IFN therapy and recombinant IL-29 offers protection against severe gastrointestinal aGvHD

Graft-versus-host disease (GvHD) limits the benefits offered by allogeneic stem cell transplantation (allo-SCT) or bone marrow transplantation (BMT) in patients with high-risk malignancies. One of the key target sites for GvHD development is the gastrointestinal (GI) mucosa. This site acts as a protective interface between the luminal microbiota and immune cell populations, and any damage to this interface promotes immune suppression and acute GvHD (aGvHD) development.

Interferons (IFNs) play different roles in the GI tract, with type III IFNs (IFNλ) identified as vital in controlling immune responses at mucosal surfaces. Therefore, Henden et al,¹ have recently defined the role of type III IFNλ and pegylated IL-29 in protecting intestinal stem cells (ISC), which can be used as a therapy in GI-GvHD patients prior to BMT to prevent loss of ISC and mucosal integrity.

Methods

• Tamoxifen-dependent Cre recombinase activity was induced in female mice (≥6 weeks) using 1 mg/day, intraperitoneally for 5 days, 2 weeks before transplant.
• GvHD was induced through transplantation of 5 × 10⁶ T cells and 10 × 10⁶ unmanipulated bone marrow (BM) cell grafts.
• T-cell depleted BM was used for non-GvHD grafts.
• 1 × 10⁶ BCR-ABL nup98hoxA9 or MLL-AF9 transformed leukemia cells were introduced with the graft for graft-versus-leukemia (GvL) experiments.
• Anti-NK1.1 antibody (PK136) or a mouse IgG control was given intraperitoneally on Day −1 (1 mg/mouse), Day 3, and Day 6 (0.5 mg/mouse) for NK cell depletion.
• 5 µg/day of PEG-rIL-29 treatment was delivered intraperitoneally either 3 days prior to tissue harvest or on Days −2, −1, and 0 of transplantation.
• Flow cytometry and antibody staining were used for assessment.
• Organoids were grown from crypt preparations harvested from the small intestine or colon of naïve wild type (WT) or Ifnlr1^−/− mice.
• Organoids grown from single cell sorted preparations of Lgr5⁺ cells were obtained by a modification of the crypt isolation method to generate single cell suspensions.

Results

IFNλ signaling in recipient tissue determines GvHD severity

• The protective role of IFNλ was identified from histopathology results from Ifnlr1^−/− (homozygous mutant lacking the IFN receptor gene) mice on Day 7 posttransplant. These Ifnlr1^−/− mice had impaired barrier function in the GI tract compared with WT mice, and systemic dysregulation of IFNλ and IL-6. A reduction in systemic and small intestine levels of IL-28 was observed after irradiation, with systemic levels remaining low after BMT, and small intestine levels increasing during GvHD. Severe GvHD was observed in Ifnlr1^−/− recipients.

Secretion of IFNλ and Ifnlr1-signaling offers protection against GvHD in multiple tissues

• Ifnlr1-signaling deficiency in hematopoietic and nonhematopoietic compartments in chimeric mice caused enhanced GvHD histopathology in the colon.
• Higher levels of serum IFNγ and IL-6 were also observed in recipients.
• Moreover, over 5−10-fold higher expression of Ifnlr1 mRNA was observed in naïve GI mucosa compared with the liver and spleen, which suggests direct IFNλ-mediated protection within the epithelium of the GI tract.

Ifnlr1-signaling in recipient NK cells controls the rate of donor T-cell expansion after BMT

• Rapid donor derived T-cell expansion was observed in the spleen and colon of Ifnlr1^−/− recipients on Day 4, post-BMT. Additionally, higher levels of CD4⁺/CD8⁺ T cells were observed in Ifnlr1^−/− recipients on Day 7, post-BMT. However, equivalent CD4⁺/CD8⁺ T-cell production was also observed when stimulated by WT and Ifnlr1^−/− dendritic cells. Furthermore, there was no increase in the proportion of donor CD4⁺/CD8⁺ T cells expressing IFNγ. Thus, the increase observed in serum IFNγ in Ifnlr1^−/− recipients was due to increased overall T-cell expansion.
• The impact of antibody depletion of recipient NK cells with anti-NK1.1 was compared between WT and mutant recipients in in vivo cytotoxicity assays. An increase in donor T-cell engraftment and a dysregulation in systemic IFNγ were observed in WT, whilst a reduction in its impact was observed in Ifnlr1^−/− mice. However, this enhanced anti-donor response in WT was lost on depletion of NK cells in recipients.
• In vitro testing where conditional deletion with NKp46^Cre.Ifnlr1^fl/fl mice was carried out, depicted loss of IFNλ receptor responses in NK cells, which is responsible for a reduced cytotoxic function in Ifnlr1^−/− recipients, and the absence of IFNλ signaling in NK cells reduced cell survival.
• RNA sequencing analysis of NK cells with deleted IFNλ receptors demonstrated differential gene expression affecting pathways including apoptosis. This shows that Ifnlr1-signaling in recipient NK cells is involved in controlling the rate of donor T-cell expansion after BMT.

Impact of microbiome on aGvHD

• 16S ribosomal sequencing and fecal microbial content analysis was performed to identify the impact of microbiota on the aGvHD phenotype in co-housed and separately housed WT and Ifnlr1^−/− BMT mice. Significant differences observed in the beginning were diminished among the two groups after 4 weeks. This indicates that an altered microbiome in mutant mice did not contribute towards aGvHD enhancement.

Effect of Ifnlr1-signaling in Lgr5⁺ intestinal stem cells

• To identify if Ifnlr1-signlling plays a vital role in maintenance and proliferation of GI epithelia and ISC, crypts containing ISC were isolated from the colon of naïve Ifnlr1^−/− and WT mice. Ifnlr1^−/− crypt donors generated fewer and smaller organoids.
• To confirm that loss of Ifnlr1-signaling in Lgr5⁺ ISC was relevant to the Ifnlr1^−/− GvHD phenotype, Lgr5-EGFPIRES^creERT2.Ifnlr1^fl/fl (Lgr5^Cre.Ifnlr1^fl/fl) mice were generated. Tamoxifen-induced conditional deletion of Ifnlr1 in ISC in Cre⁺ mice resulted in severe GI aGvHD, and fewer organoids grown 7 days posttransplantation compared to Cre⁻ controls.
• These results suggest that Ifnlr1-signaling responses in Lgr5⁺ ISC dictate Ifnlr1^−/− GvHD phenotype.

IFNλ treatment produces a proliferative phenotype in GI stem cells

• The therapeutic efficacy of IFNλ was assessed through generation of colon organoids from mice receiving PEG-rIL-29 or PBS for 3 days prior to crypt harvest. Although there was no consistent increase in size, an increase in numbers of primary colon organoids grown from PEG-rIL-29-treated mice was seen.
• PEG-rIL-29 resulted in a higher growth of ISC in the colon compared with the small intestine when tested in Lgr5-EGFPIRES^creERT2 reporter mice.
• It was also observed that PEG-rIL-29 functionality was independent of type-1 IFN receptor and IL-22.
• Distinct transcriptional signatures were observed in Lgr5⁺ and Lgr5⁻ compartments with PBS and PEG-rIL-29 treatment as observed during RNA sequencing transcriptional analysis of CD45.2-negative, EpCAM-positive, and Lgr5-positive and negative fractions.
• IFN cytokines and IFN regulatory factors supported PEG-rIL-29-induced transcriptional response regulating the proliferation in Lgr5⁺ cells. This suggests an enhanced proliferative capacity of Lgr5⁺ cells.

IFNλ treatment protects from GvHD within the GI tract

• The BALB/c → B6 and B6 → B6D2F1 systems were tested to evaluate the prophylactic effect of PEG-rIL-29 on GvHD from Day −2 to day 0 of transplantation. PEG-rIL-29 treatment resulted in a prolonged survival in both models, and GI-GvHD histopathology was reduced. It was observed that PEG-rIL-29 treatment did not inhibit early hematopoietic engraftment, even in the absence of donor T cells.
• Furthermore, PEG-rIL-29 pretreatment appeared to enhance epithelial proliferation post-BMT when examined in vitro. A significantly higher Ki67 expression was observed in the colon and the small intestine epithelial cells, including crypt bases, post-BMT in PEG-rIL-29 pretreated cells.
• To identify the PEG-rIL-29 treatment effect on ISC numbers in the absence of NK cells, a BALB/c → B6 mice model was used in NK cell-depleted recipients. Results from multispectral imaging revealed an increase in ISC numbers and no change in Paneth cell numbers, proximity of ISC to Paneth cells, or numbers of infiltrating T cells in the GI tract.
• The data functionally validate that the upregulation of proliferative pathways in Lgr5⁺ cells, seen in the RNA sequence analysis, was mediated by PEG-rIL-29.
• Furthermore, it was also confirmed that PEG-rIL-29 treatment neither had any impact on graft-versus-leukemia (GvL) using GFP-expressing BCR-ABL nup98HoxA9 acute myeloid leukemia (AML), nor did it influence leukemia growth, or leukemic death in mice, relative to PBS controls.

Conclusion

• IFNλ plays an important role in the immune defence at the mucosal barriers. It improves the epithelial barrier integrity by increasing proliferation and regeneration of mature GI epithelia through Lgr5⁺ ISC, which contributes to the protection from GI aGvHD.
• IFNλ prevents GI aGvHD through both hematopoietic and nonhematopoietic recipient cells.

• Absence of Ifnlr1-signaling induces impaired NK cell function, which in turn accelerates donor T-cell engraftment.  Thus, IFNλ-signaling in recipient NK cells is desired to limit GvHD target tissue damage.
• IFNλ-dependent improvements in the intestinal epithelial barrier and ISC function, at least in part via effects on gut epithelium, were observed.

 IFNλ-mediated protection of the GI epithelia in allogeneic BMT can be explored to identify therapeutic potential, as PEG-rIL-29 administration limited ISC loss and protected from severe GI aGvHD.