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. 2009 Sep 17;114(12):2417-26.
doi: 10.1182/blood-2008-12-189266. Epub 2009 Jul 15.

Safety and immunologic effects of IL-15 administration in nonhuman primates

Carolina Berger  1 , Michael Berger, Robert C Hackman, Michael Gough, Carole Elliott, Michael C Jensen, Stanley R Riddell
Affiliations

Safety and immunologic effects of IL-15 administration in nonhuman primates

Carolina Berger et al. Blood. .

Abstract

The administration of cytokines that modulate endogenous or transferred T-cell immunity could improve current approaches to clinical immunotherapy. Interleukin-2 (IL-2) is used most commonly for this purpose, but causes systemic toxicity and preferentially drives the expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cells, which can inhibit antitumor immunity. IL-15 belongs to the gamma(c) cytokine family and possesses similar properties to IL-2, including the ability to induce T-cell proliferation. Whereas IL-2 promotes apoptosis and limits the survival of CD8(+) memory T cells, IL-15 is required for the establishment and maintenance of CD8(+) T-cell memory. However, limited data are available to guide the clinical use of IL-15. Here, we demonstrate in nonhuman primates that IL-15 administration expands memory CD8(+) and CD4(+) T cells, and natural killer (NK) cells in the peripheral blood, with minimal increases in CD4(+)CD25(+)Foxp3(+) regulatory T cells. Daily administration of IL-15 resulted in persistently elevated plasma IL-15 levels and transient toxicity. Intermittent administration of IL-15 allowed clearance of IL-15 between doses and was safe for more than 3 weeks. These findings demonstrate that IL-15 has profound immunomodulatory properties distinct from those described for IL-2, and suggest that intermittent administration of IL-15 should be considered in clinical studies.

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Figures

Figure 1
Figure 1
Effect of daily subcutaneous IL-15 in 2 immunocompetent macaques. (A) Schedule of IL-15 administration (15 μg/kg per day). Each formula image represents a single daily dose of IL-15 (15 μg/kg), and the formula imageindicates a single daily dose of IL-15 (5 μg/kg). (B-C) Absolute numbers of WBCs, lymphocytes, and neutrophils (ANCs) in the peripheral blood before, during, and after IL-15 therapy. Absolute cell numbers of (B) WBCs (▵) and lymphocytes (●) per microliter of peripheral blood or (C) ANCs (◇) per microliter of peripheral blood and level of hemoglobin (■ Hb; gd/L) were determined on the indicated days. The arrows indicate the duration of the IL-15 administration. (D) Photomicrographs of hematoxylin and eosin–stained BM biopsy sections obtained after 7 days of treatment display hypoplasia and moderate focal hemorrhage (top, 5× objective). At higher magnification (middle, 40× objective), there is also fat necrosis and stromal edema. The scattered cells are erythroid precursors, lymphocytes, and macrophages. Normal clusters of maturing myeloid cells are absent (bottom, 100× objective). Photomicrographs were obtained with a Leica DFC320 camera on a Leica DM3000 microscope and processed with the Leica Application Suite version 3.1.0 (Leica Microsystems GmbH). Images were processed using Photoshop 7.0 software (Adobe Systems). Bars represent 100 micrometers. (E) Schedule of IL-15 administration for macaque 05078 (15 μg/kg per day). Each formula image represents a single daily dose of IL-15. (F) Absolute cell numbers of WBCs (▵) and lymphocytes (●) per microliter of peripheral blood before, during, and after IL-15 therapy. (G) ANCs (◇) per microliter of peripheral blood and level of hemoglobin (■ Hb; gd/L) before, during, and after IL-15 therapy. formula image indicates the duration of the IL-15 administration. (H) Photomicrographs of hematoxylin and eosin–stained marrow biopsy sections obtained after the IL-15 treatment as described in panel D. Bars represent 100 micrometers.
Figure 2
Figure 2
Analysis of lymphocyte subsets after daily administration of IL-15. (A-B) Samples of PBMCs were obtained from macaque 02269 (A) or macaque 05078 (B) before, during, and after the IL-15 treatment. Aliquots of PBMCs were stained with mAbs binding to CD3, CD4, CD8, or CD16, and examined by flow cytometry. The data for CD3+CD4+ (▵), CD3+CD8+ (●), and CD16+ (♦) are shown as the absolute cell number of each subset per microliter of blood at the indicated days. (C) Representative sample of PBMCs obtained from macaque 05078 at the end of the IL-15 treatment. Aliquots of PBMCs were stained with mAbs binding to CD3, CD4, CD8, and a TCRγδ-specific mAb, and examined by flow cytometry. (Left panel) Cells are gated on CD3+ T cells. The values indicate the proportion of each of the T-cell subsets (in percentage). (Right panel) TCRγδ T cells within the PBMC population. (D) The data for CD3+CD4−CD8− cells (◇) are shown as the absolute cell number of each subset per microliter of blood at the indicated days. (E-F) Expression of Ki-67. Aliquots of PBMCs from macaque 02269 (E) or macaque 05078 (F) were obtained before, during, or after the IL-15 treatment and were examined for intracellular expression of Ki-67 within the CD3+CD4+, CD3+CD8+, or CD3+CD4−CD8− T-cell compartment, and within the CD16+ NK cell subset. The fraction of Ki-67–expressing cells in percentage (%) in each of the subsets on the indicated days before, during, and after the IL-15 treatment is shown. formula image indicates the duration of the IL-15 administration.
Figure 3
Figure 3
Effect of daily IL-15 on absolute number and Ki-67 staining of CD8+ and CD4+ TN, TCM, and TEM subsets. (A-B) CD8+ TN, TCM, and TEM subsets. Samples of PBMCs from macaque 02269 (A) or macaque 05078 (B) at the indicated days before, during, and after the IL-15 treatment were stained with mAbs that bind to CD3, CD8, or CD95, and CCR7 to distinguish TN, TCM, and TEM subsets, and examined by flow cytometry. The data for each T-cell subset is shown as the fold increase of the absolute numbers per microliter of blood on the indicated day relative to the absolute cell numbers per microliter of the T-cell subset enumerated on the start of the treatment. (C-D) Expression of Ki-67 by CD8+ T-cell subsets. The data show the fraction of cells in percentage (%) within the CD8+ TN, TCM, and TEM subsets that express Ki-67 on the indicated days. (E-F) CD4+ TN, TCM, and TEM subsets. Samples of PBMCs from macaque 02269 (E) or macaque 05078 (F) at the indicated days before, during, and after the IL-15 treatment were stained with mAbs that bind to CD3, CD4, or CD95, and CCR7 to distinguish TN, TCM, and TEM subsets, and examined by flow cytometry. The data for each T-cell subset are shown as the fold increase of the absolute numbers per microliter of blood on the indicated day relative to the absolute cell numbers per microliter of the T-cell subset enumerated on the start of the treatment. (G-H) Expression of Ki-67 by CD4+ T-cell subsets. The data show the fraction of cells in percentage (%) within the CD4+ TN, TCM, and TEM subsets that express Ki-67 on the indicated days. → indicates the duration of the IL-15 treatment.
Figure 4
Figure 4
Effects of intermittent administration of IL-15 in immunocompetent macaques. (A) Schedule and dose of administration. Each formula image represents a single dose of IL-15 given every 3 days. (B) Absolute cell numbers of WBCs, lymphocytes, neutrophils (ANCs), as well as T-cell subsets, and NK cells per microliter of peripheral blood of macaques 97067, K00043, 02279, M05118, and T02392. Aliquots of the PBMCs were obtained from each of the macaques before, during, and after the IL-15 treatment, and stained with mAbs to CD3, CD4, CD8, and CD16. The data for CD3+CD8+, CD3+CD4+, and CD16+ cells are shown as the absolute cell number of each subset per microliter of peripheral blood at the indicated days. (C) Ki-67 expression. The percentage (%) of Ki-67+ cells within the CD3+CD8+, CD3+CD4+ T-cell, and CD16+ NK cell subsets is shown at the indicated days before, during, and after the IL-15 treatment of macaques 97067, K00043, 02279, M05118, and T02392. The vertical bar represents the mean.
Figure 5
Figure 5
Analysis of CD8+ TN, TCM, and TEM subsets during intermittent IL-15 administration. (A) Samples of PBMCs were obtained from macaques 97067, K00043, 02279, M05118, and T02392 before, during, and after the treatment with IL-15. Cells were stained with mAbs binding to CD3, CD8, or CD95, and CCR7 and analyzed by flow cytometry to distinguish TN (CD95lowCCR7+), TCM (CD95+CCR7+), and TEM (CD95+CCR7−) subsets. The fold increase of each subset at the indicated time of treatment is shown. The vertical bar represents the mean. formula image indicates the duration of the IL-15 treatment. The last posttreatment PBMC sample from K00043 was obtained on day 283. (B-C) Expression of Ki-67 by CD8+ T-cell subsets. (B) Representative data of macaque 02279. Gating of CD8+ TN, TCM, and TEM subsets stained for Ki-67 expression. PBMCs were stained with mAb specific for CD3, CD8, CD95, and CCR7, permeabilized, and stained with an antibody that binds to Ki-67, and analyzed by flow cytometry. Cells are gated on CD3+CD8+ T cells. The inset value in the upper right quadrant indicates the proportion of Ki-67+ T cells (in percentage). (C) The percentage (%) of Ki-67+ T cells within the CD8+ TN, TCM, and TEM subsets is shown at the indicated time of IL-15 treatment of macaques 97067, K00043, 02279, M05118, and T02392. The vertical bar represents the mean. formula image indicates the duration of the IL-15 treatment.
Figure 6
Figure 6
Effect of IL-15 on circulating CMV-specific CD8+ memory T cells. (A) Samples of PBMCs were obtained from CMV-immune macaques 97067, K00043, 02279, and T02392 at the indicated time before, during, or after intermittent IL-15 administration. The samples were stimulated with the CMV peptide, and examined by CFC for the presence of IFN-γ–producing CMV-specific CD8+ T cells. Controls consisted of PBMCs cultured in medium alone to subtract background levels. The absolute number of CMV-specific CD8+ T cells per microliter of peripheral blood was calculated based on the frequency of T cells that produced IFN-γ after CMV-peptide stimulation at each time point and the absolute number of CD8+ T cells. The fold increase of the absolute number of CMV-specific CD8+ T cells per microliter of peripheral blood compared with the start of the treatment is shown. The vertical bar represents the mean. formula image indicates the duration of the IL-15 administration. *K00043 and T02392: no sample was available from week 8. (B-C) Representative data are shown for macaque K00043. PBMCs obtained before and on day 6 of the IL-15 treatment were stimulated with media alone or with CMV peptide, and examined by CFC for expression of IFN-γ and for Ki-67-expression, respectively. (B) The samples are gated on lymphocytes or (C) on peptide-stimulated CMV+ CD8+ IFN-γ+ T cells.
Figure 7
Figure 7
Absolute and relative numbers of Foxp3+ regulatory CD4+ T cells in immunocompetent macaques during IL-15 therapy. Samples of PBMCs obtained from macaques 97067, K00043, 02279, M05118, and T02392 were stained with mAbs binding to CD3, CD4, and CD25. The cells were then fixed and permeabilized, stained with an anti-Foxp3 antibody, and examined by flow cytometry. (A) The mean (± SEM) of the absolute cell number of CD4+Foxp3+ T cells per microliter of peripheral blood in the 5 macaques receiving intermittent IL-15 treatment at the indicated time is shown. (B-C) The fold increase of the absolute number of (B) CD4+CD25+Foxp3+ T cells per microliter or (C) CD4+ T cells per microliter in the peripheral blood compared with the start of the treatment is shown at the indicated time before, during, and after the respective intermittent IL-15 administration. Data are shown from macaques 97067, K00043, 02279, M05118, and T02392. The vertical bar represents the mean. formula image indicates the duration of the IL-15 administration.
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