ABSTRACT
Although several promising approaches for the treatment of relapsed/refractory diffuse large B-cell lymphoma (rrDLBCL) have been approved recently, it remains unclear which patients will ultimately achieve long-term responses. Circulating tumor (ct)DNA sequencing has emerged as a valuable tool to assess minimal residual disease (MRD). Correlations between MRD and outcomes have been shown in previously untreated DLBCL, but data on the repeated assessment of MRD in the dynamic course of rrDLBCL is limited. Here, we present an approach leveraging cost- and time-sensitivity of digital droplet (dd)PCR to repeatedly assess MRD in rrDLBCL and present proof-of-principle for its ability to predict outcomes.
Subject(s)
Lymphoma, Large B-Cell, Diffuse , Neoplasm, Residual , Polymerase Chain Reaction , Humans , Neoplasm, Residual/diagnosis , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/diagnosis , Lymphoma, Large B-Cell, Diffuse/drug therapy , Polymerase Chain Reaction/methods , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Recurrence , Prognosis , Circulating Tumor DNA/genetics , Male , Female , Drug Resistance, Neoplasm/genetics , Biomarkers, Tumor , Middle Aged , Treatment OutcomeABSTRACT
CD8αα(+) intraepithelial lymphocytes (IELs) are instrumental in maintaining the epithelial barrier in the intestine. Similar to natural killer cells and other innate lymphoid cells, CD8αα(+) IELs constitutively express the T-box transcription factor T-bet. However, the precise role of T-bet for the differentiation or function of IELs is unknown. Here we show that mice genetically deficient for T-bet lacked both TCRαß(+) and TCRγδ(+) CD8αα(+) IELs and thus are more susceptible to chemically induced colitis. Although T-bet was induced in thymic IEL precursors (IELPs) as a result of agonist selection and interleukin-15 (IL-15) receptor signaling, it was dispensable for the generation of IELPs. Subsequently, T-bet was required for the IL-15-dependent activation, differentiation, and expansion of IELPs in the periphery. Our study reveals a function of T-bet as a central transcriptional regulator linking agonist selection and IL-15 signaling with the emergence of CD8αα(+) IELs.
Subject(s)
CD8 Antigens/biosynthesis , Interleukin-15/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, gamma-delta/genetics , T-Box Domain Proteins/genetics , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Colitis/chemically induced , Colitis/immunology , Epithelial Cells/immunology , Interleukin-15/immunology , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Intestines/cytology , Intestines/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Interleukin-15/immunology , Signal Transduction/immunology , T-Box Domain Proteins/biosynthesisABSTRACT
At mucosal surfaces, the immune system should not initiate inflammatory immune responses to the plethora of antigens constantly present in the environment, but should remain poised to unleash a potent assault on intestinal pathogens. The transcriptional programs and regulatory factors required for immune cells to switch from homeostatic (often tissue-protective) function to potent antimicrobial immunity are poorly defined. Mucosal retinoic-acid-receptor-related orphan receptor-γt-positive (RORγt(+)) innate lymphoid cells (ILCs) are emerging as an important innate lymphocyte population required for immunity to intestinal infections. Various subsets of RORγt(+) ILCs have been described but the transcriptional programs controlling their specification and fate remain largely unknown. Here we provide evidence that the transcription factor T-bet determines the fate of a distinct lineage of CCR6(-)RORγt(+) ILCs. Postnatally emerging CCR6(-)RORγt(+) ILCs upregulated T-bet and this was controlled by cues from the commensal microbiota and interleukin-23 (IL-23). In contrast, CCR6(+)RORγt(+) ILCs, which arise earlier during ontogeny, did not express T-bet. T-bet instructed the expression of T-bet target genes such as interferon-γ (IFN-γ) and of the natural cytotoxicity receptor NKp46. Mice genetically lacking T-bet showed normal development of CCR6(-)RORγt(+) ILCs, but they could not differentiate into NKp46-expressing RORγt(+) ILCs (that is, IL-22-producing natural killer (NK-22) cells) and failed to produce IFN-γ. The production of IFN-γ by T-bet-expressing CCR6(-)RORγt(+) ILCs was essential for the release of mucus-forming glycoproteins required to protect the epithelial barrier during Salmonella enterica infection. Salmonella infection also causes severe enterocolitis that is at least partly driven by IFN-γ. Mice deficient for T-bet or depleted of ILCs developed only mild enterocolitis. Thus, graded expression of T-bet in CCR6(-)RORγt(+) ILCs facilitates the differentiation of IFN-γ-producing CCR6(-)RORγt(+) ILCs required to protect the epithelial barrier against Salmonella infections. Co-expression of T-bet and RORγt, which is also found in subsets of IL-17-producing T-helper (T(H)17) cells, may be an evolutionarily conserved transcriptional program that originally developed as part of the innate defence against infections but that also confers an increased risk of immune-mediated pathology.
Subject(s)
Cell Lineage , Immunity, Innate/immunology , Lymphocytes/cytology , Lymphocytes/immunology , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Receptors, CCR6/deficiency , T-Box Domain Proteins/metabolism , Animals , Antigens, Ly/genetics , Cell Differentiation , Cells, Cultured , Enterocolitis/immunology , Enterocolitis/metabolism , Enterocolitis/pathology , Epithelium/immunology , Epithelium/metabolism , Epithelium/microbiology , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-23/immunology , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Intestinal Mucosa/microbiology , Lymphocytes/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mucus/metabolism , Natural Cytotoxicity Triggering Receptor 1/genetics , Receptors, CCR6/metabolism , Salmonella Infections/immunology , Salmonella Infections/metabolism , Salmonella typhimurium/immunology , Salmonella typhimurium/pathogenicityABSTRACT
The intestine is a major immune organ with several specialized lymphoid structures and immune cells. Among these are thymus-derived natural intraepithelial lymphocytes (IELs) that lack expression of the classical co-receptors CD4 or CD8αß (double negative (DN)). Natural IELs are both αß+ and γδ+ T cells that play important roles in the maintenance of the epithelial barrier at steady state and during inflammation. The transcription factor T-bet is essential for the peripheral development of natural IELs, but its role during thymic development has remained less clear. Here we show that a T-bet gradient in DN TCRαß+NK1.1- thymocytes (IEL precursors (IELPs)) determines IEL fate in natural TCRαß+ IELs. Employing T-bet ZsGreen reporter mice in in vitro cultures and in vivo transfer experiments, we demonstrate that with increasing expression of T-bet, DN TCRαß+NK1.1- thymocytes are gradually restricted to a DN IEL fate. Furthermore, we show that the natural TCRαß+ IELs seed the intestine within the first month of life. This in turn is preceded by the appearance of T-bet- and T-bet+ IELPs that egress from the thymus in a sphingosine-1-phosphate (S1P)-dependent manner. In summary, the use of T-bet reporter mice has enabled us to identify and refine an immediate and clearly committed postselection precursor of natural TCRαß+ IELs.