Gene Expression Profiling of Bronchoalveolar Lavage Cells Preceding a Clinical Diagnosis of Chronic Lung Allograft Dysfunction

Weigt, S. Samuel; Wang, Xiaoyan; Palchevskiy, Vyacheslav; Gregson, Aric L.; Patel, Neil; Derhovanessian, Ariss; Shino, Michael Y.; Sayah, David M.; Birjandi, Shirin; Lynch, Joseph P.; Saggar, Rajan; Ardehali, A.; Ross, David J.; Palmer, Scott M.; Elashoff, David; Belperio, John A.

doi:10.1371/journal.pone.0169894

Cited by 32 publications

(20 citation statements)

References 44 publications

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“…Briefly, raw count values were used as input into deSeq2, and variance stabilizing transformation was used to transform data. Data were batch-and patient corrected using Limma 62 . A cut-off of >100 counts across the samples was used to filter out low expressed genes.…”

Section: Methodsmentioning

confidence: 99%

Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood

Marquardt

Scharenberg

Mold

et al. 2019

Preprint

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29The concept of adaptive-like "memory" NK cells has been extensively investigated in 30 recent years. In humans, NK cells with adaptive-like features have been identified in 31 peripheral blood and liver of cytomegalovirus (CMV)-seropositive individuals. The 32 human lung is a major target organ for infections and is also a significant reservoir for 33 CMV. However, it remains unknown whether adaptive-like NK cells can be found in 34 this organ. Using RNAseq and flow cytometry analysis, we here identify a novel 35 adaptive-like KIR + NKG2C + NK cell subset with a CD49a + CD56 bright CD16tissue-36 resident (tr)NK cell phenotype in human lung and in peripheral blood. Adaptive-like 37 lung trNK cells were found to be present independently of adaptive-like CD56 dim CD16 + 38 NK cells, to be hyperresponsive towards target cells, and to exhibit signs of metabolic 39 reprogramming. In conclusion, adaptive-like trNK cells constitute a novel subset of 40 human lung NK cells, likely contributing to unique defense mechanisms in context of 41 infection at this site. 42 4 Results 99 Adaptive-like NK cells can be identified in human lung 100We first set out to investigate whether expansions of KIR + NKG2C + adaptive-101 like NK cells could be identified in the human lung. The majority of NK cells in the 102 lung are phenotypically similar to NK cells found in peripheral blood (CD69 -103 CD56 dim CD16 + ), suggesting that these cells may recirculate between this organ and 104 peripheral blood 20 . Accordingly, KIR + NKG2C + CD56 dim CD16 + NK cells could be 105 identified not only in peripheral blood but also in the lung (Fig. 1a). Surprisingly, KIR 106 and NKG2C were also co-expressed on CD56 bright CD16lung NK cells, with varying 107 frequencies between donors (Fig. 1b, c) (see Supplementary Fig. 1a for the gating 108 strategy to identify KIR + NKG2C + CD56 bright CD16and CD56 dim CD16 + NK cells). In 109 several donors the frequencies of KIR + NKG2C + CD56 bright CD16 -NK cells in human 110 lung vastly exceeded those previously described in the liver 7 , with up to 98% of 111 CD56 bright CD16lung NK cells co-expressing KIR and NKG2C (Fig. 1 a, b). 112Next, we assessed phenotypic features of KIR + NKG2C + CD56 bright CD16lung 113 NK cells in an unbiased manner. Uniform manifold approximation and expression 114 (UMAP) analysis revealed a distinct subset of cells with an expression pattern 115 consistent with adaptive-like NK cells found in peripheral blood and liver, including 116 low expression of Siglec-7 and CD161, and high expression of NKG2C, KIRs, and 117 CD2 7,8,23,24 (Fig. 1d). Unlike KIR + NKG2C + CD56 dim CD16 + NK cells, 118 KIR + NKG2C + CD56 bright CD16 -NK cells expressed lower levels of CD45RA and 119 NKp80, and higher levels of CD8 (Fig. 1d). In addition to these expression patterns, 120 manual analysis of individual samples additionally confirmed low expression of ILT2 121 and FcεR1g, as compared to KIR + NKG2C + CD56 dim CD16 + lung NK cells (Fig. 1e, f). 122Furthermore, KIR + NKG2C + CD56 bright CD16lu...

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Section: Methodsmentioning

confidence: 99%

Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood

Marquardt

Scharenberg

Mold

et al. 2019

Preprint

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“…Bronchoalveolar lavage (BAL) offers a larger sampling area, lower risk and provides a reliable source of RNA. Researchers identified a distinct BAL transcriptome profile in patients who went on to develop CLAD, and the change in gene expression appeared to predate its detectable clinical features, with a similar profile seen in patients with acute rejection . Transcriptomics may therefore have a role in the earlier diagnosis of rejection and identification of upstream therapeutic targets.…”

Section: Transcriptomics: a Primermentioning

confidence: 99%

Transcriptomics and single‐cell RNA‐sequencing

et al. 2018

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The past four decades have yielded advances in molecular biology allowing detailed characterization of the cellular genome and the transcriptome: the complete set of RNA species transcribed by a cell or tissue. Through transcriptomics and next‐generation sequencing, we can now attain an unprecedented level of detail in understanding cellular phenotypes through examining the genes expressed in specific physiological and pathological states. In this review, we provide an overview of transcriptomics and RNA‐sequencing in the analysis of whole tissue and single cells. We describe the techniques and pitfalls involved in the isolation and sequencing of single cells, and what additional benefits this application can provide. Finally, we look to how these technologies are being applied in pulmonary research, and how they may translate in the near future into clinical practice.

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“…This is particularly concerning when compared with other solid organ transplants such as liver, kidney, and heart that have 5-year survival rates of at least 70% (1)(2)(3)(4)(5)(6). Lung transplant recipients also have much higher rates of rejection, the main risk factor for limited lung allograft survival (7)(8)(9)(10)(11)(12)(13)(14)(15). Rejection is classically considered a consequence of an immune response to donor alloantigens that results in allograft dysfunction (7)(8)(9)(10)(11)(12)(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

“…Lung transplant recipients also have much higher rates of rejection, the main risk factor for limited lung allograft survival (7)(8)(9)(10)(11)(12)(13)(14)(15). Rejection is classically considered a consequence of an immune response to donor alloantigens that results in allograft dysfunction (7)(8)(9)(10)(11)(12)(13)(14)(15). However, many of the molecular factors involved in the initiation of the alloresponse that leads to rejection remain largely unknown.…”

Section: Introductionmentioning

confidence: 99%

CCR4 expression on host T cells is a driver for alloreactive responses and lung rejection

Palchevskiy¹,

Xue²,

Kern³

et al. 2019

JCI Insight

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Gene Expression Profiling of Bronchoalveolar Lavage Cells Preceding a Clinical Diagnosis of Chronic Lung Allograft Dysfunction

Cited by 32 publications

References 44 publications

Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood

Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood

Transcriptomics and single‐cell RNA‐sequencing

CCR4 expression on host T cells is a driver for alloreactive responses and lung rejection

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