Becca Asquith

Mitochondrial DNA (mtDNA) mutations have been linked to ageing. We find that the mtDNA mutations detected in routine bulk sequencing are greatly outnumbered by a class of concealed mutations that, despite being in a proliferative tissue, show little negative selection. Combining new scATAC-seq experiments with existing scRNA-seq, we map mtDNA mutations across 2.7 million cells from 311 individuals, covering 11 proliferative cell types and 5 tissues. We find that low-prevalence mutations (in ≤ 1% of assayed cells), concealed from routine bulk sequencing, accumulate markedly with age, with mutations unique to single cells (cryptic) predominating. We find a near universal accumulation rate across cell types and tissues, with, by age 50, approximately 40% of cells having at least one mutation affecting 30% of cellular mtDNA or more. At a correlative level we find that human age can be predicted from these concealed mutations but there is evidence of, possibly physiological, inter-individual variation. These low-prevalence mutations (unlike mutations tracking larger clones) show limited negative selection and coincide with gene-expression changes marking dysfunction.

T stem cell-like memory cells (T_SCM cells) are considered to be essential for the maintenance of immune memory. The T_SCM population has been shown to have the key properties of a stem cell population: multipotency, self-renewal and clonal longevity. Here we show that no single population has all these stem cell properties, instead the properties are distributed. We show that the human T_SCM population consists of two distinct cell subpopulations which can be distinguished by the level of their CD95 expression (CD95int and CD95hi). Crucially, using long-term in vivo labelling of human volunteers, we establish that these are distinct populations rather than transient states of the same population. These two subpopulations have different functional profiles ex vivo, different transcriptional patterns, and different tissue distributions. They also have significantly different TREC content indicating different division histories and we find that the frequency of CD95hi T_SCM increases with age. Most importantly, CD95hi and CD95int T_SCM cells also have very different dynamics in vivo with CD95hi cells showing considerably higher proliferation but significantly reduced clonal longevity compared with CD95int T_SCM. While both T_SCM subpopulations exhibit considerable multipotency, no single population of T_SCM cells has both the properties of self-renewal and clonal longevity. Instead, the “stemness” of the T_SCM population is generated by the complementary dynamic properties of the two subpopulations: CD95int T_SCM which have the property of clonal longevity and CD95hi T_SCM which have the properties of expansion and self-renewal. We suggest that together, these two populations function as a stem cell population.

Stable isotope labelling is one of the best methods currently available for quantifying cell dynamics in vivo, particularly in humans where the absence of toxicity makes it preferable over other techniques such as CFSE or BrdU. Interpretation of stable isotope labelling data (as for BrdU and CFSE) necessitates simplifying assumptions. Here we investigate the impact of three of the most commonly used simplifying assumptions: (i) that the cell population of interest is closed, (ii) that the population of interest is kinetically homogeneous, and (iii) that the population is spatially homogeneous and suggest pragmatic ways in which the resulting errors can be reduced.

Inhibitory killer cell immunoglobulin-like receptors (iKIRs) are a family of inhibitory receptors that are expressed by natural killer (NK) cells and late-stage differentiated T cells. There is accumulating evidence that iKIRs regulate T cell-mediated immunity. Recently, we reported that T cell-mediated control was enhanced by iKIRs in chronic viral infections. We hypothesized that in the context of autoimmunity, where an enhanced T cell response might be considered detrimental, iKIRs would have an opposite effect. We studied Type 1 diabetes (T1D) as a paradigmatic example of autoimmunity. In T1D, variation in the Human Leucocyte Antigen (HLA) genes explains up to 50% of the genetic risk, indicating that T cells have a major role in T1D etiopathogenesis. To investigate if iKIRs affect this T cell response, we asked whether HLA associations were modified by iKIR genes. We conducted an immunogenetic analysis of a case-control T1D dataset (N = 11,961) and found that iKIR genes, in the presence of genes encoding their ligands, have a consistent and significant effect on protective HLA class II genetic associations. Our results were validated in an independent data set. We conclude that iKIRs significantly decrease HLA class II protective associations and suggest that iKIRs regulate CD4⁺ T cell responses in T1D.

In addition to their central role during innate responses, NK cells regulate adaptive immunity through various mechanisms. A wide array of innate receptors has been involved in the NK cell regulatory function. However, the clinical implications of these regulatory pathways are poorly understood. Here, we review the experimental evidence on the effects of NK cells on T cells and their positive and negative consequences for disease outcome during T cell responses in humans.

Dendritic cells (DC) are specialized mononuclear phagocytes that link innate and adaptive immunity. They comprise two principal subsets: plasmacytoid DC (pDC) and conventional DC (cDC). Understanding the generation, differentiation, and migration of cDC is critical for immune homeostasis. Through human in vivo deuterium-glucose labeling, we observed the rapid appearance of AXL+ Siglec6+ DC (ASDC) in the bloodstream. ASDC circulate for ∼2.16 days, while cDC1 and DC2 circulate for ∼1.32 and ∼2.20 days, respectively, upon release from the bone marrow. Interestingly, DC3, a cDC subset that shares several similarities with monocytes, exhibits a labeling profile closely resembling that of DC2. In a human in vivo model of cutaneous inflammation, ASDC were recruited to the inflammatory site, displaying a distinctive effector signature. Taken together, these results quantify the ephemeral circulating lifespan of human cDC and propose functions of cDC and their precursors that are rapidly recruited to sites of inflammation.

Inhibitory killer cell immunoglobulin-like receptors (iKIRs) are a family of inhibitory receptors that are expressed by natural killer cells and late-stage differentiated T cells. There is accumulating evidence that iKIRs regulate T cell-mediated immunity. Recently, we reported that T cell-mediated control was enhanced by iKIRs in chronic viral infections. We hypothesized that in the context of autoimmunity, where an enhanced T cell response might be considered detrimental, iKIRs would have an opposite effect. We studied Type 1 diabetes (T1D) as a paradigmatic example of autoimmunity. In T1D, variation in the Human Leucocyte Antigen (HLA) genes explains up to 50% of the genetic risk, indicating that T cells have a major role in T1D etiopathogenesis. To investigate if iKIRs affect this T cell response we asked whether HLA associations were modified by iKIR genes. We conducted an immunogenetic analysis of a case-control T1D dataset (N= 11,961) and found that iKIR genes, in the presence of genes encoding their ligands, have a consistent and significant effect on protective HLA class II genetic associations. Our results were validated in an independent data set. We conclude that iKIRs significantly decrease HLA class II protective associations and suggest that iKIRs regulate CD4+ T cell responses in T1D.

Background: Accurate quantification of rare genes from limited clinical samples is crucial for research purposes but is technically challenging. In particular, nucleic acid extraction for quantification of gene targets may lead to target loss. Here, we report the development and validation of a novel crude lysate ddPCR assay for the absolute quantification of rare genes, TRECs in our case, from infrequent cells, that removes the need for DNA extraction, hence minimizing the target loss. Methods: The analytical validation was performed on PBMCs extracted from the blood of healthy donors. Standard ddPCR was first optimized to detect TREC copies/cell and then applied to a crude lysate ddPCR assay. The assay was optimized by varying several steps. The optimised assay was directly compared to standard ddPCR and the performance of the assay quantified. Results: The newly developed assay showed good agreement with the standard ddPCR assay in the range from 0.0003 to 0.01 TRECs/cell. The assay had a limit of quantification of <0.0003 TRECs/cell and a limit of detection of <0.0001 TRECs/cell; this performance is favourable compared to standard ddPCR. The intra-assay variation was low. This method can also be applied to fixed and permeabilized cells. Conclusions: The newly developed crude lysate ddPCR assay for quantifying rare targets from limited samples has high accuracy, specificity, and reproducibility; additionally, it eliminates the need for DNA extraction for absolute quantification. The assay has the potential to be used for quantification of other trace targets from small samples.

Author summary: Stable isotope labelling is one of the best available methods for quantifying cell dynamics in vivo, particularly in humans where the absence of toxicity makes it preferable over other techniques such as CFSE or BrdU. Interpretation of stable isotope labelling data necessitates simplifying assumptions. Here we investigate the impact of three of the most commonly used simplifying assumptions (that the cell population of interest is closed, that the population of interest is kinetically homogeneous, and that the population is spatially homogeneous) and suggest pragmatic ways in which the resulting errors can be reduced.

BACKGROUND. There is increasing evidence, in transgenic mice and in vitro, that inhibitory killer cell immunoglobulin-like receptors (iKIRs) can modulate T cell responses. Furthermore, we have previously shown that iKIRs are an important determinant of T cell–mediated control of chronic viral infection and that these results are consistent with an increase in the CD8⁺ T cell lifespan due to iKIR-ligand interactions. Here, we tested this prediction and investigated whether iKIRs affect T cell lifespan in humans in vivo. METHODS. We used stable isotope labeling with deuterated water to quantify memory CD8⁺ T cell survival in healthy individuals and patients with chronic viral infections. RESULTS. We showed that an individual’s iKIR-ligand genotype was a significant determinant of CD8⁺ T cell lifespan: in individuals with 2 iKIR-ligand gene pairs, memory CD8⁺ T cells survived, on average, for 125 days; in individuals with 4 iKIR-ligand gene pairs, the memory CD8⁺ T cell lifespan doubled to 250 days. Additionally, we showed that this survival advantage was independent of iKIR expression by the T cell of interest and, further, that the iKIR-ligand genotype altered the CD8⁺ and CD4⁺ T cell immune aging phenotype. CONCLUSIONS. Together, these data reveal an unexpectedly large effect of iKIR genotype on T cell survival. FUNDING. Wellcome Trust; Medical Research Council; EU Horizon 2020; EU FP7; Leukemia and Lymphoma Research; National Institute of Health Research (NIHR) Imperial Biomedical Research Centre; Imperial College Research Fellowship; National Institutes of Health; Jefferiss Trust.