In Vivo Depletion of Lymphotoxin-Alpha Expressing Lymphocytes Inhibits Xenogeneic Graft-versus-Host-Disease

Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic cell transplantation and is largely mediated by activated donor lymphocytes. Lymphotoxin (LT)-α is expressed by subsets of activated T and B cells, and studies in preclinical models demonstrated that targeted depletion of these cells with a mouse anti-LT-α monoclonal antibody (mAb) was efficacious in inhibiting inflammation and autoimmune disease. Here we demonstrate that LT-α is also upregulated on activated human donor lymphocytes in a xenogeneic model of GVHD and targeted depletion of these donor cells ameliorated GVHD. A depleting humanized anti-LT-α mAb, designated MLTA3698A, was generated that specifically binds to LT-α in both the soluble and membrane-bound forms, and elicits antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Using a human peripheral blood mononuclear cell transplanted SCID (Hu-SCID) mouse model of GVHD, the anti-human LT-α mAb specifically depleted activated LT-expressing human donor T and B cells, resulting in prolonged survival of the mice. A mutation in the Fc region, rendering the mAb incapable of mediating ADCC, abolished all in vitro and in vivo effects. These data support a role for using a depleting anti-LT-α antibody in treating immune diseases such as GVHD and autoimmune diseases

Expansion and expression of surface LT on human lymphocytes following transfer into SCID animals.

<p>CFSE-labeled human PBMCs were transferred into SCID mice via intrasplenic injection, then proliferation (<b>A–C</b>) or surface LT expression (<b>D–G</b>) was determined by flow cytometry. At indicated time points after transfer, spleen cells were harvested and human lymphocyte populations were identified on the basis of CFSE and specific cell marker staining. Proliferation of CD4⁺ T cells (<b>A</b>), CD8⁺ T cells (<b>B</b>) and CD19⁺ B cells (<b>C</b>). Surface LT expression on CFSE-labeled bulk transferred human PBMCs (<b>D</b>), or CFSE⁺ gated CD4⁺ T cells (<b>E</b>), CD8⁺ T cells (<b>F</b>) and CD19⁺ B cells (<b>G</b>). In each experiment, 2–3 spleens were pooled to provide sufficient cell numbers for data collection. Data are representative of staining for 1 pool out of 3 per experiment. A minimum of 3 experiments were performed for each cell type.</p

LT-α-specific mAb depletes LT-expressing human lymphocytes in Hu-SCID GVHD model.

<p>Spleens were harvested from SCID mice two days following intrasplenic injection of human PBMCs, after one day treatment with anti-LT-α MLTA3698A, anti-LT-α-FcMT or isotype control mAb. <b>A</b>. LT staining on total CFSE-labeled transferred cells and enumeration of percentage of total human cells expressing surface LT. <b>B</b>–<b>D</b>. LT expression and quantitation of mAb treatment effects on specific human lymphocyte populations. Spleen cells were gated on CD4⁺ cells (<b>B</b>), CD8⁺ cells (<b>C</b>) or CD19⁺ cells (<b>D</b>). In each experiment, 2–3 spleens were pooled to provide sufficient cell numbers for data collection. Data show staining for 1 pool out of 3 per experiment, and are representative of a minimum of 2 experiments for each cell type.</p

LT expression on human immune cell populations.

<p>Human CD4⁺ T cell expression of LT. <b>A</b>. Sorted CD4⁺ T cells were labeled with CFSE and proliferation and LT expression was monitored at indicated time points following stimulation with anti-CD3 and anti-CD28. <b>B</b>, <b>C</b>. Co-expression of LT with T cell activation markers CD25 (<b>B</b>) or CD45RO (<b>C</b>) on CD4⁺ gated cells. <b>D</b>, <b>E</b>. CD8⁺ T cell expression of LT. Co-expression of LT with CD25 (<b>D</b>) or CD45RO (<b>E</b>) on CD8⁺ gated cells. For CD4⁺ and CD8⁺ T cell activation, PBMCs were stimulated with anti-CD3 and anti-CD28 mAbs for 2 days. <b>F</b>. Human B cell expression of LT. B cells were stimulated with anti-IgM and BAFF for two days, then LT expression determined on CD19⁺ B cells with CD69 as a marker for activation. <b>G</b>. Human monocyte expression of LT. CD14⁺ monocytes were stimulated with LPS, with activation status assessed on the basis of HLA-DR up-regulation.</p

Efficacy of anti-LT-α mAb in Hu-SCID model of GVHD.

<p>SCID mice were sublethally irradiated, randomized, then immediately treated with anti-LT-α MLTA3698A (filled circles), anti-LT-α-FcMT (open circles), CTLA-4.Ig (open triangles) or isotype control Ab (open squares). 4 hr after initial treatment, human PBMC were transferred via i.v. injection. Treatment was continued with twice-weekly dosing throughout the study. Two independent studies were performed with similar results.</p

Binding, blocking and depleting properties of anti-LT-α mAb.

<p><b>A</b>. Anti-LT-α MLTA3698A (filled circles) LT-βR.Ig (open squares) and TNFRII.Ig (open triangles) binding to LT-α1β2 in ELISA binding assays. <b>B</b>. ELISA binding to LT-α3. <b>C</b>. Blockade of LT-βR.Fc binding to LT-α1β2 in ELISA competition assays with anti-LT-α MLTA3698A (filled circles), LT-βR.Ig (open squares) or TNFRII.Ig (open triangles). <b>D</b>. Blockade of TNFRII.Fc binding to LT-α3 in ELISA competition assays. ADCC activity of anti-LT-α mAb against LT-expressing cells. LT expression on 293-hLT-αβ cells (<b>E</b>) and activated human CD4⁺ T cells (<b>F</b>) was detected using anti-LT-α MLTA3698A (dark gray shaded histograms), anti-LT-α-FcMT mAb (solid line). Isotype control antibody staining is indicated by light-shaded histograms. Data are representative of at least five experiments. ADCC activity against 293-hLT-αβ (<b>G</b>) or activated CD4⁺ T cell (<b>H</b>) targets mediated by MLTA3698A (filled circles) or anti-LT-α-FcMT(open circles). Isotype control mAb is indicated by open squares. Data are representative of three experiments.</p

Integrative Biology Approach Identifies Cytokine Targeting Strategies for Psoriasis

Cytokines are critical checkpoints of inflammation. The treatment of human autoimmune disease has been revolutionized by targeting inflammatory cytokines as key drivers of disease pathogenesis. Despite this, there exist numerous pitfalls when translating preclinical data into the clinic. We developed an integrative biology approach combining human disease transcriptome data sets with clinically relevant in vivo models in an attempt to bridge this translational gap. We chose interleukin-22 (IL-22) as a model cytokine because of its potentially important proinflammatory role in epithelial tissues. Injection of IL-22 into normal human skin grafts produced marked inflammatory skin changes resembling human psoriasis. Injection of anti-IL-22 monoclonal antibody in a human xenotransplant model of psoriasis, developed specifically to test potential therapeutic candidates, efficiently blocked skin inflammation. Bioinformatic analysis integrating both the IL-22 and anti-IL-22 cytokine transcriptomes and mapping them onto a psoriasis disease gene coexpression network identified key cytokine-dependent hub genes. Using knockout mice and small-molecule blockade, we show that one of these hub genes, the so far unexplored serine/threonine kinase PIM1, is a critical checkpoint for human skin inflammation and potential future therapeutic target in psoriasis. Using in silico integration of human data sets and biological models, we were able to identify a new target in the treatment of psoriasis