Zbtb20 identifies and controls a thymus-derived population of regulatory T cells that play a role in intestinal homeostasis.

The expression of BTB-ZF transcription factors such as ThPOK in CD4+ T cells, Bcl6 in T follicular helper cells, and PLZF in natural killer T cells defines the fundamental nature and characteristics of these cells. Screening for lineage-defining BTB-ZF genes led to the discovery of a subset of T cells that expressed Zbtb20. About half of Zbtb20+ T cells expressed FoxP3, the lineage-defining transcription factor for regulatory T cells (Tregs). Zbtb20+ Tregs were phenotypically and genetically distinct from the larger conventional Treg population. Zbtb20+ Tregs constitutively expressed mRNA for interleukin-10 and produced high levels of the cytokine upon primary activation. Zbtb20+ Tregs were enriched in the intestine and specifically expanded when inflammation was induced by the use of dextran sodium sulfate. Conditional deletion of Zbtb20 in T cells resulted in a loss of intestinal epithelial barrier integrity. Consequently, knockout (KO) mice were acutely sensitive to colitis and often died because of the disease. Adoptive transfer of Zbtb20+ Tregs protected the Zbtb20 conditional KO mice from severe colitis and death, whereas non-Zbtb20 Tregs did not. Zbtb20 was detected in CD24hi double-positive and CD62Llo CD4 single-positive thymocytes, suggesting that expression of the transcription factor and the phenotype of these cells were induced during thymic development. However, Zbtb20 expression was not induced in "conventional" Tregs by activation in vitro or in vivo. Thus, Zbtb20 expression identified and controlled the function of a distinct subset of Tregs that are involved in intestinal homeostasis.

Increased Activity of a NK-Specific CAR-NK Framework Targeting CD3 and CD5 for T-Cell Leukemias.

NK effector cells expressing a CAR construct may be used to target T-lineage markers. In this work, we compared the activity of a NK-specific CAR-NK and a CAR-T framework when expressed on NK effector cells to target CD3 and CD5 in T-cell malignancies. Our results show that CD3-CAR-T is more active than CD5-CAR-T to eliminate malignant T cells in vitro, however, CD3-CAR-T were less efficient to eliminate tumor cells in vivo, while CD5-CAR-T had antitumor activity in a diffuse xenograft model. Lack of in vivo efficacy correlated with downregulation of CD3 levels in target T cells after coculture with CD3-CAR effector cells. The CAR-NK framework greatly improved the efficacy of CARs leading to increased degranulation, cytokine secretion and elimination of the tumor xenograft by CD5-CAR-NK effector cells. Finally, all CAR constructs were similarly effective to eliminate malignant T cells in vitro. Our results show that the NK-CAR framework improves the activity of CARs in NK cells and that CD5 would be a better target than CD3 for T-cell malignancies, as dynamic downregulation of target expression may affect in vivo efficacy.

From Hematopoietic Stem Cell Transplantation to Chimeric Antigen Receptor Therapy: Advances, Limitations and Future Perspectives.

Chimeric antigen receptor (CAR) T-cell therapy was envisioned as a mechanism to re-direct effector T-cells to eliminate tumor cells. CARs are composed of the variable region of an antibody that binds a native cancer antigen coupled to the signaling domain of a TCR and co-stimulatory molecules. Its success and approval by the U.S. Food and Drug Administration for the treatment of B-cell malignancies revolutionized the immunotherapy field, leading to extensive research on its possible application for other cancer types. In this review, we will focus on the evolution of CAR-T cell therapy outlining current technologies as well as major obstacles for its wide application. We will highlight achievements, the efforts to increase efficacy and to evolve into an off-the-shelf treatment, and as a possible future treatment for non-cancer related diseases.

Siglec-6 is a target for chimeric antigen receptor T-cell treatment of chronic lymphocytic leukemia.

The only current curative treatment for chronic lymphocytic leukemia (CLL) is allogenic hematopoietic stem cell transplantation. Chimeric antigen receptor treatment targeting CD19 for CLL achieved some complete responses, suggesting the need for alternative or combinational therapies to achieve a more robust response. In this work, we evaluated CAR-T cells specific for Siglec-6, an antigen expressed in CLL, as a novel CAR-T cell treatment for CLL. We found that detection of SIGLEC6 mRNA and Siglec-6 protein is highly restricted to placenta and immune cells in other tissues and it is not expressed in hematopoietic stem cells. We generated CAR-T cells specific for Siglec-6 based on the sequence of the fully human anti-Siglec-6 antibody (JML1), which was identified in a CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells in vitro and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels in vitro and in vivo. Our results identify a novel CAR-T cell therapy for CLL and establish Siglec-6 as a possible target for immunotherapy.

Safety and clinical activity of PD-L1 blockade in patients with aggressive recurrent respiratory papillomatosis.

BACKGROUND: Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV)-driven disorder that causes substantial morbidity and can lead to fatal distal airway obstruction and post-obstructive pneumonias. Patients require frequent surgical debridement of disease, and no approved systemic adjuvant therapies exist. METHODS: A phase II study was conducted to investigate the clinical activity and safety of programmed death-ligand 1 (PD-L1) blockade with avelumab in patients with RRP. RESULTS: Twelve patients were treated. All patients with laryngeal RRP displayed improvement in disease burden, and 5 of 9 (56%) displayed partial responses. None of 4 patients with pulmonary RRP displayed a response. Using each patient's surgical history as their own control, patients required fewer surgical interventions after avelumab treatment (p = 0.008). A subset of partial responders developed HPV-specific reactivity in papilloma-infiltrating T-cells that correlated with reduced HPV viral load and an increased Tissue Inflammation Signature. CONCLUSIONS: Avelumab demonstrated safety and clinical activity in patients with laryngeal RRP. Further study of immune checkpoint blockade for RRP, possibly with longer treatment duration or in combination with other immunotherapies aimed at activating antiviral immunity, is warranted. TRIAL REGISTRATION: NCT, number NCT02859454 , registered August 9, 2016.

Zbtb1 controls NKp46+ ROR-gamma-T+ innate lymphoid cell (ILC3) development.

Innate lymphoid cells (ILCs) play a central role conferring protection at the mucosal frontier. In this study, we have identified a requirement of the transcription factor Zbtb1 for the development of RORγt+ ILCs (ILC3s). Zbtb1-deficient mice lacked NKp46+ ILC3 cells in the lamina propria of the small and large intestine. This requirement of Zbtb1 was cell intrinsic, as NKp46+ ILC3s were not generated from Zbtb1-deficient progenitors in bone marrow chimeras and Zbtb1-deficient RORγt+ CCR6-NKp46- ILC3s didn't generate NKp46+ ILC3s in co-cultures with OP9-DL1 stroma. In correlation with this impairment, Zbtb1-deficient ILC3 cells failed to upregulate T-bet expression, and to acquire IFN-γ production characteristic of NKp46+ cells. Finally, absence of NKp46+ILC3 cells combined with the absence of T-cells in Zbtb1-deficient mice, led to a transient susceptibility to C. rodentium infections. Altogether, these results establish that Zbtb1 is essential for the development of NKp46+ ILC3 cells.

Zbtb1 prevents default myeloid differentiation of lymphoid-primed multipotent progenitors.

Zbtb1 is a transcription factor that prevents DNA damage and p53-mediated apoptosis in replicating immune progenitors, affecting lymphoid as well as myeloid development when hematopoietic progenitors are in competition in mixed bone marrow chimeras. However, Zbtb1-deficient mice do not have an apparent myeloid deficiency. We report here that Zbtb1-deficient lymphoid-primed multipotent progenitors (LMPPs) are biased to develop towards the myeloid fate in detriment of lymphoid development, contributing to the apparent unaffected myeloid development. Zbtb1 expression was maintained during lymphoid development of LMPP cells but downregulated during myeloid development. Deficiency of Zbtb1 in LMPP cells was sufficient to direct a myeloid fate in lymphoid-inducing conditions and in the absence of myeloid cytokines as shown by upregulation of a myeloid gene signature and the generation of myeloid cells in vitro. Finally, biased myeloid differentiation of Zbtb1-deficient LMPP cells was not due to increased p53-dependent apoptosis as it was not reverted by transgenic Bcl2 expression or p53 deficiency. Altogether, our results show that Zbtb1 expression prevents activation of a default myeloid program in LMPP cells, ensuring the generation of lymphoid cells.

Zbtb1 Safeguards Genome Integrity and Prevents p53-Mediated Apoptosis in Proliferating Lymphoid Progenitors.

Expression of the transcription factor Zbtb1 is required for normal lymphoid development. We report in the present study that Zbtb1 maintains genome integrity in immune progenitors, without which cells undergo increased DNA damage and p53-mediated apoptosis during replication and differentiation. Increased DNA damage in Zbtb1-mutant (ScanT) progenitors was due to increased sensitivity to replication stress, which was a consequence of inefficient activation of the S-phase checkpoint response. Increased p53-mediated apoptosis affected not only lymphoid but also myeloid development in competitive bone marrow chimeras, and prevention of apoptosis by transgenic Bcl2 expression and p53 deficiency rescued lymphoid as well as myeloid development from Zbtb1-mutant progenitors. Interestingly, however, protection from apoptosis rescued only the early stages of T cell development, and thymocytes remained arrested at the double-negative 3 developmental stage, indicating a strict requirement of Zbtb1 at later T cell developmental stages. Collectively, these results indicate that Zbtb1 prevents DNA damage in replicating immune progenitors, allowing the generation of B cells, T cells, and myeloid cells.

PLZF Controls the Development of Fetal-Derived IL-17+Vγ6+ γδ T Cells.

Expression of promyelocytic leukemia zinc finger (PLZF) protein directs the effector differentiation of invariant NKT (iNKT) cells and IL-4(+) γδ NKT cells. In this study, we show that PLZF is also required for the development and function of IL-17(+) γδ T cells. We observed that PLZF is expressed in fetal-derived invariant Vγ5(+) and Vγ6(+) γδ T cells, which secrete IFN-γ and IL-17, respectively. PLZF deficiency specifically affected the effector differentiation of Vγ6(+) cells, leading to reduced numbers of mature CD27(-)CD44(+) phenotype capable of secreting IL-17. Although PLZF was not required for Vγ5(+) γδ T cells to develop, when these cells were reprogrammed into IL-17-secreting cells in Skint-1 mutant mice, they required PLZF for their effector maturation, similarly to Vγ6(+) γδ T cells. The impaired effector differentiation of PLZF-deficient Vγ6(+) γδ T cells was not due to increased apoptosis and it was related to reduced proliferation of immature CD27(+)CD44(-) Vγ6(+) γδ T cells, which was required for their differentiation into mature CD27(-)CD44(+) IL-17-secreting cells. Thus, the present study identifies that PLZF function is not restricted to NKT or IL-4(+) T cells, but it also controls the development of IL-17(+) γδ T cells.

Ectopic T cell receptor-α locus control region activity in B cells is suppressed by direct linkage to two flanking genes at once.

The molecular mechanisms regulating the activity of the TCRα gene are required for the production of the circulating T cell repertoire. Elements of the mouse TCRα locus control region (LCR) play a role in these processes. We previously reported that TCRα LCR DNA supports a gene expression pattern that mimics proper thymus-stage, TCRα gene-like developmental regulation. It also produces transcription of linked reporter genes in peripheral T cells. However, TCRα LCR-driven transgenes display ectopic transcription in B cells in multiple reporter gene systems. The reasons for this important deviation from the normal TCRα gene regulation pattern are unclear. In its natural locus, two genes flank the TCRα LCR, TCRα (upstream) and Dad1 (downstream). We investigated the significance of this gene arrangement to TCRα LCR activity by examining transgenic mice bearing a construct where the LCR was flanked by two separate reporter genes. Surprisingly, the presence of a second, distinct, reporter gene downstream of the LCR virtually eliminated the ectopic B cell expression of the upstream reporter observed in earlier studies. Downstream reporter gene activity was unaffected by the presence of a second gene upstream of the LCR. Our findings indicate that a gene arrangement in which the TCRα LCR is flanked by two distinct transcription units helps to restrict its activity, selectively, on its 5'-flanking gene, the natural TCRα gene position with respect to the LCR. Consistent with these findings, a TCRα/Dad1 locus bacterial artificial chromosome dual-reporter construct did not display the ectopic upstream (TCRα) reporter expression in B cells previously reported for single TCRα transgenes.

PLZF induces the spontaneous acquisition of memory/effector functions in T cells independently of NKT cell-related signals.

The broad complex, tramtrack, bric-a-brac-zinc finger (BTB-ZF) transcription factor promyelocytic leukemia zinc finger (PLZF) is required for development of the characteristic innate/effector functions of NKT cells. In this study, we report the characterization and functional analysis of transgenic mouse T cells with forced expression of PLZF. PLZF expression was sufficient to provide some memory/effector functions to T cells without the need for Ag stimulation or proliferation. The acquisition of this phenotype did not require the proliferation typically associated with T cell activation. Furthermore, PLZF transgenic cells maintained a diverse TCR repertoire, indicating that there was no preferential expansion of specific clones. Functionally, PLZF transgenic CD4 and CD8 lymphocytes were similar to wild type memory cells, in that they had similar requirements for costimulation and exhibited a similar pattern of cytokine secretion, with the notable exception that transgenic T cells produced significantly increased levels of IL-17. Whereas transgene-mediated PLZF expression was not sufficient to rescue NKT cell development in Fyn- or signaling lymphocytic activation-associated protein (SAP)-deficient mice, the acquisition of memory/effector functions induced by PLZF in conventional T cells was independent of Fyn and SAP. These data show that PLZF is sufficient to promote T cell effector functions and that PLZF acts independently of SAP- and Fyn-mediated signaling pathways.

A novel TCR transgenic model reveals that negative selection involves an immediate, Bim-dependent pathway and a delayed, Bim-independent pathway.

A complete understanding of negative selection has been elusive due to the rapid apoptosis and clearance of thymocytes in vivo. We report a TCR transgenic model in which expression of the TCR during differentiation occurs only after V(D)J-like recombination. TCR expression from this transgene closely mimics expression of the endogenous TCRalpha locus allowing for development that is similar to wild type thymocytes. This model allowed us to characterize the phenotypic changes that occurred after TCR-mediated signaling in self-reactive thymocytes prior to their deletion in a highly physiological setting. Self-reactive thymocytes were identified as being immature, activated and CD4(lo)CD8(lo). These cells had upregulated markers of negative selection and were apoptotic. Elimination of Bim reduced the apoptosis of self-reactive thymocytes, but it did not rescue their differentiation and the cells remained at the immature CD4(lo)CD8(lo) stage of development. These cells upregulate Nur77 and do not contribute to the peripheral T cell repertoire in vivo. Remarkably, development past the CD4(lo)CD8(lo) stage was possible once the cells were removed from the negatively selecting thymic environment. In vitro development of these cells occurred despite their maintenance of high intracellular levels of Nur77. Therefore, in vivo, negatively selected Bim-deficient thymocytes are eliminated after prolonged developmental arrest via a Bim-independent pathway that is dependent on the thymic microenvironment. These data newly reveal a layering of immediate, Bim-dependent, and delayed Bim-independent pathways that both contribute to elimination of self-reactive thymocytes in vivo.

Beta-catenin/Tcf determines the outcome of thymic selection in response to alphabetaTCR signaling.

Thymic maturation of T cells depends on the intracellular interpretation of alphabetaTCR signals by processes that are poorly understood. In this study, we report that beta-catenin/Tcf signaling was activated in double-positive thymocytes in response to alphabetaTCR engagement and impacted thymocyte selection. TCR engagement combined with activation of beta-catenin signaled thymocyte deletion, whereas Tcf-1 deficiency rescued from negative selection. Survival/apoptotis mediators including Bim, Bcl-2, and Bcl-x(L) were alternatively influenced by stabilization of beta-catenin or ablation of Tcf-1, and Bim-mediated beta-catenin induced thymocyte deletion. TCR activation in double-positive cells with stabilized beta-catenin triggered signaling associated with negative selection, including sustained overactivation of Lat and Jnk and a transient activation of Erk. These observations are consistent with beta-catenin/Tcf signaling acting as a switch that determines the outcome of thymic selection downstream the alphabetaTCR cascade.

Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.

Adaptable TCR avidity thresholds for negative selection.

Central tolerance plays a significant role in preventing autoimmune diseases by eliminating T cells with high and intermediate avidity for self. To determine the manner of setting the threshold for deletion, we created a unique transgenic mouse strain with a diverse T cell population and globally increased TCR avidity for self-peptide/MHC complexes. Despite the adaptations aimed at reducing T cell reactivity (reduced TCR levels and increased levels of TCR signaling inhibitor CD5), transgenic mice displayed more severe experimental allergic encephalomyelitis and lupus. The numbers and activity of natural (CD4(+)CD25(+)) regulatory T cells were not altered. These findings demonstrate that the threshold for deletion is adaptable, allowing survival of T cells with higher avidity when TCR avidity is globally increased.

The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions.

Invariant natural killer T cells (iNKT cells) have an innate immunity-like rapidity of response and the ability to modulate the effector functions of other cells. We show here that iNKT cells specifically expressed the BTB-zinc finger transcriptional regulator PLZF. In the absence of PLZF, iNKT cells developed, but they lacked many features of innate T cells. PLZF-deficient iNKT cells accumulated in lymph nodes rather than in the liver, did not express NK markers and did not have the characteristic activated phenotype. PLZF-deficient iNKT cells failed to secrete large amounts of interleukin 4 and interferon-gamma after activation; however, some cells produced either interleukin 4 or interferon-gamma but not both. PLZF, therefore, is an iNKT cell-specific transcription factor that is necessary for full functionality.

Beta-catenin stabilization stalls the transition from double-positive to single-positive stage and predisposes thymocytes to malignant transformation.

Activation of beta-catenin has been causatively linked to the etiology of colon cancer. Conditional stabilization of this molecule in pro-T cells promotes thymocyte development without the requirement for pre-TCR signaling. We show here that activated beta-catenin stalls the developmental transition from the double-positive (DP) to the single-positive (SP) thymocyte stage and predisposes DP thymocytes to transformation. beta-Catenin-induced thymic lymphomas have a leukemic arrest at the early DP stage. Lymphomagenesis requires Rag activity, which peaks at this developmental stage, as well as additional secondary genetic events. A consistent secondary event is the transcriptional up-regulation of c-Myc, whose activity is required for transformation because its conditional ablation abrogates lymphomagenesis. In contrast, the expression of Notch receptors as well as targets is reduced in DP thymocytes with stabilized beta-catenin and remains low in the lymphomas, indicating that Notch activation is not required or selected for in beta-catenin-induced lymphomas. Thus, beta-catenin activation may provide a mechanism for the induction of T-cell-acute lymphoblastic leukemia (T-ALL) that does not depend on Notch activation.

c-Myc mediates pre-TCR-induced proliferation but not developmental progression.

Constitutive and cell-autonomous signals emanating from the pre-T-cell receptor (pre-TCR) promote proliferation, survival and differentiation of immature thymocytes. We show here that induction of pre-TCR signaling resulted in rapid elevation of c-Myc protein levels. Cre-mediated thymocyte-specific ablation of c-Myc in CD25(+)CD44(-) thymocytes reduced proliferation and cell growth at the pre-TCR checkpoint, resulting in thymic hypocellularity and a severe reduction in CD4(+)CD8(+) thymocytes. In contrast, c-Myc deficiency did not inhibit pre-TCR-mediated differentiation or survival. Myc(-/-) double-negative (DN) 3 cells progressed to the double-positive (DP) stage and up-regulated TCRalphabeta surface expression in the absence of cell proliferation, in vivo as well as in vitro. These observations indicate that distinct signals downstream of the pre-TCR are responsible for proliferation versus differentiation, and demonstrate that c-Myc is only required for pre-TCR-induced proliferation but is dispensable for developmental progression from the DN to the DP stage.