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Cytotoxicity assay with sorted PD1- CD8 T cells

Exhausted T cells have been described in cancer patients and murine tumor models largely based on their expression of various inhibitory receptors. Understanding of the functional attributes of these cells is limited. Here, we report that among CD8+ T cells in commonly used syngeneic tumor models, the coexpression of inhibitory receptors PD-1, LAG3, and TIM3 defined a group of highly activated and functional effector cells. Coexpression of these receptors further enriched for antigen-specific cells with increased T-cell receptor clonality. Anti–PD-L1 treatment increased the number and activation of these triple-positive CD8+ T cells without affecting the density of PD-1− cells. The intratumoral density of CD8+ T cells coexpressing inhibitory receptors negatively correlated with tumor burden. The density ratio and pretreatment phenotype of CD8+ T cells coexpressing inhibitory receptors was positively correlated with response across a variety of tumor models. Our results demonstrate that coexpression of inhibitory receptors is not a signifier of exhausted T cells, but rather can define a group of activated and functional effector cells in syngeneic tumor models. In the cancer setting, these cells could represent a heterogeneous population of not only exhausted but also highly activated cells responsive to treatment.

Cytotoxicity assay with sorted PD1+LAG3+TIM3+ CD8 T cells

Additional material and methods

Supplemental Figures Showing: Fig. S1: CD8 T cells co-expressing PD1, LAG3 and TIM3 are present at different frequencies in MC38 and EMT6 tumors and represent activated cells. Fig. S2: CD8 T cells co-expressing inhibitory receptors are enriched in antigen-experienced and activated cells relative to the PD1- subset. Fig. S3: Majority of CD8 T cells in blood of MC38 and EMT6 tumor bearing mice lack PD1 and T-bet expression. Fig. S4: Anti-PD-L1 treatment increases the activity and cytolytic potential of PD1+ CD8 T cell subsets in MC38 and EMT6 tumors. Fig. S5: JC and 4T1 tumors do not respond to anti-PD-L1 treatment. Fig. S6: CD8 T cells co-expressing PD1, LAG3 and TIM3 are highly activated in JC and 4T1 tumors. Table S1: List of flow cytometry markers used for univariate linear regression analysis in Supplementary table 2. Table S2: Top parameters correlated with response to anti-PD-L1.

Cytotoxicity assay with sorted PD1- CD8 T cells

FACS parameter database

Exhausted T cells have been described in cancer patients and murine tumor models largely based on their expression of various inhibitory receptors. Understanding of the functional attributes of these cells is limited. Here, we report that among CD8

Peripheral T lymphocytes share many functional properties with hematopoietic stem cells (HSCs), including long-term maintenance, quiescence, and latent proliferative potential. In addition, peripheral T cells retain the capacity for further differentiation into a variety of subsets, much like HSCs. While the similarities between T cells and HSC have long been hypothesized, the potential common genetic regulation of HSCs and T cells has not been widely explored. We have studied the T cell-intrinsic role of Zfx, a transcription factor specifically required for HSC maintenance. We report that T cell-specific deletion of Zfx caused age-dependent depletion of naïve peripheral T cells. Zfx-deficient T cells also failed to undergo homeostatic proliferation in a lymphopenic environment, and showed impaired antigen-specific expansion and memory response. In addition, the invariant natural killer T cell compartment was severely reduced. RNA-Seq analysis revealed that the most dysregulated genes in Zfx-deficient T cells were similar to those observed in Zfx-deficient HSC and B cells. These studies identify Zfx as an important regulator of peripheral T cell maintenance and expansion and highlight the common molecular basis of HSC and lymphocyte homeostasis.

Foxp3

SummaryTumor-propagating cells in acute leukemia maintain a stem/progenitor-like immature phenotype and proliferative capacity. Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) originate from different lineages through distinct oncogenic events such as MLL fusions and Notch signaling, respectively. We found that Zfx, a transcription factor that controls hematopoietic stem cell self-renewal, controls the initiation and maintenance of AML caused by MLL-AF9 fusion and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx prevents differentiation and activates gene sets characteristic of immature cells of the respective lineages. In addition, endogenous Zfx contributes to gene induction and transformation by Myc overexpression in myeloid progenitors. Key Zfx target genes include the mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescues the propagation of Zfx-deficient AML. These results show that distinct leukemia types maintain their undifferentiated phenotype and self-renewal by exploiting a common stem-cell-related genetic regulator.

C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.

Loss of function of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) is associated with a wide spectrum of cancers. We report that tamoxifen-induced BAP1 deletion in adult mice resulted in severe thymic atrophy. BAP1 was critical for T cell development at several stages. In the thymus, BAP1 was required for progression through the pre-T cell receptor checkpoint. Peripheral T cells lacking BAP1 demonstrated a defect in homeostatic and antigen-driven expansion. Deletion of BAP1 resulted in suppression of E2F target genes and defects in cell cycle progression, which was dependent on the catalytic activity of BAP1, but did not require its interaction with host cell factor-1 (HCF-1). Loss of BAP1 led to increased monoubiquitination of histone H2A at Lys119 (H2AK119ub) throughout the T cell lineage, in particular in immature thymocytes, but did not alter trimethylation of histone H3 at Lys27 (H3K27me3). Deletion of BAP1 also abrogated B cell development in the bone marrow. Our findings uncover a nonredundant function for BAP1 in maintaining the lymphoid lineage.

The development, homeostasis, and function of B lymphocytes involve multiple rounds of B-cell receptor (BCR)–controlled proliferation and prolonged maintenance. We analyzed the role of transcription factor Zfx, a recently identified regulator of hematopoietic stem cell maintenance, in B-cell development and homeostasis. Panhematopoietic or B cell–specific deletion of Zfx in the bone marrow blocked B-cell development at the pre-BCR selection checkpoint. Zfx deficiency in peripheral B cells caused accelerated B-cell turnover, depletion of mature recirculating B cells, and delayed T-dependent antibody responses. In addition, the numbers and function of B-1 cell lineage were reduced. Zfx-deficient B cells showed normal proximal BCR signaling, but impaired BCR-induced proliferation and survival in vitro. This was accompanied by aberrantly enhanced and prolonged integrated stress response and by delayed induction of cyclin D2 and Bcl-xL proteins. Thus, Zfx restrains the stress response and couples antigen receptor signaling to cell expansion and maintenance during B-cell development and peripheral homeostasis. These results identify a novel transcriptional regulator of the B-cell lineage and highlight the common genetic control of stem cell maintenance and lymphocyte homeostasis.

SummaryStem cells (SC) exhibit a unique capacity for self-renewal in an undifferentiated state. It is unclear whether the self-renewal of pluripotent embryonic SC (ESC) and of tissue-specific adult SC such as hematopoietic SC (HSC) is controlled by common mechanisms. The deletion of transcription factor Zfx impaired the self-renewal but not the differentiation capacity of murine ESC; conversely, Zfx overexpression facilitated ESC self-renewal by opposing differentiation. Furthermore, Zfx deletion abolished the maintenance of adult HSC but did not affect erythromyeloid progenitors or fetal HSC. Zfx-deficient ESC and HSC showed increased apoptosis and SC-specific upregulation of stress-inducible genes. Zfx directly activated common target genes in ESC and HSC, as well as ESC-specific target genes including ESC self-renewal regulators Tbx3 and Tcl1. These studies identify Zfx as a shared transcriptional regulator of ESC and HSC, suggesting a common genetic basis of self-renewal in embryonic and adult SC.

Mammalian hematopoiesis is based on the activity of multipotent hematopoietic stem cells (HSC) that undergo continuous self-renewal throughout the adult life. The self-renewal represents a unique property of adult HSC, and appears distinct from the proliferation of hematopoietic progenitors or from the initial specification and expansion of embryonic HSC. Despite recent progress, little is known about the molecular mechanisms governing HSC self-renewal. Zfx is a broadly expressed zinc finger-containing transcriptional activator that is highly conserved in vertebrates. Using conditional gene targeting in mice, we demonstrate that Zfx is an essential and specific regulator of HSC self-renewal. The expression of Zfx was elevated in HSC compared to progenitors and differentiated cells. An inducible deletion of Zfx from the adult bone marrow (BM) resulted in the specific loss of the HSC population. The short-term proliferation and lodging of Zfx-deficient HSC in the BM were normal, suggesting a specific defect of long-term self-renewal. The deletion of Zfx in pre-established BM chimeras completely abrogated HSC maintenance as reflected by the rapid loss of HSC contribution to hematopoiesis. Furthermore, a constitutive pan-hematopoietic deletion of Zfx spared embryonic HSC in the fetal liver, yet resulted in the loss of adult BM HSC. In contrast, adult erythromyeloid progenitors or differentiated cells were not affected by the absence of Zfx. Genome-wide expression analysis identified candidate target genes of Zfx, some of which were controlled by Zfx in HSC but not in their differentiated progeny. Finally, several immediate-early and/or stress-inducible genes were upregulated specifically in Zfx-deficient HSC, suggesting that the latter undergo increased stress-related signaling. Thus, functional and gene expression analysis establishes Zfx as a novel specific regulator of adult HSC self-renewal. Further studies of the target genes and pathways controlled by Zfx should provide novel insights into the molecular basis of HSC function.