GM-CSF and IL-2 induce specific cellular immunity and provide protection against Epstein-Barr virus lymphoproliferative disorder

Abstract

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is a potentially life-threatening complication in immune-deficient patients. We have used the severe combined immune deficient (SCID) mouse engrafted with human leukocytes (hu-PBL-SCID) to evaluate the use of human cytokines in the prevention of EBV-LPD in vivo. Daily low-dose IL-2 therapy can prevent EBV-LPD in the hu-PBL-SCID mouse, but protection is lost if murine natural killer (NK) cells are depleted. Here we demonstrate that combined therapy with human GM-CSF and low-dose IL-2 is capable of preventing EBV-LPD in the hu-PBL-SCID mouse in the absence of murine NK cells. Lymphocyte depletion experiments showed that human NK cells, CD8(+) T cells, and monocytes were each required for the protective effects of GM-CSF and IL-2 combination therapy. This treatment resulted in a marked expansion of human CD3(+)CD8(+) lymphocytes in vivo. Using HLA tetramers complexed with EBV immunodominant peptides, a subset of these lymphocytes was found to be EBV-specific. These data establish that combined GM-CSF and low-dose IL-2 therapy can prevent the immune deficiencies that lead to fatal EBV-LPD in the hu-PBL-SCID mouse depleted of murine NK cells, and they point to a critical role for several human cellular subsets in mediating this protective effect.

Figure 1

Combined therapy with human GM-CSF and low-dose IL-2 significantly improves survival of hu-PBL-SCID mice in the absence of murine NK cells. Survival curves are shown for hu-PBL-SCID mice treated with daily subcutaneous injections of 500 IU of PEG-IL-2 (open triangles), 3 μg GM-CSF intraperitoneal injections every other day + IL-2 + ASGM-1 weekly (open circles), GM-CSF + ASGM-1 (filled triangles), or IL-2 + ASGM-1 (filled circles) after intraperitoneal injection of 50 × 10⁶ human PBMCs.

Figure 2

hu-PBL-SCID mice treated with IL-2 therapy demonstrate distinctly different populations of isolated human leukocytes in their spleens at week 4 compared with hu-PBL-SCID mice treated with GM-CSF and IL-2. Flow cytometric analysis of splenocytes from SCID mice 4 weeks after intraperitoneal injection of 50 × 10⁶ human PBMCs and the initiation of treatment with either daily subcutaneous injections of 500 IU PEG-IL-2 + ASGM-1 weekly (a, c, e, g, and i) or 3 μg GM-CSF intraperitoneally every other day + IL-2 + ASGM-1 (b, d, f, h, and j). Each group included ten animals, with comparable human Ig levels. (a and b) Forward scatter (size index) versus reactivity with anti-human CD45-FITC to distinguish cells of human origin. (c–j) Human leukocyte subset analysis of this CD45⁺ population.

Figure 3

Combination therapy with GM-CSF and IL-2, but not IL-2 alone, induces expansion of EBV-specific T cells in vivo. Flow cytometric analysis of splenocytes from SCID mice engrafted with PBMCS from an EBV-seropositive donor (HLA A1/A1, B8/B8), and treated weekly with ASGM-1 and daily with either IL-2 alone (upper panels) or GM-CSF and IL-2 (lower panels). HLA tetramers complexed with immunodominant (a) lytic (RAK) or (b) latent (FLR) EBV peptides were used to identify expanded populations of human CD3⁺ subsets with EBV-specificity.