C/EBPα Is Dispensable for the Ontogeny of PD-1+ CD4+ Memory T Cells but Restricts Their Expansion in an Age-Dependent Manner

<div><p>Ageing and cancer is often associated with altered T cell distributions and this phenomenon has been suggested to be the main driver in the development of immunosenescence. Memory phenotype PD-1+ CD4+ T cells accumulate with age and during leukemic development, and they might account for the attenuated T cell response in elderly or diseased individuals. The transcription factor C/EBPα has been suggested to be responsible for the accumulation as well as for the senescent features of these cells including impaired TCR signaling and decreased proliferation. Thus modulating the activity of C/EBPα could potentially target PD-1+ CD4+ T cells and consequently, impede the development of immunosenescence. To exploit this possibility we tested the importance of C/EBPα for the development of age-dependent PD-1+ CD4+ T cells as well as its role in the accumulation of PD-1+ CD4+ T cells during leukemic progression. In contrast to earlier suggestions, we find that loss of C/EBPα expression in the lymphoid compartment led to an increase of PD-1+ CD4+ T cells specifically in old mice, suggesting that C/EBPα repress the accumulation of these cells in elderly by inhibiting their proliferation. Furthermore, C/EBPα-deficiency in the lymphoid compartment had no effect on leukemic development and did not affect the accumulation of PD-1+ CD4+ T cells. Thus, in addition to contradict earlier suggestions of a role for C/EBPα in immunosenescence, these findings efficiently discard the potential of using C/EBPα as a target for the alleviation of ageing/cancer-associated immunosenescence.</p></div

Increase in PD-1+ CD4+ T cells during ageing and in development of AML.

<p>(A) Spleen cells from 2 months old and 14 months old mice were stained with antibodies against CD4 and PD-1. (B) Quantification of the data in (A) is presented as mean +/− SD, (young: n = 3, old: n = 7). (C) PD-1- CD4+ and PD-1+ CD4+ splenic T cells from 14 months old mice were analyzed for expression of <i>Cebpa</i> normalized to <i>β-actin</i> by qRT-PCR. Data are presented as mean +/− SEM, (n = 7). (D) Spleens from 3 months old mice were stained for CD4, PD-1, CD44 and CD62L. A representative example is shown (n = 5). (E) The spleens from healthy (age-matched, non-transplanted) and leukemic mice were analyzed for PD-1+ CD4+ T cells. **P<0.01; n.s.: not significant.</p

C/EBPα inhibits proliferation of CD4+ T cells in old mice.

<p>(A and B) Sorted PD-1- CD4+ and PD-1+ CD4+ T cells from 2 months old <i>Cebpa</i>^fl/fl and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> mice were assessed for transcripts for the indicated genes by qRT-PCR. The relative expression were normalized to β-actin and presented as mean of <i>Cebpa</i>^fl/fl n = 7 and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> n = 8+/− SEM. (C) CFSE labeled splenocytes from 2 months old <i>Cebpa</i>^fl/fl and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> mice were cultured with or without CD3 and CD28 antibodies and after 72 hours after the splenocytes were stained with CD4 antibody and assayed by flow cytometry. Black and grey lines indicate non-stimulated and stimulated cells, respectively. The numbers of cell divisions as given by the Proliferation feature of FlowJo are shown. (D) Quantification of CD4+ T cells in cell cycle 0–4. (<i>Cebpa</i>^fl/fl n = 3, <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> n = 3). (E and F) Analysis of proliferation of CD4+ T cells in the spleen of 10 to 15 months old <i>Cebpa</i>^fl/fl (n = 8) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 12) mice. The contour plot and histograms are examples from <i>Cebpa</i>^fl/fl mice. Mean +/− SD; *P<0.05; **P<0.01; n.s.: not significant.</p

C/EBPα is dispensable for the differentiation of lymphoid cells in young mice.

<p>(A and B) Analysis of DN1-DN4 T cells and (C and D) CD4+ and/or CD8+ T cells in thymi from 2 months old <i>Cebpa</i>^fl/fl (n = 4) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 5) mice. (E and F) Analysis of mature hematopoietic lineages in spleens from 2 months old <i>Cebpa</i>^fl/fl (n = 3) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 4) mice. (G and H) Analysis of the mature hematopoietic lineages in BMs from 2 months old <i>Cebpa</i>^fl/fl (n = 5) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 6) mice. (I and J) Analysis of the PD-1+ CD4+ T cells in spleens from 2 months old <i>Cebpa</i>^fl/fl (n = 3) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 4) mice. (K and L) Analysis of the PD-1+ CD4+ T cells in BMs from 2 months old <i>Cebpa</i>^fl/fl (n = 5) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 6) mice. (M–O) Analysis of CD44 and CD62L subsets within PD-1- and PD1+ CD4+ T cells in spleens from 3 months old <i>Cebpa</i>^fl/fl (n = 5) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 5) mice. The contour plots are examples from <i>Cebpa</i>^fl/fl mice. Mean +/− SD; n.s. = not significant.</p

C/EBPα restricts the formation of PD-1+ CD4+ T cells in spleens of old mice.

<p>(A and B) Analysis of PD-1+ CD4+ T cells in spleens from 14 months old <i>Cebpa</i>^fl/fl (n = 7) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 8) mice. (C and D) Analysis of mature hematopoietic lineages in spleens from 14 months old <i>Cebpa</i>^fl/fl (n = 7) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 8) mice. (E and F) Analysis of the PD-1+ CD4+ T cells as well as the mature hematopoietic lineages (G and H) in BMs from 14 months old <i>Cebpa</i>^fl/fl (n = 7) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 8) mice. The contour plots are examples from <i>Cebpa</i>^fl/fl mice. Mean +/− SD; *P<0.05; **P<0.01; n.s.: not significant.</p

The DSM-5 criteria, level of arousal and delirium diagnosis: inclusiveness is safer

Background: Delirium is a common and serious problem among acutely unwell persons. Alhough linked to higher rates of mortality, institutionalisation and dementia, it remains underdiagnosed. Careful consideration of its phenomenology is warranted to improve detection and therefore mitigate some of its clinical impact. The publication of the fifth edition of the Diagnostic and Statistical Manual of the American Psychiatric Association (DSM-5) provides an opportunity to examine the constructs underlying delirium as a clinical entity. Discussion: Altered consciousness has been regarded as a core feature of delirium; the fact that consciousness itself should be physiologically disrupted due to acute illness attests to its clinical urgency. DSM-5 now operationalises ‘consciousness’ as ‘changes in attention’. It should be recognised that attention relates to content of consciousness,but arousal corresponds to level of consciousness. Reduced arousal is also associated with adverse outcomes. Attention and arousal are hierarchically related; level of arousal must be sufficient before attention can be reasonably tested. Summary: Our conceptualisation of delirium must extend beyond what can be assessed through cognitive testing (attention) and accept that altered arousal is fundamental. Understanding the DSM-5 criteria explicitly in this way offers the most inclusive and clinically safe interpretation.status: publishe