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Embryonic development is characterized by rapidly dividing cells, cellular plasticity and a highly vascular microenvironment. These features are similar to those of tumour tissue, in that malignant cells are characterized by their ability to proliferate and exhibit cellular plasticity. The tumour microenvironment also often includes immunosuppressive features. Reciprocal communication between various cellular subpopulations enables fetal and tumour tissues to proliferate, migrate and escape immune responses. Fetal-like reprogramming has been demonstrated in the tumour microenvironment, indicating extraordinary cellular plasticity and bringing an additional layer of cellular heterogeneity. More importantly, some of these features are also present during inflammation. This Perspective discusses the similarity between embryogenesis, inflammation and tumorigenesis, and describes the mechanisms of oncofetal reprogramming that enable tumour cells to escape from immune responses, promoting tumour growth and metastasis.

The spatial organisation of cells defines the biological functions of tissue ecosystems from development to disease. Recently, an array of technologies have been developed to query gene expression in a spatial context. These include techniques such as employing barcoded oligonucleotides, single-molecule fluorescence in situ hybridization (smFISH), and DNA nanoball (DNB)-patterned arrays. However, resolution and efficiency vary across platforms and technologies. To obtain spatially relevant biological information from spatially resolved transcriptomics, we combined the Stereo-seq workflow with CRISPRclean technology to develop the STOmics-GenX pipeline. STOmics-GenX not only allowed us to reduce genomic, mitochondrial, and ribosomal reads, but also lead to a ∼2.1-fold increase in the number of detected genes when compared to conventional Stereo-seq (STOmics). Additionally, the STOmics-GenX pipeline resulted in an improved detection of cell type specific genes, thereby improving cellular annotations. Most importantly, STOmics-GenX allowed for enhanced detection of clinically relevant biomarkers such as Alpha-fetoprotein (AFP), enabling the identification of two spatially distinct subsets of hepatocytes in hepatocellular carcinoma tissue. Thereby, combining CRISPRclean technology with STOmics not only allowed improved gene detection but also paved the way for spatial precision oncology by improved detection of clinically relevant biomarkers.

Loss of T cell immunogenicity due to mutations in virally encoded epitopes is a well-described adaptation strategy to limit host anti-viral immunity. Another described, but less understood, adaptation strategy involves the selection of mutations within epitopes that retain immune recognition, suggesting a benefit for the virus despite continued immune pressure (termed non-classical adaptation). To understand this adaptation strategy, we utilized a single cell transcriptomic approach to identify features of the HIV-specific CD8+ T cell responses targeting non-adapted (NAE) and adapted (AE) forms of epitopes containing a non-classical adaptation. T cell receptor (TCR) repertoire and transcriptome were obtained from antigen-specific CD8+ T cells of chronic (n=7) and acute (n=4) HIV-infected subjects identified by either HLA class I tetramers or upregulation of activation markers following peptide stimulation. CD8+ T cells were predominantly dual tetramer+, confirming a large proportion of cross-reactive TCR clonotypes capable of recognizing the NAE and AE form. However, single-reactive CD8+ T cells were identified in acute HIV-infected subjects only, providing the potential for the selection of T cell clones over time. The transcriptomic profile of CD8+ T cells was dependent on the autologous virus: subjects whose virus encoded the NAE form of the epitope (and who transitioned to the AE form at a later timepoint) exhibited an ‘effective’ immune response, as indicated by expression of transcripts associated with polyfunctionality, cytotoxicity and apoptosis (largely driven by the genes GZMB, IFNɣ, CCL3, CCL4 and CCL5). These data suggest that viral adaptation at a single amino acid residue can provide an alternative strategy for viral survival by modulating the transcriptome of CD8+ T cells and potentially selecting for less effective T cell clones from the acute to chronic phase.

Cellular immune responses to Gag correlate with improved HIV control. The full extent of cellular immune responses comprises both the number of epitopes recognized by CD4(+) and CD8(+) T cells and the diversity of the T cell receptor (TCR) repertoire directed against each epitope. The optimal diversity of the responsive TCR repertoire is unclear. Therefore, we evaluated the TCR diversity of CD4(+) and CD8(+) T cells responding to HIV-1 Gag to determine if TCR diversity correlates with clinical or virologic metrics. Previous studies of TCR repertoires have been limited primarily to CD8(+) T cell responses directed against a small number of well-characterized T cell epitopes restricted by specific human leukocyte antigens. We stimulated peripheral blood mononuclear cells from 21 chronic HIV-infected individuals overnight with a pool of HIV-1 Gag peptides, followed by sorting of activated CD4(+) and CD8(+) T cells and TCR deep sequencing. We found Gag-reactive CD8(+) T cells to be more oligoclonal, with a few dominant TCRs comprising the bulk of the repertoire, compared with the highly diverse TCR repertoires of Gag-reactive CD4(+) T cells. HIV sequencing of the same donors revealed that high CD4(+) T cell TCR diversity was strongly associated with lower HIV Gag genetic diversity. We conclude that the TCR repertoire of Gag-reactive CD4(+) T helper cells displays substantial diversity without a clearly dominant circulating TCR clonotype, in contrast to a hierarchy of dominant TCR clonotypes in the Gag-reactive CD8(+) T cells, and may serve to limit HIV diversity during chronic infection. IMPORTANCE Human T cells recognize portions of viral proteins bound to host molecules (human leukocyte antigens) on the surface of infected cells. T cells recognize these foreign proteins through their T cell receptors (TCRs), which are formed by the assortment of several available V, D, and J genes to create millions of combinations of unique TCRs. We measured the diversity of T cells responding to the HIV Gag protein. We found that the CD8(+) T cell response is primarily made up of a few dominant unique TCRs, whereas the CD4(+) T cell subset has a much more diverse repertoire of TCRs. We also found there was less change in the virus sequences in subjects with more diverse TCR repertoires. HIV has a high mutation rate, which allows it to evade the immune response. Our findings describe the characteristics of a virus-specific T cell response that may allow it to limit viral evolution.

Human immunodeficiency virus (HIV) can adapt to an individual’s T cell immune response via genomic mutations that affect antigen recognition and impact disease outcome. These viral adaptations are specific to the host’s human leucocyte antigen (HLA) alleles, as these molecules determine which peptides are presented to T cells. As HLA molecules are highly polymorphic at the population level, horizontal transmission events are most commonly between HLA-mismatched donor/recipient pairs, representing new immune selection environments for the transmitted virus. In this study, we utilised a deep sequencing approach to determine the HIV quasispecies in 26 mother-to-child transmission pairs where the potential for founder viruses to be pre-adapted is high due to the pairs being haplo-identical at HLA loci. This scenario allowed the assessment of specific HIV adaptations following transmission in either a non-selective immune environment, due to recipient HLA mismatched to original selecting HLA, or a selective immune environment, mediated by matched donor/recipient HLA. We show that the pattern of reversion or fixation of HIV adaptations following transmission provides insight into the replicative cost, and likely compensatory networks, associated with specific adaptations in vivo. Furthermore, although transmitted viruses were commonly heavily pre-adapted to the child’s HLA genotype, we found evidence of de novo post-transmission adaptation, representing new epitopes targeted by the child’s T cell response. High-resolution analysis of HIV adaptation is relevant when considering vaccine and cure strategies for individuals exposed to adapted viruses via transmission or reactivated from reservoirs., Author summary Highly mutable pathogens utilise genetic variations within T cell epitopes as a mechanism of immune escape (viral adaptation). The diversity of the human leucocyte antigen (HLA) molecules that present viral targets to T cells in human populations partially protects against rapid population-level accumulation of human immunodeficiency virus (HIV) adaptations through horizontal transmissions. In contrast, vertical transmissions occur between haplo-identical mother/child pairs, and potentially include adaptive changes through father-mother-child transmission, representing a pathway to complete pre-adaptation to HLA alleles in child hosts over only two transmission events. We utilised next-generation sequencing to examine HIV evolution in the unique setting of vertical HIV transmission. We predict the in vivo replicative cost and immune benefit of specific HIV adaptations that could be used to inform vaccine design and cure strategies to combat viral immune adaptation.