Linjie Guo

The ATM–p53 pathway suppresses aneuploidy-induced tumorigenesis

The spindle assembly checkpoint (SAC) is essential for proper sister chromatid segregation. Defects in this checkpoint can lead to chromosome missegregation and aneuploidy. An increasing body of evidence suggests that aneuploidy can play a causal role in tumorigenesis. However, mutant mice that are prone to aneuploidy have only mild tumor phenotypes, suggesting that there are limiting factors in the aneuploidy-induced tumorigenesis. Here we provide evidence that p53 is such a limiting factor. We show that aneuploidy activates p53 and that loss of p53 drastically accelerates tumor development in two independent aneuploidy models. The p53 activation depends on the ataxia-telangiectasia mutated (ATM) gene product and increased levels of reactive oxygen species. Thus, the ATM-p53 pathway safeguards not only DNA damage but also aneuploidy.

Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy

Advances in the design of chimeric antigen receptors (CARs) have improved the antitumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. We proposed that CAR expression in Vα24-invariant natural killer T (NKT) cells can build on the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKT cells were engineered to express a CAR against the GD2 ganglioside (CAR.GD2), which is highly expressed by neuroblastoma (NB). We compared CAR.GD2 constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB costimulatory endodomains. CAR.GD2 expression rendered NKT cells highly cytotoxic against NB cells without affecting their CD1d-dependent reactivity. We observed a striking T helper 1-like polarization of NKT cells by 4-1BB-containing CARs. Importantly, expression of both CD28 and 4-1BB endodomains in the CAR.GD2 enhanced in vivo persistence of NKT cells. These CAR.GD2 NKT cells effectively localized to the tumor site had potent antitumor activity, and repeat injections significantly improved the long-term survival of mice with metastatic NB. Unlike T cells, CAR.GD2 NKT cells did not induce graft-versus-host disease. These results establish the potential of NKT cells to serve as a safe and effective platform for CAR-directed cancer immunotherapy.

IL-15 protects NKT cells from inhibition by tumor-associated macrophages and enhances antimetastatic activity

Vα24-invariant NKT cells inhibit tumor growth by targeting tumor-associated macrophages (TAMs). Tumor progression therefore requires that TAMs evade NKT cell activity through yet-unknown mechanisms. Here we report that a subset of cells in neuroblastoma (NB) cell lines and primary tumors expresses membrane-bound TNF-α (mbTNF-α). These proinflammatory tumor cells induced production of the chemokine CCL20 from TAMs via activation of the NF-κB signaling pathway, an effect that was amplified in hypoxia. Flow cytometry analyses of human primary NB tumors revealed selective accumulation of CCL20 in TAMs. Neutralization of the chemokine inhibited in vitro migration of NKT cells toward tumor-conditioned hypoxic monocytes and localization of NKT cells to NB grafts in mice. We also found that hypoxia impaired NKT cell viability and function. Thus, CCL20-producing TAMs served as a hypoxic trap for tumor-infiltrating NKT cells. IL-15 protected antigen-activated NKT cells from hypoxia, and transgenic expression of IL-15 in adoptively transferred NKT cells dramatically enhanced their antimetastatic activity in mice. Thus, tumor-induced chemokine production in hypoxic TAMs and consequent chemoattraction and inhibition of NKT cells represents a mechanism of immune escape that can be reversed by adoptive immunotherapy with IL-15-transduced NKT cells.

Lymphotoxin Pathway-Directed, Autoimmune Regulator-Independent Central Tolerance to Arthritogenic Collagen

Ectopic expression of peripherally restricted Ags by medullary thymic epithelial cells (mTECs) is associated with negative selection. Autoimmune regulator (AIRE) is considered to be the master regulator of these Ags. We show in this study that the ectopic expression of type II collagen (CII) in mTECs and the corresponding central tolerance to CII are AIRE independent but lymphotoxin dependent. The failure to properly express CII in mTECs of Lta−/− and Ltbr−/− mice leads to overt autoimmunity to CII and exquisite susceptibility to arthritis. These findings define the existence of additional pathways of ectopic peripheral Ag expression, parallel to and independent of AIRE, which may cover an extended spectrum of peripheral Ags in the thymus.

CD62L+ NKT cells have prolonged persistence and antitumor activity in vivo

Vα24-invariant natural killer T cells (NKTs) localize to tumors and have inherent antitumor properties, making them attractive chimeric antigen receptor (CAR) carriers for redirected cancer immunotherapy. However, clinical application of CAR-NKTs has been impeded, as mechanisms responsible for NKT expansion and the in vivo persistence of these cells are unknown. Here, we demonstrated that antigen-induced expansion of primary NKTs in vitro associates with the accumulation of a CD62L+ subset and exhaustion of CD62L- cells. Only CD62L+ NKTs survived and proliferated in response to secondary stimulation. When transferred to immune-deficient NSG mice, CD62L+ NKTs persisted 5 times longer than CD62L- NKTs. Moreover, CD62L+ cells transduced with a CD19-specific CAR achieved sustained tumor regression in a B cell lymphoma model. Proliferating CD62L+ cells downregulated or maintained CD62L expression when activated via T cell receptor alone or in combination with costimulatory receptors. We generated HLAnull K562 cell clones that were engineered to express CD1d and costimulatory ligands. Clone B-8-2 (HLAnullCD1dmedCD86high4-1BBLmedOX40Lhigh) induced the highest rates of NKT expansion and CD62L expression. B-8-2-expanded CAR-NKTs exhibited prolonged in vivo persistence and superior therapeutic activities in models of lymphoma and neuroblastoma. Therefore, we have identified CD62L as a marker of a distinct NKT subset endowed with high proliferative potential and have developed artificial antigen-presenting cells that generate CD62L-enriched NKTs for effective cancer immunotherapy.

Deficiency in activation-induced cytidine deaminase promotes systemic autoimmunity in lpr mice on a C57BL/6 background

Activation‐induced deaminase (AID) is a prerequisite for immunoglobulin (Ig) class‐switch recombination and somatic hypermutation, which is critical for antibody affinity maturation. IgM and IgG autoantibodies are characteristic of the systemic autoimmune disorders such as lupus. However, the relative contributions of hypermutated high‐affinity IgG antibodies and germline‐encoded IgM antibodies to systemic autoimmunity are not defined fully. The role of AID in autoimmunity is unclear. The current study used AID‐deficient mice to investigate the role of AID in the development and pathogenesis of murine lupus. C57BL/6 mice deficient in both Fas and AID were generated. Compared to their AID‐competent littermates, AID−/− lymphoproliferative (lpr) mice produced significantly elevated levels of IgM autoreactive antibodies with enhanced germinal centre (GC) response, developed more advanced splenomegaly and exhibited more severe glomerulonephritis. Thus, AID may play an important role in the negative regulation of systemic autoimmune manifestations in murine lupus. The results also indicate that hypermutated high‐affinity IgG antibodies are not necessary for the development of autoimmune syndrome in lpr mice on a C57BL/6 background.

Redirecting T cells to glypican-3 with 4-1BB zeta chimeric antigen receptors results in Th1 polarization and potent antitumor activity

T cells engineered to express CD19-specific chimeric antigen receptors (CARs) have shown breakthrough clinical successes in patients with B-cell lymphoid malignancies. However, similar therapeutic efficacy of CAR T cells in solid tumors is yet to be achieved. In this study we systematically evaluated a series of CAR constructs targeting glypican-3 (GPC3), which is selectively expressed on several solid tumors. We compared GPC3-specific CARs that encoded CD3ζ (Gz) alone or with costimulatory domains derived from CD28 (G28z), 4-1BB (GBBz), or CD28 and 4-1BB (G28BBz). All GPC3-CARs rendered T cells highly cytotoxic to GPC3-positive hepatocellular carcinoma, hepatoblastoma, and malignant rhabdoid tumor cell lines in vitro. GBBz induced the preferential production of Th1 cytokines (interferon γ/granulocyte macrophage colony-stimulating factor) while G28z preferentially induced Th2 cytokines (interleukin-4/interleukin-10). Inclusion of 4-1BB in G28BBz could only partially ameliorate the Th2-polarizing effect of CD28. 4-1BB induced superior expansion of CAR T cells in vitro and in vivo. T cells expressing GPC3-CARs incorporating CD28, 4-1BB, or both induced sustained tumor regressions in two xenogeneic tumor models. Thus, GBBz CAR endows T cells with superior proliferative potential, potent antitumor activity, and a Th1-biased cytokine profile, justifying further clinical development of GBBz CAR for immunotherapy of GPC3-positive solid tumors.

Overexpression of CXC Chemokine Ligand 14 Exacerbates Collagen-Induced Arthritis

CXCL14 is a relatively new chemokine with unidentified receptor and undefined function. Recently, we found that CXCL14 is upregulated in arthritic joints in a mouse model of autoimmune arthritis, collagen-induced arthritis. To examine the role of CXCL14 in the development and pathogenesis of autoimmune arthritis, we have generated transgenic (Tg) mice that overexpress CXCL14 under control of phosphoglycerate kinase promoter. The results showed that CXCL14-Tg mice developed more severe arthritis compared with wild-type controls. The draining lymph nodes of CXCL14-Tg mice were significantly enlarged and contained an increased number of activated T cells, particularly the CD44+CD62Llow effector memory cells. In addition, T cells from CXCL14-Tg mice exhibited an enhanced proliferative response against collagen II and produced higher levels of IFN-γ but not IL-4 or IL-17. CXCL14-Tg mice also had elevated levels of IgG2a autoantibodies. These findings indicated that CXCL14 plays an important role in the autoimmune arthritis, which may have an implication in understanding the pathogenic mechanisms of rheumatoid arthritis in humans and, ultimately, therapeutic interference.

IgM plays an important role in induction of collagen-induced arthritis

IgM is one major type of B cell receptor (BCR) expressed on most of the B cells from immature to mature stages. During normal B cell ontogeny, signals transduced through the IgM BCR play an important role in regulating B cell maturation and survival at multiple checkpoints. In addition, IgM BCR is also required for antigen‐dependent differentiation and activation of B cells. However, whether IgM BCR‐mediated signalling is important for the pathogenesis of autoimmune diseases remains elusive. Using IgM‐deficient mice, we examined the effect of absence of IgM on the development of collagen‐induced arthritis (CIA), an animal model of rheumatoid arthritis (RA). Compared to their wild‐type littermates, IgM‐deficient mice were either resistant to arthritis induction or developed significantly less severe arthritis. There was a significant decrease of autoantibody production in IgM‐deficient mice, particularly IgG2a antibodies, which is believed to be pathogenic in CIA. Thus, although IgM–/– mice have relatively normal B cell development with IgD BCR replacing IgM BCR, the absence of IgM‐mediated signals has a profound impact on the development of CIA, indicating that IgM plays an important role in the development and pathogenesis of autoimmune arthritis and IgM‐mediated signalling is critical in the generation of pathogenic autoreactive antibodies.

Inhibition of clonal expansion by Foxp3 expression as a mechanism of controlled T‐cell responses and autoimmune disease

The essential role of the transcription factor Foxp3 in the development and function of Treg has been well documented. The role of Foxp3 in non‐Treg, however, is not fully understood. Emerging evidence indicates that Foxp3 expression is not confined to CD4+CD25+ Treg. The present study shows that in Foxp3 transgenic (Foxp3‐Tg) mice, in which the transgene is driven by the lck distal promoter, CD4+CD25− T cells that express the Foxp3 transgene do not upregulate the expression of CD25−, GITR, or CTLA‐4, and do not have suppressive function; however, the Foxp3‐Tg+CD4+CD25− T cells exhibit significantly reduced proliferative response to TCR engagement. Foxp3‐Tg mice are resistant to collagen‐induced arthritis via reduced cellular proliferation of activated T cells. These findings indicate that Foxp3 upregulation in activated non‐Treg may be a mechanism to suppress immune responses by reduced clonal expansion of activated T cells.