Impact of Cytomegalovirus Infection and Genetic Background on the Frequencies of Peripheral Blood Suppressor Cells in Human Twins.

Frequencies and proportions of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in peripheral blood may be informative biomarkers for certain disease states. The influence of genetics and lifetime pathogen exposures on Treg and MDSC frequencies is largely unexplored. Cytomegalovirus (CMV) establishes a latent infection and causes an accumulation of late-differentiated CD8+ memory T cells, commonly associated with a lower frequency of naive cells. Here, analyzing peripheral blood mononuclear cells by multicolor flow cytometry, we found a tendency towards lower frequencies of CD4+CD25+FoxP3+ Tregs in CMV-seropositive than -seronegative middle-aged individuals (p = 0.054), whereas frequencies of lineage-negative CD14+HLA-DR-MDSCs were significantly lower in CMV-seropositive participants (p = 0.005). Assessing associations with the presence of antibodies against different CMV structural proteins, rather than merely assigning seropositivity or seronegativity, failed to yield any closer associations. Examining Treg subsets revealed at most a minor role of the individual's genetic background, based on an analysis of monozygotic (MZ, n = 42) versus dizygotic (DZ, n = 39) twin pairs from the Danish Twin Registry. The same was true for MDSCs. These initial results suggest that an immunological history of exposures is more important than genetics in determining overall human suppressor cell levels.

Genetic Influence on the Peripheral Differentiation Signature of Vδ2+ γδ and CD4+ αß T Cells in Adults.

Adaptive as well as innate immune traits are variously affected by environmental and genetic influences, but little is known about the impact of genetics on the diversity, differentiation and functionality of γδ T cells in humans. Here, we analyzed a cohort of 95 middle-aged twins from the Danish Twin Registry. The differentiation status of peripheral αß and γδ T cells was assessed by flow cytometry based on the surface expression of CD27, CD28 and CD45RA. Our data confirm the established associations of latent cytomegalovirus (CMV) infection with an accumulation of late differentiated memory T cells in the αß compartment as well as in the Vδ1+ γδ T cell subset. A comparison of differentiation phenotypes of γδ and αß T cells that were not affected by CMV seropositivity identified a significant correlation of early differentiated (ED) Vδ2+ and intermediate differentiated (ID) CD4+ T cells in monozygotic (MZ), but not in dizygotic (DZ) co-twins. Thus, our data suggest a genetic influence on the differentiation of γδ and αß T cell subsets.

New insights into the pathomechanism of cyclic neutropenia.

Cyclic neutropenia (CyN) is a hematologic disorder in which peripheral blood absolute neutrophil counts (ANCs) show cycles of approximately 21-day intervals. The majority of CyN patients harbor ELANE mutations, but the mechanism of ANC cycling is unclear. We performed analysis of bone marrow (BM) subpopulations in CyN patients at the peak and the nadir of the ANC cycle and detected high proportions of BM hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) at the nadir of the ANC cycle, as compared with the peak. BM HSPCs produced fewer granulocyte colony-forming unit colonies at the ANC peak. To investigate the mechanism of cycling, we found that mRNA expression levels of ELANE and unfolded protein response (UPR)-related genes (ATF6, BiP (HSPA5), CHOP (DDIT3), and PERK (EIF2AK3)) were elevated, but antiapoptotic genes (Bcl-2 (BCL2) and bcl-xL (BCL2L1)) were reduced in CD34+ cells tested at the ANC nadir. Moreover, HSPCs revealed increased levels of reactive oxygen species and gH2AX at the ANC nadir. We suggest that in CyN patients, some HSPCs escape the UPR-induced endoplasmic reticulum (ER) stress and proliferate in response to granulocyte colony-stimulating factor (G-CSF) to a certain threshold at which UPR again affects the majority of HSPCs. There is a cyclic balance between ER stress-induced apoptosis of HSPCs and compensatory G-CSF-stimulated HSPC proliferation followed by granulocytic differentiation.

CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients.

A Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 µg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. "Maturation arrest," the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and "maturation arrest" corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients' primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.

LMO2 activation by deacetylation is indispensable for hematopoiesis and T-ALL leukemogenesis.

Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.

CCR4+ Regulatory T Cells Accumulate in the Very Elderly and Correlate With Superior 8-Year Survival.

CD4(+) regulatory T cells (Tregs) are a distinct population of T cells involved in maintaining peripheral tolerance to self-antigens. Several studies have shown increased frequency and number of Tregs in the elderly. Whether such an increase has any clinical relevance has not been addressed. Here, we have analyzed circulating Tregs in 114 donors between the ages of 18 and 89 years and assessed their implications for survival of the very elderly. In line with previously published data, we observed higher proportions of Tregs in the elderly. Expression of chemokine receptor 4 (CCR4) by Tregs has been shown to characterize antigen-primed activated Tregs with immediate suppressive function. Thus we further analyzed Tregs expressing or lacking this chemokine receptor. There were more CCR4(+) and CCR4(-) Tregs in the elderly than the young. Finally, using a subset of 48 elderly donors participating in the Leiden 85-plus study we documented that people with greater median frequencies of CCR4(+) Tregs enjoyed a better 8-year survival rate than those with lower frequencies of these cells. Our data, demonstrating for the first time a positive correlation between increased frequency of Tregs and survival in the elderly, imply an increasing importance of controlling inappropriate immune responses and inflammation as we grew old.

Latent infection with cytomegalovirus is associated with poor memory CD4 responses to influenza A core proteins in the elderly.

Influenza remains a major pathogen in older people. Infection with CMV and the accumulation of late-differentiated T cells associated with it have been implicated in poor Ab responsiveness to influenza vaccination in the elderly, most of whom are CMV positive. However, whether CMV infection also affects memory T cell responses to influenza remains unknown. To investigate this, we assessed T cell responses to influenza A matrix protein and nucleoprotein ex vivo in 166 Dutch individuals (mean age 62.2 y, range 42-82) and validated the results in a second cohort from North America (mean age 73.1 y, range 65-81, n = 28). We found that less than half of the CMV-infected older subjects mounted a CD4 T cell response to influenza Ags, whereas ∼80% of uninfected elderly did so. A similar proportion of younger subjects possessed influenza A virus-responsive CD4 T cells, and, interestingly, this was the case whether they were CMV-infected. Thus, the effect of CMV was only seen in the older donors, who may have been exposed to the virus for decades. The percentage of donors with CD8 responses to influenza A virus was lower than those with CD4; this was not influenced by whether the subjects were CMV seropositive or seronegative. CMV-seropositive responders had significantly higher frequencies of late-differentiated CD4 T-cells (CD45RA(+/-)CCR7(-)CD27(-)CD28(-)) compared with CMV-infected nonresponders. These data add to the accumulating evidence that infection with CMV has profound but heterogeneous effects on responses to the products of other viruses and have implications for the design of influenza vaccines, especially in the elderly.

Lower proportion of naïve peripheral CD8+ T cells and an unopposed pro-inflammatory response to human Cytomegalovirus proteins in vitro are associated with longer survival in very elderly people.

The low percentages of naïve T cells commonly observed in elderly people are thought to be causally associated with mortality, primarily from infectious disease, and are taken as a hallmark of "immunosenescence". Whether low levels of naive cells actually do associate with mortality has, however, not been tested in longitudinal studies. Here, we present correlations between peripheral T-cell phenotypes and 8-year survival in individuals from the population-based prospective Leiden 85-plus Study. Counter-intuitively, we found that a lower frequency of naïve CD8+ T cells (characterized as CD45RA+CCR7+CD27+CD28+) at baseline (>88 years) correlated with significantly better survival, while there was a tendency for the reciprocal accumulation of late-differentiated effector memory cells (CD45RA-CCR7-CD27-CD28-) also to associate with better survival. These findings suggest that better retention of memory cells specific for previously encountered antigens may provide a survival advantage in this particular population. Given the prevalence of Cytomegalovirus (CMV) and its reported association with immunosenescence, we tested whether memory for this potential pathogen was relevant to survival. We found that individuals mounting an exclusively pro-inflammatory ex vivo response (TNF, IFN-γ, IL-17) to the major CMV target molecules pp65 and IE1 had a significant survival advantage over those also having anti-inflammatory responses (IL-10). These findings suggest that higher levels of naïve T cells may not necessarily be associated with a survival advantage and imply that the nature of immunosurveillance against CMV may be crucial for remaining longevity, at least in the very elderly.

Cytomegalovirus-associated accumulation of late-differentiated CD4 T-cells correlates with poor humoral response to influenza vaccination.

Influenza vaccination is less effective in the elderly compared to the young. Studies that have attempted to identify immune parameters correlating with satisfactory vaccine responses have yielded inconclusive results. Here, we correlate the distribution of different circulating CD4+ and CD8+ T-cell phenotypes with the humoral response to vaccination with Intanza, an intradermal seasonal vaccine, in 54 individuals of different ages. Subjects were stratified according to age (below or over 60) and presence of a latent infection with Cytomegalovirus (CMV). CMV-seropositivity was significantly associated with a lower response rate to the vaccine in people over but not below 60 yr of age. Unlike reported data, late-differentiated (CD45RA+CCR7-CD27-CD28-) CD4+, but not CD8+ T-cells associated with a poorer vaccine response. Thus, latent CMV infection has a deleterious effect on influenza antibody responses in the elderly, which might be mediated through CD4 T-cells lacking CCR7, CD27 and CD28 and re-expressing CD45RA.

Infection with cytomegalovirus but not herpes simplex virus induces the accumulation of late-differentiated CD4+ and CD8+ T-cells in humans.

Human cytomegalovirus (CMV) establishes persistent, usually asymptomatic, infection in healthy people. Because CMV infection is associated with the presence of lower proportions of peripheral naïve CD8+ T-cells and a higher fraction of late-differentiated CD8+ cells, commonly taken as biomarkers of age-associated compromised adaptive immunity ('immunosenescence'), we asked whether chronic exposure to any persistent virus mediates these effects. Herpes simplex virus (HSV) is also a widespread herpesvirus that establishes lifelong persistence, but, unlike CMV, its impact on the distribution of T-cell subsets has not been established. Here, we analysed T-cell subsets in 93 healthy people aged 42-81 years infected or not infected with CMV and/or HSV. Individuals harbouring CMV were confirmed to possess lower frequencies of naïve CD8+ T-cells (defined as CD45RA+CCR7+CD27+CD28+) and greater proportions of late-differentiated effector memory (CD45RA-CCR7-CD27-CD28-) and so-called TEMRA (CD45RA+CCR7-CD27-CD28-) CD4 and CD8 subsets, independent of HSV seropositivity. In CMV-seronegative donors, HSV did not affect T-cell subset distribution significantly. We conclude that these hallmarks of age-associated alterations to immune signatures are indeed observed in the general population in people infected with CMV and not those infected with a different persistent herpesvirus.

Hallmark features of immunosenescence are absent in familial longevity.

Seropositivity for CMV is one of the parameters of the "immune risk profile" associated with mortality in longitudinal studies of the very elderly and may accelerate immunosenescence. Thus, any genetic factors influencing human longevity may be associated with susceptibility to CMV and CMV-accelerated immunosenescence. To test this, we analyzed long-lived families in the Leiden Longevity Study (LLS) in which offspring enjoy a 30% reduced standardized mortality rate, possibly owing to genetic enrichment. Serum C-reactive protein levels and the frequency of different T cell subsets were compared between 97 LLS offspring and 97 controls (their partners, representing the normal population). We also determined the capacity of T cells to respond against immunodominant Ags from CMV in a smaller group of LLS subjects and controls. CMV infection was strongly associated with an age-related reduction in the frequency of naive T cells and an accumulation of CD45RA-re-expressing and late-differentiated effector memory T cells in the general population, but not in members of long-lived families. The latter also had significantly lower C-reactive protein levels, indicating a lower proinflammatory status compared with CMV-infected controls. Finally, T cells from a higher proportion of offspring mounted a proliferative response against CMV Ags, which was also of greater magnitude and broader specificity than controls. Our data suggest that these rare individuals genetically enriched for longevity are less susceptible to the characteristic CMV-associated age-driven immune alterations commonly considered to be hallmarks of immunosenescence, which might reflect better immunological control of the virus and contribute to their decreased mortality rate.

Pretreatment frequency of circulating IL-17+ CD4+ T-cells, but not Tregs, correlates with clinical response to whole-cell vaccination in prostate cancer patients.

The aim of this study was to determine the prognostic implications of the pretreatment level of Th17 cells compared with regulatory T-cell status in prostate cancer patients receiving active whole cell immunotherapy. Ten-color flow cytometry was used to analyze IL-17-producing CD4(+) T-cells in the peripheral blood of hormone-resistant non-bone metastatic prostate cancer patients prior to immunotherapy with an allogeneic whole-cell vaccine. Surface expression of the chemokine receptors CCR4 and CCR6 was used to further subdivide IL-17-producing cells into subsets with distinct homing properties. The frequency of circulating regulatory T-cells (Tregs), defined as CD3(+)CD4(+)CD127(lo)Foxp3(+)CD25(+) was compared in the same patients. The frequency of CCR4(-)IL-17(+)CD4(+) T-cells prevaccination inversely correlated with time to disease progression (TTP) in 23 prostate cancer patients. Furthermore, responder (R) patients with statistically significant reductions in PSA velocity (PSAV) in response to the immunotherapy (n = 9), showed a Th17 profile similar to healthy male controls and significantly different from non-responder (NR) patients (n = 14) (i.e., those without any significant reduction in PSAV). In contrast, the frequency of Tregs in peripheral blood in PSA-R (n = 11) and -NR (n = 14) patients was similar (but in both cases, significantly higher than in age-matched healthy men). Accordingly, there was no significant correlation between frequency of Tregs and TTP in these late-stage prostate cancer patients undergoing active immunotherapy. These data imply an important role for IL-17-producing helper T-cells in cancer immunology and highlight their potential use as a pretreatment screen to ensure appropriate treatment is offered to hormone-resistant prostate cancer patients.