by bioluminescence imaging (BLI). By day 4 after transfer, a prominent signal was observed in the spleen and lymph node (LN) sites. Between days 7 and 10, the NKT migrated to skin, while still remaining present in high numbers in LNs, but decreasing to low levels in spleen. The total photons emitted per mouse reached a peak at approximately 25 days after transplantation, followed by a steady decline. The NKT expansion was more vigorous than that of luc+CD4+CD25+ regulatory T cells (Tregs), which also peak around day 25, but do not show extensive migration to skin. Expansion of NKT was far less than conventional (CD4+ and CD8+) T cells (Tcon), with approximately 10 times more photons/mouse being emitted from 5x105 luc+Tcon as from 5.5x105 NKT. The NKT did not cause GVHD where Tcon rapidly resulted in acute GVHD and animal mortality. To assess the impact of donor-type NKT on GVHD induction by Tcon, we co-transferred various doses of highly purified wt NKT at day 0 with 5x106 TCD-BM, followed by 5x105 luc+Tcon at day 2. Weight and survival of groups were monitored, as well as the proliferation of Tcon by BLI. In groups receiving only Tcon, 50% of the mice died within 2 weeks, and 90% died by day 80. Remarkably, if 2.5x104 sorted NKT were transferred, 100% of the mice survived past day 100. To achieve the same effect with Tregs, 2.5x105 Tregs were required. Mice treated with 2.5x104 NKT lost more weight at early time points than those receiving 2.5x105 Tregs, but both groups recovered from this weight loss and did not exhibit other signs of GVHD (hair loss, hunched back, diarrhea, etc). Interestingly, 2.5x104 NKT caused only a slight reduction in Tcon proliferation, whereas 2.5x105 Treg strongly reduced Tcon proliferation. Surprisingly, when the dose of NKT was increased to 5x104, survival was only 62%, and when increased to 1x105 cells, only 50% of mice survived past day 100. To determine how NKT reduce GVHD, we examined intracellular levels of various cytokines in Tcon with or without 2.5x104 NKT, following HCT. At 8 days after HCT, mice receiving NKT had reductions in the number of IL-17-positive cells (CD4: 2.6% to 0.84%; CD8: 2.5% to 0.66%), and TNFα+ cells (CD4: 45% to 27%; CD8 36% to 24%) in cells from LNs. By day 11, IL-17-positive cells had declined to undetectable levels and TNF levels between groups were equivalent. IFNg levels, which were high in both NKT treated and untreated groups at day 8 (85%–95%), decreased significantly in NKT treated mice by day 11 (CD4: 40%; CD8: 43%), but were still abundant in Tcon only mice (CD4: 78%; CD8: 80%). Together these data indicate that NKT cells persist in vivo upon adoptive transfer and suppress GVHD by decreasing the percentage of Tcon secreting pro-inflammatory cytokines.

Description: Natural Killer (NK) cells have the ability to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT) during allogeneic bone marrow transplantation (BMT). Previous studies in allogeneic BMT models have shown NK cell trafficking to and proliferation in lymphoid organs and GVHD target organs, which are also sites of donor T cell trafficking. This study aims to investigate the impact of NK cells on alloreactive, GVHD-inducing donor T cells. Interleukin-2 activated allogeneic NK cells isolated from C57Bl6 (H–2b) or FVB (H–2q) animals were transplanted along with T cell-depleted bone marrow into lethally irradiated BALB/c (H–2d) mice, followed 2 days later by luciferase-expressing CD4+ and CD8+ conventional T cells from the same donor strain (NK+Tcon group). Control mice received lethal irradiation and T cell-depleted bone marrow on day 0, and luciferase-expressing T cells on day 2 after transplant (Tcon group). Bioluminescence imaging of NK+Tcon mice revealed a significantly lower T cell bioluminescent signal (p=0.03 for FVB into BALB/c on day 6) than from Tcon mice. CFSE proliferation analysis of alloreactive T cells on day 3 after transplant showed no significant change in the percent of donor T cells that have divided in the spleen, and only a slight decrease in the percent of T cells that have divided in the lymph nodes when NK cells are present. However, at this timepoint 82% of the proliferating cells have divided past the third generation, in contrast to 64% in the NK+Tcon mice. Donor T cells in both groups become equally activated in vivo, expressing similar levels of the early-activation marker CD69. T cells re-isolated from NK+Tcon animals at day 5 stained 2 to 10-fold higher for the TUNEL apoptosis marker than those from Tcon mice in the mesenteric and peripheral lymph nodes, respectively (p

Description: Natural Killer (NK) cells have the ability to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT) following murine allogeneic bone marrow transplantation (BMT). Prior studies have shown that NK cells suppress GVHD by eliminating recipient dendritic cells. To assess additional potential mechanisms of GVHD suppression we evaluated the impact of donor NK cells on GVHD-inducing donor T cells. Interleukin-2 activated allogeneic NK cells isolated from C57Bl6 (H-2b) or FVB (H-2q) animals were transplanted along with T cell-depleted bone marrow (TCD-BM) into lethally irradiated BALB/c (H-2d) mice, followed 2 days later by luciferase-expressing CD4+ and CD8+ conventional T cells (Tcon) from the same donor strain (Tcon+NK group). Control mice received TCD-BM on day 0, and luciferase-expressing T cells on day 2 after transplant (Tcon group). Bioluminescence imaging of Tcon+NK mice revealed a significantly lower T cell bioluminescent signal compared to Tcon mice (p=0.01 on day 5 post T cell transplant). We assessed the impact of NK cells on donor T cell activation and proliferation. CFSE proliferation analysis of alloreactive CD4 and CD8 T cells reisolated on day 4 post transplant showed a decreased percentage of dividing donor T cells in the Tcon+NK group. A reduced percentage of T cells in the Tcon+NK group as compared to the Tcon group expressed the T cell activation marker CD25 (11% and 49%, respectively, among donor CD4) and a reduced percentage of T cells from the Tcon+NK group down-regulated CD62L. Reisolated donor T cell numbers were reduced in the Tcon+NK mice compared to Tcon control mice. The impact of donor NK cells on donor Tcon function was addressed by intracellular cytokine staining. Fewer donor T cells reisolated from the spleen and lymph nodes of Tcon+NK mice produced the proinflammatory cytokines IFN-γ and IL-2 on day 3 after transplant. These observations can be explained by an NK cell-mediated induction of apoptosis in the donor Tcon. T cells reisolated from the peripheral lymph nodes of Tcon+NK animals at day 4 post transplant stained higher for the TUNEL apoptosis marker than those from Tcon mice (p

Description: Based on their ability to control T-cell homeostasis, Foxp3+CD4+CD25+ regulatory T cells (Tregs) are being considered for treatment of autoimmune disorders and acute graft-versus-host disease (aGVHD). When combining Tregs with the immunosuppressant rapamycin (RAPA), we observed reduced alloreactive conventional T-cell (Tconv) expansion and aGVHD lethality compared with each treatment alone. This synergistic in vivo protection was paralleled by intact expansion of polyclonal Tregs with conserved high FoxP3 expression. In contrast to Tconv, activation of Tregs with alloantigen and interleukin-2 preferentially led to signal transducer and activator of transcription 5 (STAT5) phosphorylation and not phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activity. Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative regulator of the PI3K/Akt/mTOR pathway, remained high in Tregs but not Tconv during stimulation. Conversely, targeted deletion of PTEN increased susceptibility of Tregs to mTOR inhibition by RAPA. Differential impact of RAPA as a result of reduced usage of the mTOR pathway in Tregs compared with conventional T cells explains the synergistic effect of RAPA and Tregs in aGVHD protection, which has important implications for clinical trials using Tregs.

Description: FoxP3+CD4+CD25+ regulatory T-cells (Treg) have been shown to effectively reduce the severity of experimental acute graft-versus-host disease (aGvHD) while sparing graft-versus-leukemia activity. These findings, in concert with the observation that human and murine Treg share functional characteristics, have fueled interest in clinical trials to control aGvHD. Recent data indicates that the immunosuppressant rapamycin (RAPA) in contrast to cyclosporine A does not interfere with in vivo function of Treg and could enhance Treg expansion in vitro by a yet unknown mechanism. To investigate the impact of mTOR inhibition on proliferating Treg and Tconv, both cell types were exposed to CD3/CD28 Mabs in the presence of different RAPA concentrations in vitro. Phosphorylation of mTOR downstream products p70S6K1 and 4E-BP1 were assessed by western blot and flow cytometry. Inhibition of the phosphorylation of p70S6K1 and 4E-BP1 was observed in both populations in the presence of RAPA. Interestingly, Treg were more resistant to mTOR inhibition as compared to Tconv and displayed significantly higher phosphorylated products in the presence of RAPA at 10 nM (MFI Treg vs Tconv, p