Role of TET proteins in B cells and Treg cells

DNA demethylation is promoted by TET enzymes, a family of three proteins -TET1, TET2 and TET3- which oxidize methyl group of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). TET proteins have been previously associated to cancer. In particular, Tet2 loss of function is strongly associated to haematopoietic malignancies in humans, such as B cell and T cell lymphoma. In this dissertation we explore the role of TET proteins in B and Treg cells. To do this we rely in the use of in vivo and in vitro models. We generated a mouse model with Tet2/3 deletion, CD19 Tet2/3 DKO mice. The TET deficient mice showed an expansion of Germinal Center B cells and developed B cell lymphoma, as early as 6 weeks of age; coupled to this, we observed an increase of G-quaruplexes and R-loops and increase of DNA damage. Mice generated with the adiddtional deletion of DNMT1, the enzyme responsible to maintain DNA methylation and also a G-quadruplex binding protein, on top of the Tet2/3 deletion, showed an inhibition of Germinal Center B cells and a significant delay on B cell lymphoma. We also explored the role of TET proteins in Treg cells using genomic and epigenomic approaches in in vivo and in vitro models. The Tet2/3 deficient Treg cells showed a loss of Treg-specific molecular features, and an impairement of IL-2/STAT5 signaling pathway. In our in vitro system, we induced Treg (iTreg) cells with TGF and treated them with Vitamin C, a potent activator of TET proteins and other dioxygenases. Oposite to what we observed in vivo, boosting TET activity with Vitamin C enhanced IL-2 responsiveness in iTreg cells by increasing IL2R expression and STAT5 activity

Innate-like functions of natural killer T cell subsets result from highly divergent gene programs

Natural killer T cells (NKT cells) have stimulatory or inhibitory effects on the immune response that can be attributed in part to the existence of functional subsets of NKT cells. These subsets have been characterized only on the basis of the differential expression of a few transcription factors and cell-surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic level and epigenomic level and by single-cell RNA sequencing. Our data indicated that despite their similar antigen specificity, the functional NKT cell subsets were highly divergent populations with many gene-expression and epigenetic differences. Therefore, the thymus 'imprints' distinct gene programs on subsets of innate-like NKT cells that probably impart differences in proliferative capacity, homing, and effector functions.</p

17q21 asthma-risk variants switch CTCF binding and regulate IL-2 production by T cells

Asthma and autoimmune disease susceptibility has been strongly linked to genetic variants in the 17q21 haploblock that alter the expression of ORMDL3; however, the molecular mechanisms by which these variants perturb gene expression and the cell types in which this effect is most prominent are unclear. We found several 17q21 variants overlapped enhancers present mainly in primary immune cell types. CD4(+) T cells showed the greatest increase (threefold) in ORMDL3 expression in individuals carrying the asthma-risk alleles, where ORMDL3 negatively regulated interleukin-2 production. The asthma-risk variants rs4065275 and rs12936231 switched CTCF-binding sites in the 17q21 locus, and 4C-Seq assays showed that several distal cis-regulatory elements upstream of the disrupted ZPBP2 CTCF-binding site interacted with the ORMDL3 promoter region in CD4(+) T cells exclusively from subjects carrying asthma-risk alleles. Overall, our results suggested that T cells are one of the most prominent cell types affected by 17q21 variants.</p

TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8+ T cell exhaustion.

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive (exhausted or dysfunctional) within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8+ T cell exhaustion. We show that the high-mobility group (HMG)-box transcription factors TOX and TOX2, as well as members of the NR4A family of nuclear receptors, are targets of the calcium/calcineurin-regulated transcription factor NFAT, even in the absence of its partner AP-1 (FOS-JUN). Using a previously established CAR T cell model, we show that TOX and TOX2 are highly induced in CD8+ CAR+ PD-1high TIM3high (exhausted) tumor-infiltrating lymphocytes (CAR TILs), and CAR TILs deficient in both TOX and TOX2 (Tox DKO) are more effective than wild-type (WT), TOX-deficient, or TOX2-deficient CAR TILs in suppressing tumor growth and prolonging survival of tumor-bearing mice. Like NR4A-deficient CAR TILs, Tox DKO CAR TILs show increased cytokine expression, decreased expression of inhibitory receptors, and increased accessibility of regions enriched for motifs that bind activation-associated nuclear factor κB (NFκB) and basic region-leucine zipper (bZIP) transcription factors. These data indicate that Tox and Nr4a transcription factors are critical for the transcriptional program of CD8+ T cell exhaustion downstream of NFAT. We provide evidence for positive regulation of NR4A by TOX and of TOX by NR4A, and suggest that disruption of TOX and NR4A expression or activity could be promising strategies for cancer immunotherapy

TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8+ T cell exhaustion

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive ("exhausted" or "dysfunctional") within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8(+) T cell exhaustion. We show that the high-mobility group (HMG)-box transcription factors TOX and TOX2, as well as members of the NR4A family of nuclear receptors, are targets of the calcium/calcineurin-regulated transcription factor NFAT, even in the absence of its partner AP-1 (FOS-JUN). Using a previously established CAR T cell model, we show that TOX and TOX2 are highly induced in CD8(+) CAR(+)PD-1(high) TIM3(high) ("exhausted") tumor-infiltrating lymphocytes (CAR TIL5), and CAR TIL5 deficient in both TOX and TOX2 (Tox DKO) are more effective than wild-type (WT), TOX-deficient, or TOX2-deficient CAR TIL5 in suppressing tumor growth and prolonging survival of tumor-bearing mice. Like NR4A-deficient CAR TIL5, Tox DKO CAR TIL5 show increased cytokine expression, decreased expression of inhibitory receptors, and increased accessibility of regions enriched for motifs that bind activation-associated nuclear factor kappa B (NF kappa B) and basic region-leucine zipper (bZIP) transcription factors. These data indicate that Tox and Nr4a transcription factors are critical for the transcriptional program of CD8(+) T cell exhaustion downstream of NFAT. We provide evidence for positive regulation of NR4A by TOX and of TOX by NR4A, and suggest that disruption of TOX and NR4A expression or activity could be promising strategies for cancer immunotherapy.

Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer

Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103(hi) tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.</p