Understanding and Modulating Immunity With Cell Reprogramming

Abstract

Cell reprogramming concepts have been classically developed in the fields of developmental and stem cell biology and are currently being explored for regenerative medicine, given its potential to generate desired cell types for replacement therapy. Cell fate can be experimentally reversed or modified by enforced expression of lineage specific transcription factors leading to pluripotency or attainment of another somatic cell type identity. The possibility to reprogram fibroblasts into induced dendritic cells (DC) competent for antigen presentation creates a paradigm shift for understanding and modulating the immune system with direct cell reprogramming. PU.1, IRF8, and BATF3 were identified as sufficient and necessary to impose DC fate in unrelated cell types, taking advantage of Clec9a, a C-type lectin receptor with restricted expression in conventional DC type 1. The identification of such minimal gene regulatory networks helps to elucidate the molecular mechanisms governing development and lineage heterogeneity along the hematopoietic hierarchy. Furthermore, the generation of patient-tailored reprogrammed immune cells provides new and exciting tools for the expanding field of cancer immunotherapy. Here, we summarize cell reprogramming concepts and experimental approaches, review current knowledge at the intersection of cell reprogramming with hematopoiesis, and propose how cell fate engineering can be merged to immunology, opening new opportunities to understand the immune system in health and disease.

Keywords: antigen presentation; cancer immunotherapy; cell fate reprogramming; dendritic cell; hematopoiesis; regenerative medicine; transcription factor; transdifferentiation.

Figure 1

Experimental approaches for cell fate reprogramming. Nuclear transfer, cell fusion, and enforced expression of defined factors have revealed the plasticity of cell identity. Adult cell commitment can be experimentally reverted or modified by exposing a cell nucleus to unidentified or defined factors. In SCNT, a nucleus of an adult cell is transferred into an enucleated metaphase-II oocyte. The somatic cell nucleus is reprogrammed to totipotency by the action of zygotic factors. Cell fate can also be reverted or modified by cell fusion. Two cells are fused to generate a multinucleated heterokaryon, where nuclear factors shuttle across nuclei. Nuclear fusion gives rise to a tetraploid hybrid cell that is able to proliferate. Cell fate conversion can be accomplished by defined factors, including cell type-specific transcription factors, epigenetic modifiers, microRNAs and small molecules, acting in combination to impose pluripotency or alternative somatic cell identities.

Figure 2

Programming hematopoietic cell fates with transcription factors. Overview of reprogramming approaches in the hematopoietic system, with transcription factors classified as keepers, facilitators, and instructors. Keepers are factors that maintain cell fate and repress alternative lineages, as determined by gene knockout experiments, and are depicted by red loop arrows. Transcription factor facilitators of lineage reprogramming within the hematopoietic system, as determined through enforced expression experiments, are indicated by green lines showing the lineage of origin (solid circle), direction of reprogramming (green arrows), and lineage outcome (open circle). Blue boxes depict combinatorial expression of instructive transcription factors leading to reprogramming of non-hematopoietic cells into hematopoietic cells. Instructive factors reported in multiple studies are highlighted on top. Abbreviations: HE, hemogenic endothelium; HSPC, hematopoietic stem and progenitor cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; Mk, megakaryocyte; Er, erythrocyte; Gr, granulocyte; Mo, monocyte; MΦ, macrophage; cDC2, conventional dendritic cell type 2; cDC1, conventional dendritic cell type 1; pDC, plasmacytoid dendritic cell; NK, natural killer; ILC, innate lymphoid cell.

Figure 3

Clec9a-tdTomato reporter allows the identification of dendritic cell-instructive transcription factors. (A) Using double transgenic mouse embryonic fibroblasts (MEF) expressing Cre recombinase under the control of Clec9a promoter and Rosa26-lox-stop-tdTomato, we identified PU.1 (P), IRF8 (I), and BATF3 (B) to program induced dendritic cells (iDC) affiliated with the conventional DC type 1 (cDC1) fate. (B) Heat map showing gene expression of C-type lectin receptors across hematopoietic cell lineages (GSE127267). Forty-two receptors expressed in DCs are highlighted in red. DC, dendritic cell; NK, natural killer. (C) Heat maps showing differential gene expression of C-type lectin receptors (upper panel), where nine genes enriched in cDC1s are highlighted in red, and genes associated with antigen cross-presentation (bottom panel) in mouse cDC1, cDC2, and plasmacytoid DCs (pDC) (GSE103618). Red indicates increased expression, whereas blue indicates decreased expression over the mean. (D) Expression levels of Clec9a and Clec1a and (E) B2m, Tap1, Tapbp, and Psmb8 during DC reprogramming at days 3 (d3), 7 (d7), and 9 (d9). Log values of census counts are shown. Horizontal lines correspond to median values. (F) Flow cytometry analysis of B2M surface expression in tdTomato-positive cells generated by transduction with PU.1, IRF8, and BATF3 after 9 days. MEF transduced with M2rtTA were included as control. Adapted from Rosa et al. (147).

Figure 4

Understanding gene regulatory networks and lineage heterogeneity with cell reprogramming. (A) Two models have been proposed for instructive transcription factor (represented as A, B and C) binding to chromatin: (1) cooperative binding among multiple transcription factors at open chromatin regions leading to a gradual increase in site accessibility (left panel), and (2) pioneer transcription factors, as a unique class of transcriptional regulators with the capacity to bind nucleosomal DNA (closed chromatin, right panel). Experimentally, these models can be interrogated at the onset of lineage reprogramming in fibroblasts by expressing instructive transcription factors in combination (top panel) or individually (bottom panel) followed by genome location studies. (B) Combined expression of PU.1, IRF8, and BATF3 in fibroblasts generates conventional dendritic cells type 1 (cDC1)-like cells capturing part of the DC lineage heterogeneity. DC cell states are depicted as positions in a matrix and cDC1 are highlighted in blue. We hypothesize that direct lineage reprogramming offers an exciting way to distinguish stable from dynamic DC attractor states according to their dependence on transcription factor combination and stoichiometry (dashed lines). (C) Heat map showing single cell gene expression of Cd207 and Cx3cr1 during reprogramming of mouse embryonic fibroblasts (MEF) to induced DCs (iDC) at days 3 (d3), 7 (d7), and 9 (d9) (GSE103618). (D) Gene set enrichment analysis between d9 iDC and splenic cDC1 was performed using migratory and resident DC gene sets (168), and (E) between d9 iDC and MEF, and d9 iDC and cDC1 (GSE103618), with TLR-induced or homeostatic maturation gene sets (169). NES, normalized enrichment score; FDR q, false discovery rate q value. Adapted from Rosa et al. (147).

Figure 5

Direct reprogramming for immune-therapeutic applications. Direct lineage reprogramming allows the identification of instructive combinations of factors required to impose immune cell fates in unrelated somatic cells such as fibroblasts. These combinations can be repurposed to derive immune cells from expandable cell sources, such as iPSCs or hematopoietic stem and progenitor cells (HSPC). Forward reprogramming mediated by instructors represents a strategy to generate large quantities of homogeneous populations of immune cells for therapeutic applications. It may also be possible to impose functional reprogramming, or confer immune functional properties with instructors while retaining some features of the starting cell type.