Recombinant Human IL-12 Promotes NK Cell Migration: Increased CXCL10, Co-Receptors IL-12Rβ2 and CXCR3, and Bright CD56 NK Cells In Healthy Human Subjects

Interleukin-12 (IL-12) is a heterodimeric cytokine with pleiotropic effects as a potent immunoregulatory molecule and a hematopoietic stimulatory factor. Studies in irradiated non-human primates (NHP) have shown the ameliorating effects of recombinant human IL-12 (HemaMax^TM, rHuIL-12) in a) regenerating hematopoietic tissue following radiation-induced bone marrow ablation, b) lowering the incidence of hemorrhage, and c) decreasing the risk of infections, collectively leading to an increase in NHP survival following lethal radiation exposure. A Phase 1b study was performed in healthy human subjects to evaluate the safety of rHuIL-12 following a single subcutaneously (sc) administered unit dose of 12μg to 32 healthy human subjects. Placebo was administered to 8 subjects. Peripheral blood samples were collected before and after rHuIL-12 administration and up to study day 14. Immunophenotyping of blood cell populations was conducted by Fluorescence Activated Cell Sorting (FACS).

rHuIL-12 administration resulted in a transient decrease in peripheral blood mean CD45⁺ leukocytes, CD45⁺ lymphocytes, CD45⁺CD16⁺CD56⁺ NK cells, and CD45⁺CD34⁺ hematopoietic progenitor cells. Nadirs were reached on day 2, 24 hours after rHuIL-12 administration, for lymphocytes (54.2% reduction from baseline), CD16⁺CD56⁺ NK cells (80.4% reduction from baseline), and CD34⁺ hematopoietic progenitor cells (37.9% reduction from baseline). Cell levels returned to approximately baseline levels on day 5 for CD16⁺CD56⁺ NK cells, by day 7 for lymphocytes and CD34⁺ cells and by day 9 for CD45⁺leukocytes. Placebo was without effect. Plasma concentrations of IFNγ (Interferon γ) and the chemokine CXCL10 (Interferon γ-induced protein 10), as determined by ELISA, were increased on days 3 and 4 in rHuIL-12-treated subjects. Placebo was without effect.

Cell surface expression of IL-12Rβ2, a subunit of the heterodimeric receptor for IL-12, was also studied by flow cytometry. IL-12Rβ2⁺ NK (CD16⁺CD56⁺) cells showed a decrease in levels on day 2, with a reduction of 80.4% from baseline. In addition, the mean fluorescence intensity (MFI) was determined for bright CD56, on NK cells. There was an increase in CD56 MFI for the rHuIL-12-treated subjects, while no effect was seen in the placebo treated subjects. The peak of expression occurred on day 3 (an increase of 55.5%) and had returned to baseline by day 11.

Increased CD56 expression is found on a NK subset uniquely equipped to traffic to sites of innate and adaptive immune responses. IL-12, initially called NK cell stimulating factor, can regulate NK cell recruitment to tissues by inducing migration and interaction with endothelial cells. The transient change in IL-12Rβ2⁺ NK (CD16⁺CD56⁺) cells after rHuIL-12 administration, together with the increase in bright CD56⁺NK cells, suggests that NK cells may be leaving the peripheral blood and migrating into the tissues, fulfilling their role of immune surveillance.

Using human peripheral blood mononuclear cells (PBMC) obtained from healthy donors rHuIL-12 was shown by qPCR to induce IFNγ and CXCL10 mRNA expression. Furthermore rHuIL-12 induced a transient upregulation of co-receptors IL-12Rβ2 and CXCR3 (the Gα_iprotein-coupled receptor for CXCL10) on lymphocytes, as determined by FACS.

Our data indicate that rHuIL-12 administration to healthy human subjects induces IL-12Rβ2⁺, CD16⁺CD56⁺ NK cell migration from the peripheral blood into the tissue compartment, through a mechanism facilitated by IFNγ-induced CXCL10 chemokine and its receptor CXCR3. Further it is likely that this mechanism is the basis for the transient decrease of CD45⁺ leukocytes, CD45⁺ lymphocytes, CD45⁺CD16⁺CD56⁺ NK cells, and CD45⁺CD34⁺hematopoietic progenitor cells, described above, following rHuIL-12 administration to healthy human subjects.

This project has been funded in part with Federal funds from the Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, Office of the Secretary, Department of Health and Human Services, under Contract No. HHSO100201100037C.