Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation

Abstract

Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL-activated NK cells (aNK-DLI) following HLA-matched, T-cell-depleted (1-2 × 10(4) T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3(+)-depleted, CD56(+)-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL(+)IL-15Rα(+) artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.

Figures

Figure 1

Protocol treatment and NK expansion schema. (A) Treatment schema. Asterisk denotes time point that was delayed to days (D) 15 and 24 to meet Food and Drug Administration–mandated regulatory requirements for subjects 1 and 2, respectively. (B) Processing of filgrastim-mobilized peripheral blood stem cells to yield a T cell–depleted PBSC graft and a T cell–depleted, activated NK donor leukocyte infusion (aNK-DLI). G-CSF, granulocyte macrophage colony-stimulating factor; TCD, T-cell–depleted; Wk, week.

Figure 2

Gross and histologic evidence of aGVHD. (A-I) Subject 1. Photographs of skin lesions (A-C) show palmar and solar erythema, and edema and bullous lesions in the lower extremities. Photomicrographs (all 200×) of hematoxylin and eosin (H&E) (D), anti-CD3 (E), and anti-CD56 (F) stained sections of skin involved with GVHD. There is basal vacuolization of the epidermis, apoptosis of epidermal keratinocytes, and infiltration of T cells and NK cells around dermal vessels and along the dermal–epidermal junction. Exocytosis—infiltration of lymphocytes into the epidermis—is also seen. (G-I) Photographs taken during flexible sigmoidoscopy that demonstrate severely congested, erythematous, eroded, granular, and inflamed mucosa in the rectum and distal sigmoid colon. Colonic biopsy (I) shows apoptotic crypt injury and crypt loss (H&E, ×200). (J-M) Subject 2. Photograph from colonoscopy (J) demonstrates congestion and mildly erythematous rectal mucosa. Colonic biopsy photomicrographs of H&E (K, ×400), anti-CD3 (L, ×600), and anti-CD56 (M, ×600). Crypt injury with epithelial apoptosis and crypt loss is observed, with infiltration of T and NK cells within and around injured crypts.

Figure 3

Activating receptor expression on NK cells in NK-DLI vs circulating in vivo and killing capacity of aNK-DLI. (A) Flow cytometry was used to phenotype the aNK-DLI product as well as the circulating lymphocyte subsets shown at the time of protocol enrollment (baseline), following EPOCH-F chemotherapy (pretreatment [PreTx]), and at sequential time points following HSCT. Asterisks represent time points wherein the phenotype measured differed from the product; *P < .05, **P < .005. Red, subjects who developed GVHD; black, subjects who did not develop GVHD. (B) Pooled analysis of aNK-DLI products tested for killing of TC71 Ewing sarcoma cell line without blocking fusion proteins (n = 9, designated “none”) and with the designated fusion proteins (n = 7, for each blocking antibody) to block the activating receptors noted. Asterisks denote where killing is different from that observed without fusion protein blockade (P < .05).