Alternative NF-κB Pathway in Treg Homeostasis and Function
CD4+Foxp3+ regulatory T cells (Tregs) control innate and adaptive immune responses. Aberrations in Treg homeostasis have been implicated in the development of autoimmune diseases. Whereas canonical NF-κB signaling is known to play a role in Treg development and function, the role of the alternative NF-κB components, p100 and RelB, in the homeostasis of this T cell subpopulation is largely unknown. Thus, Grinberg-Bleyer et al. (p. 2362) examined the consequence of p100 gene (nfkb2) and relb deletion in Tregs. There was a significant increase in percentage of Tregs in the spleen and peripheral lymph nodes (pLNs) in nfkb2-deficient mice relative to wild-type (WT) mice, but CD4 and CD8 T cell proportions and numbers were not affected. Selective deletion of nfkb2 in Tregs in Foxp3CREnfkb2F/F mice resulted in expansion of Treg populations in the spleen and LN. To delineate whether nfkb2 functions via cell-intrinsic and/or cell-extrinsic mechanisms, the authors performed mixed bone marrow (BM) chimera experiments, in which cell-extrinsic defects would be compensated for by the progeny of WT BM cells. In lethally irradiated WT recipients of a 1:1 mixture of congenic WT BM and Foxp3CREnfkb2F/F BM, Tregs were increased in spleen and LN, and Ki67 proliferation marker was increased in Tregs in which nfkb2 was deleted, revealing that nfkb2 functions cell-intrinsically to restrict peripheral Treg proliferation. Aged (12 mo old) Foxp3CREnfkb2F/F mice displayed signs of autoimmunity, with enhanced activation of both CD4+ and CD8+ T cells and an increase in the proportion of CD4+ and CD8+ T cells expressing IFN-γ. In vitro studies demonstrated that NF-κB2 functions as an inhibitor of RelB, as evidenced by the increase in nuclear accumulation of RelB in TCR/CD28-stimulated nfkb2−/− Tregs. In vivo, ablation of relb in the CD4crenfkb2F/F background restored a normal proportion and number of peripheral Tregs. Finally, analysis of global gene expression changes in nfkb2−/−, relb−/−, and nfkb2−/−relb−/− Tregs demonstrated that expression of genes involved in cell proliferation, antiapoptotic functions, and inflammation was increased in nfkb2−/− Tregs. However, concomitant loss of relb in these cells restored expression of several genes, providing evidence that NF-κB2 primarily acts as an inhibitor of RelB-mediated transcription. These studies reveal a novel role of the alternative NF-κB pathway in Treg homeostasis and function.
Plasmid-Based Gene Editing in Primary T Cells
Although genetically modified mouse strains are powerful tools, systems to directly edit murine lymphocytes are highly desirable as they would reduce costs and eliminate the need for extensive breeding. Despite this need, previous efforts to establish CRISPR/Cas-9–mediated gene editing in murine primary T cells are extremely limited and require extensive breeding. In this issue, Kornete et al. (p. 2489) developed an approach to ablate genes in primary T cells using commonly accessible plasmids. Transfection of both murine EL-4 thymoma and primary CD4+ T cells with a plasmid expressing Cas9-GFP and a single guide RNA targeting CD90.2 or CD45.2 achieved successful ablation of these genes in nearly 90% of cells. Combination of CD45.2 and CD90.2 gene targeting in EL-4 and primary CD4+ T cells resulted in loss of both genes in almost 50% of cells. Gene-edited primary T cells remained viable, homed to lymphoid organs, and expanded when transferred to lymphodeficient RAG-knockout hosts. Furthermore, ablation of ICOS in CD4+ T cells impaired differentiation of T follicular helper cells in response to a viral infection in lymphoreplete mice. This plasmid-based protocol also allowed the authors to develop the allele-switching assay (ASA), in which they demonstrated a successful switch from the CD90.2 to the CD90.1 allele, both of which could be quantified using specific mAbs. The ASA protocol allowed quantification of homology-directed repair (HDR) and allowed the authors to optimize HDR efficacy. The ASA also allowed identification of the epitope recognized by mAbs CD45.1 and CD45.2 and corrected a defective Foxp3 gene in mice bearing either a 2-bp insertion or a Foxp3K276X mutation. Combination of the ASA for CD90 and for CD45 revealed that two HDR events are linked in a given cell. Consistent with these observations, the authors combined repair of Foxp3-deficient CD4+ T cells with CD45.2 to CD45.1 allele switching and demonstrated that multiplexed HDR editing of a cell surface molecule can serve as a surrogate marker to enrich for cells that have simultaneously undergone HDR at a second locus. Together, these results demonstrate a versatile T cell editing approach that eliminates extensive breeding that may be used to further advance adoptive T cell technologies.
Idelalisib in Chronic Lymphocytic Leukemia
Phosphatidylinositol 3-kinase δ signaling is involved in survival, homing, and retention of malignant B cells within primary and secondary lymphoid organs. Idelalisib, a PI3Kδ inhibitor, is approved for use with rituximab in relapsed and refractory chronic lymphocytic leukemia (CLL). Palazzo et al. (p. 2304) examined the effects of idelalisib on Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cellular phagocytosis (ADCP) and assessed its in vivo efficacy in combination with rituximab or obinutuzumab, anti-CD20 mAbs currently used in a clinical setting. The effects of idelalisib on the immune effector functions of rituximab and obinutuzumab were evaluated by measuring NK cell–mediated ADCC against the B cell lymphoma line WIL2-S; idelalisib had no effect on ADCC, a finding that was confirmed using primary CLL PBMCs as targets. Similarly, the effects of idelalisib on obinutuzumab- and rituximab-mediated ADCP were assessed using primary human monocyte-derived macrophages. M2c- and M1-polarized macrophages could phagocytose WIL2-S cells in the presence of idelalisib. Furthermore, in autologous B cell depletion assays using healthy donor blood samples, B cell depletion by obinutuzumab was largely unaffected by idelalisib, whereas rituximab-mediated B cell depletion varied across donors. In primary CLL cultures, the combination of idelalisib and obinutuzumab elicited a statistically significant increase in cell death compared with either agent alone. Treatment of leukemia-bearing mice with the structurally related surrogate PI3Kδ inhibitor GS-9820 showed modest therapeutic benefit, but resulted in an effective reduction of leukemic deposits within secondary lymphoid organs. Treatment of mice with a suboptimal dose of anti-CD20 mAbs caused a rapid and significant reduction in the leukemic burden, whereas addition of GS-9820 to the treatment scheme provided a durable depletion of leukemic cells and significantly enhanced overall survival compared with Ab alone. Overall, these data demonstrate that a clinically relevant dose of idelalisib minimally interferes with rituximab or obinutuzumab-mediated immune effector functions, but extends the duration of leukemic depletion. These studies have laid the groundwork for ongoing clinical trials of obinutuzumab and idelalisib combination therapy for CLL.
- Copyright © 2018 by The American Association of Immunologists, Inc.
