Emigrant Cells Research Articles

Exhaustion of CD8+ central memory responder T cell differentiation provokes non-melanoma skin cancer in elderly kidney transplant recipients.

Immunosuppressive therapy prevents graft rejection but increases the risk of non-melanoma skin cancer (NMSC), especially in elderly kidney transplant recipients (KTR). In this study, we separately investigated the differentiation of CD8+ regulatory T cells (Tregs) and responder T cells (Tresps) between healthy KTR without NMSC, KTR developing de-novo NMSC within two years after the enrolment, and KTR with NMSC at the time of enrolment. Antigen-unexperienced CCR7+CD45RA+CD31+ recent thymic emigrant (RTE) cells differentiate via CD45RA-CD31+ memory (CD31+ memory) cells, via resting mature naïve (MN) cells or via direct proliferation into CD45RA-CD31- memory (CD31- memory) cells, consisting of both CCR7+CD45RA- central memory (CM) and CCR7-CD45RA- effector memory (EM) cells. We found that both RTE Treg and Tresp differentiation via CD31+ memory Tregs/Tresps was age-independently increased in KTR, who developed de novo NMSC during the follow-up period, causing abundant CM Treg/Tresp production, which may be crucial for cancer immunity. These changes favored a strongly increased CD8+ Treg/Tresp ratio, suggesting this ratio as a reliable marker for de-novo NMSC development in KTR. However, with age, this differentiation was replaced by increased conversion of resting MN Tregs/Tresps into CM Tregs/Tresps, which exhausted for Tresps but not for Tregs. In KTR with already existing NMSC at enrolment, differentiation was maintained via conversion and proliferation of resting MN Tregs/Tresps, which however increasingly exhausted with age, especially for Tresps. This resulted in a strong accumulation of terminally differentiated effector memory (TEMRA) Tresps in elderly individuals. Patients with NMSC recurrence showed increased proliferation of resting MN Tregs/Tresps into EM Tregs/Tresps, which tended to exhaust more rapidly, particularly for Tresps, than in patients without NMSC recurrence. In conclusion, we provide evidence that immunosuppressive therapy inhibits differentiation of CD8+ Tregs more than that of CD8+ Tresps, resulting in an exhausted Tresp profile, thus providing a possible therapeutic approach to improve poor cancer immunity in elderly KTR.

Recent Thymic Emigrants Require RBPJ-Dependent Notch Signaling to Transition into Functionally Mature Naive T Cells.

Recent thymic emigrant (RTE) cells are nascent T cells that continue their post-thymic maturation in the periphery and dominate T cell immune responses in early life and in adults having undergone lymphodepletion regimens. However, the events that govern their maturation and their functionality as they transition to mature naive T cells have not been clearly defined. Using RBPJind mice, we were able to identify different stages of RTE maturation and interrogate their immune function using a T cell transfer model of colitis. As CD45RBlo RTE cells mature, they transition through a CD45RBint immature naive T (INT) cell population that is more immunocompetent but shows a bias toward IL-17 production at the expense of IFN-γ. Additionally, the levels of IFN-γ and IL-17 produced in INT cells are highly dependent on whether Notch signals are received during INT cell maturation or during their effector function. IL-17 production by INT cells showed a total requirement for Notch signaling. Loss of Notch signaling at any stage of INT cells resulted in an impaired colitogenic effect of INT cells. RNA sequencing of INT cells that had matured in the absence of Notch signals showed a reduced inflammatory profile compared with Notch-responsive INT cells. Overall, we have elucidated a previously unknown INT cell stage, revealed its intrinsic bias toward IL-17 production, and demonstrated a role for Notch signaling in INT cell peripheral maturation and effector function in the context of a T cell transfer model of colitis.

Ex vivo activated CD4+ T cells from young calves exhibit Th2-biased effector function with distinct metabolic reprogramming compared to adult cows

As maternal passive immunity wanes at 6–8 weeks, young calves must rely on their own naïve and developing immune system for protection against pathogens. Typically, an infection in the young induces a T cell-mediated response, which skews towards a Th2 phenotype and results in a reduced effector response. Our study examines the implications this transitional period of immunocompetency has on cellular metabolism in young calves, focusing on effector function of CD4+ T cells in comparison to those from adult cows. Results from sorted CD4+ T cells from young calves and adult cows activated by α-CD3:α-CD28, show that young calves exhibit a significantly greater propensity to produce the Th2 cytokine, IL-4, in comparison to IFN-γ. Concomitantly, cells from young calves and adult cows exhibit no statistical difference in cell surface marker expression induced by α-CD3:α-CD28 stimulation. Metabolically, activated CD4+ T cells from young calves show significantly greater utilization of mitochondrial respiration, measured by oxygen consumption rate (OCR), and greater glycolytic reserve, measured by extracellular acidification rate (ECAR). However, adult cows have a significantly higher change in glycolytic rate after α-CD3:α-CD28 stimulation compared to young calves. Further, CD4+ T cells from young calves have an increased mRNA expression signature associated with glycolytic metabolism (GAPDH, HK2, FBP1, HIF1A) and Th2-associated metabolic signaling (RPTOR) in comparison to adult cows. The distinct metabolic phenotype and associated gene expression in activated CD4+ T cells may be intrinsic drivers of the Th2-biased response by young calves. Additionally, CD4+ recent thymic emigrant cells (RTEs) may further contribute to altered effector function, as they are preferential precursors to Tregs, and based on the microenvironment, have the propensity to polarize toward Th2. Evaluation of T cell master transcription regulators, as well as measuring signal joint T cell receptor excision circles between young calves and adult cows, we observed a significantly increased proportion of RTEs from sorted CD4+ T cells. In this study, we show a unique metabolic profile exhibited by activated CD4+ T cells from young calves in which mitochondrial respiration and glycolytic capacity is significantly increased compared to adult cows.

Immune system and intestinal microbiota determine efficacy of androgen deprivation therapy against prostate cancer

BackgroundProstate cancer (PC) responds to androgen deprivation therapy (ADT) usually in a transient fashion, progressing from hormone-sensitive PC (HSPC) to castration-resistant PC (CRPC). We investigated a mouse model of PC...

Immune system and intestinal microbiota determine efficacy of androgen depletion therapy against prostate cancer.

168 Background: Prostate cancer (PC) responds to androgen deprivation therapy (ADT) usually in a transient fashion, progressing from hormone-sensitive PC (HSPC) to castration-resistant PC (CRPC). While it is recognized that ADT has an immunomodulatory effect, little is known about the intestinal microbiome effect on therapeutic outcome of ADT. Methods: We investigated a mouse model of PC as well as specimens from PC patients to unravel an unsuspected contribution of thymus-derived T lymphocytes and the intestinal microbiota in the efficacy of ADT. Preclinical experiments were performed in PC-bearing mice, immunocompetent or immunodeficient. In parallel, we prospectively included 65 HSPC and CRPC patients to analyze their feces and blood specimen. Results: In PC-bearing mice, ADT increased thymic cellularity and output. PC implanted in T lymphocyte-depleted or athymic mice responded less efficiently to ADT than in immunocompetent mice. Moreover, depletion of the intestinal microbiota by oral antibiotics reduced the efficacy of ADT. PC reduced the relative abundance of Akkermansia muciniphila in the gut, and this effect was reversed by ADT. Moreover, cohousing of PC-bearing mice with tumor-free mice, or oral gavage with Akkermansia improved the efficacy of ADT. This appear to be applicable to PC patients because long-term ADT resulted in an increase of thymic output, as demonstrated by an increase in circulating recent thymic emigrant cells (sjTRECs). Moreover, as compared to HSPC controls, CRPC patients demonstrated a shift in their intestinal microbiota that significantly correlated with sjTRECs. While feces from healthy volunteers restored ADT efficacy. Conclusions: These findings suggest that reversing the intestinal dysbiosis and repairing acquired immune defects in PC patients have a potential impact on the therapeutic outcome of ADT.

Ibrutinib Treatment in CLL Interrupts CD40 Signaling Capacity and Sensitizes CLL Cells to Venetoclax

Background: For proliferation and survival, chronic lymphocytic leukemia (CLL) cells depend on interactions with cells and soluble factors present in the tumor microenvironment (TME). These interactions also increase expression of B-cell leukemia/lymphoma-2 (Bcl-2) proteins, including Bcl-XL, Mcl-1 and Bfl-1, thereby reducing drug sensitivity.In the VISION HOVON141 clinical trial, patients with relapsed or refractory CLL are treated with 2 cycles of Bruton tyrosine kinase inhibitor ibrutinib (IBR) lead-in followed by 13 cycles of IBR + Bcl-2 inhibitor venetoclax (VEN) combination before randomization. The combination of IBR + VEN may have synergistic anti-tumor effects, since IBR forces CLL cells from lymph node (LN) to the blood (PB) where they become fully dependent on Bcl-2, and succumb to VEN.To support a mechanistic basis for this premise, we investigated changes in expression of Bcl-2 proteins, effects of TME-mimicking signals, and venetoclax sensitivity before/after 2 months IBR treatment.Results: At baseline, lymph node emigrant cells (CXCR4dim/CD5high) have higher Bfl-1 expression, in addition to earlier reported Bcl-XL and Mcl-1 expression than cells immigrating back to the lymph node (CXCR4high/CD5dim)(Haselager et al., 2020). After 2 months of IBR treatment a clear reduction in all four pro-survival proteins was observed (N=17 p<0.001). Despite these changes, VEN sensitivity was not different in unstimulated PB CLL cells obtained at baseline versus at 2 months of IBR treatment (N=8; IC50=0.001µM). In contrast, CD40 stimulated CLL cells obtained at baseline are fully resistant to VEN (IC50>10µM), but unexectedly retained partial sensitivity to venetoclax after IBR treatment (IC50=0.05 µM, p-value <0.0001; Figure 1A). This suggested reduced CD40 signalling capacity and was accompanied by reduced ability to upregulate Bcl-XL, Mcl-1, and Bfl-1 expression. Indeed, cell surface expression, as well as level of CD40 activation as measured by CD95 induction, was clearly reduced after 2 months IBR. Importantly, these effects occurred in vivo, as IBR did not directly affect CD40 signaling in vitro.These data imply that under IBR treatment, when cells cannot (re-)enter LN sites, a factor is lacking that maintains or induces CD40 expression. In search of stimuli that can augment CD40 signaling capacity, it was found that BCR stimulation had no effect on CD40 expression. In contrast, TLR9 stimulation via CpG led to increased CD40 expression in CLL cells (N=7, p-value <0.0001)(Figure 1B). These findings align well with recent data which indicate a role for TLR9 signalling in vivo by unmethylated mitochondrial DNA in CLL (Kennedy et al., 2021). Taken together, these data provide a mechanistic explanation, by which a triad of signaling pathways not only involving BCR but also CD40 and TLR9 is interrupted by IBR (Figure 1C).Discussion/conclusion: IBR treatment broadly affects Bcl-2 family protein expression and CD40 signaling in vivo. The combined data indicate a novel aspect of IBR efficacy, namely its capacity to interrupt TLR9-induced CD40 upregulation, which normally primes CLL cells in the LN environment for drug resistance. This also suggests that drugs that inhibit TLR9 signaling may synergize with IBR.ReferencesHaselager, M. V., et al (2020). Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL. Blood, 136(25), 2918Kennedy, E., et al (2021). TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target. Blood, 137(22), 3064 [Display omitted] DisclosuresLevin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Westerweel: Novartis: Research Funding; Incyte: Consultancy; BMS / Celgene: Consultancy; Pfizer: Consultancy. Niemann: CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Novo Nordisk Foundation: Research Funding; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding.

T lymphocyte senescence is attenuated in Parkinson\u2019s disease

BackgroundImmune involvement is well-described in Parkinson’s disease (PD), including an adaptive T lymphocyte response. Given the increasing prevalence of Parkinson’s disease in older age, age-related dysregulation of T lymphocytes may be relevant in this disorder, and we have previously observed changes in age-associated CD8+ T cell subsets in mid-stage PD. This study aimed to further characterise T cell immunosenescence in newly diagnosed PD patients, including shifts in CD4+ and CD8+ subpopulations, and changes in markers of cellular ageing in CD8+ T lymphocytes.MethodsPeripheral blood mononuclear cells were extracted from the blood of 61 newly diagnosed PD patients and 63 age- and sex-matched controls. Flow cytometric analysis was used for immunophenotyping of CD8+ and CD4+ lymphocyte subsets, and analysis of recent thymic emigrant cells. Telomere length within CD8+ T lymphocytes was assessed, as well as the expression of the telomerase reverse transcriptase enzyme (hTERT), and the cell-ageing markers p16INK4a and p21CIP1/Waf1.ResultsThe number of CD8+ TEMRA T cells was found to be significantly reduced in PD patients compared to controls. The expression of p16INK4a in CD8+ lymphocytes was also lower in patients versus controls. Chronic latent CMV infection was associated with increased senescent CD8+ lymphocytes in healthy controls, but this shift was less apparent in PD patients.ConclusionsTaken together, our data demonstrate a reduction in CD8+ T cell replicative senescence which is present at the earliest stages of Parkinson’s disease.

Lymphocyte Subset Abnormalities in Pediatric-Onset Common Variable Immunodeficiency

Introduction: Common variable immunodeficiency (CVID) is characterized by recurrent infections, autoimmunity, lymphoproliferation, hypogammaglobulinemia, and defective antibody production. In CVID, B-cell abnormalities were described to predict end organ involvement and prognosis. Pediatric-onset CVID is much rarer than adult CVID, and lymphocyte subset abnormalities have not been thoroughly evaluated. Objective: We sought to determine lymphocyte subset abnormalities and their association with end organ involvement in pediatric-onset CVID patients. Methods: The clinical manifestations and laboratory findings including absolute numbers and percentages of B-, T-, and NK cell populations were assessed in pediatric-onset CVID patients and compared to age-matched healthy controls. The patients were divided into 2 groups according to age at assessment (pediatric CVID patients: 10–16 years, n = 9; and adult CVID patients: >16 years, n = 13). The comparisons between lymphocyte subsets and organ involvement were also evaluated. Results: Mean age at symptom onset was 18 (3–204) months. All CVID patients with pediatric onset had decreased levels of total and memory B cells, CD4⁺ T cells, CD4⁺CD45RA⁺ naive T cells, and recent thymic emigrant (RTE) cells. On the other hand, they had increases in CD8⁺CD45RO⁺ memory T cells. Interestingly, adult CVID patients demonstrated high frequencies of activated and double-negative T cells, which were unique only for this group of patients. Specific cellular abnormalities associated with the reduction in B and NK cells and increase in CD8⁺ T cells were found in patients with bronchiectasis. Moreover, in pediatric CVID patients, low serum IgA levels and decreased numbers of naive T and RTE cells were determined as risk factors for chronic diarrhea. Conclusions: Specific abnormalities in B- and T-lymphocyte compartments were identified in pediatric-onset CVID patients and appear to be associated with end organ manifestations.

Relationship between Multiple Sclerosis-Associated IL2RA Risk Allele Variants and Circulating T Cell Phenotypes in Healthy Genotype-Selected Controls.

Single nucleotide polymorphisms (SNPs) in or near the IL2RA gene, that encodes the interleukin-2 (IL-2) receptor α (CD25), are associated with increased risk of immune-mediated diseases including multiple sclerosis (MS). We investigated how the MS-associated IL2RA SNPs rs2104286 and rs11256593 are associated with CD25 expression on T cells ex vivo by multiparameter flow cytometry in paired genotype-selected healthy controls. We observed that MS-associated IL2RA SNPs rs2104286 and rs11256593 are associated with expression of CD25 in CD4+ but not CD8+ T cells. In CD4+ T cells, carriers of the risk genotype had a reduced frequency of CD25+ TFH1 cells (p = 0.001) and an increased frequency of CD25+ recent thymic emigrant cells (p = 0.006). Furthermore, carriers of the risk genotype had a reduced surface expression of CD25 in post-thymic expanded CD4+ T cells (CD31−CD45RA+), CD39+ TReg cells and in several non-follicular memory subsets. Our study found novel associations of MS-associated IL2RA SNPs on expression of CD25 in CD4+ T cell subsets. Insight into the associations of MS-associated IL2RA SNPs, as these new findings provide, offers a better understanding of CD25 variation in the immune system and can lead to new insights into how MS-associated SNPs contribute to development of MS.

Thymic output changes in children with clinical findings signaling a probable primary immunodeficiency.

Karaca N, Azarsız E, Akarcan SE, Aksu G, Kütükçüler N. Thymic output changes in children with clinical findings signaling a probable primary immunodeficiency. Turk J Pediatr 2019; 61: 885-894. Thymic maturation evaluation is inevitable for patients with clinical and laboratory findings for a primary immunodeficiency, as the T cellimmunodeficiencies are the most severe type. In this study, we aimed to show the usage of T cell surface molecule `CD31` for the evaluation of thymic output in patients (n: 66) with a large spectrum of findings signing a probable primary immunodeficiency. Besides the classical clinical and laboratory approach for these patients, T cell subpopulations as naive, memory, recent thymic emigrant cells were also investigated. The humoral immunodeficiency (34.8%), combined immunodeficiency (34.8%) and cardiopathy (7.6%) were the most frequent diagnosis groups. CD4+CD45RA+ naive T-cells percentages (p: 0.011) and absolute counts (p: 0.004) and absolute CD4+CD45RA+CD31+ RTE (recent thymic emigrant) cell counts (p: 0.007) were significantly lower in combined immunodeficiency group. Naive T-cells (p: 0.037) and RTE cells (p: 0.032) were also lower in patients who had cardiac surgery in the past. In conclusion, flow cytometric CD31+thymic naive RTE cell evaluation may provide rapid clinical information especially on T-cell immune dysfunction and CD4+CD45RA+CD31+ RTE cells may be used as an alternative to TRECs in the diagnosis of combined immunodeficiencies.

Clinical and Immunological Characterization of ICF Syndrome in Japan

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare autosomal recessive primary immunodeficiency. Hypogammaglobulinemia is a major manifestation of ICF syndrome, but immunoglobulin replacement therapy does not seem to be effective for some ICF patients. Therefore, we aimed to reassess the immunological characteristics of this syndrome. Eleven Japanese patients with ICF syndrome were enrolled. We performed whole-exome sequencing in four cases and homozygosity mapping using SNP analysis in two. We evaluated their clinical manifestations and immunological status. We newly diagnosed six ICF patients who had tentatively been diagnosed with common variable immunodeficiency. We identified two novel mutations in the DNMT3B gene and one novel mutation in the ZBTB24 gene. All patients showed low serum IgG and/or IgG2 levels and were treated by periodic immunoglobulin replacement therapy. Three of the six patients showed worse results of the mitogen-induced lymphocyte proliferation test. Analyses of lymphocyte subpopulations revealed that CD19+CD27+ memory B cells were low in seven of nine patients, CD3+ T cells were low in three patients, CD4/8 ratio was inverted in five patients, CD31+ recent thymic emigrant cells were low in two patients, and CD19+ B cells were low in four patients compared with those in the normal controls. ICF2 patients showed lower proportions of CD19+ B cells and CD16+56+ NK cells and significantly higher proportions of CD3+ T cells than ICF1 patients. T cell receptor excision circles were undetectable in two patients. Despite being treated by immunoglobulin replacement therapy, three patients died of influenza virus, fatal viral infection with persistent Epstein-Barr virus infection, or JC virus infection. One of three dead patients showed normal intelligence with mild facial anomaly. Two patients presented with autoimmune or inflammatory manifestations. Infectious episodes decreased in three patients who were started on trimethoprim-sulfamethoxazole and/or antifungal drugs in addition to immunoglobulin replacement therapy. These patients might have suffered from T cell immunodeficiency. These results indicate that patients with ICF syndrome have a phenotype of combined immunodeficiency. Thus, to achieve a better prognosis, these patients should be treated as having combined immunodeficiency in addition to receiving immunoglobulin replacement therapy.

TREG and Tcon Dynamics after Allo-HSCT: Cgvhd Is Associated to Decreased NaïVe and Stem Cell Memory Subsets with a Concomitant Increase in Terminally Differentiated T Cell Subsets

Chronic Graft versus Host disease (cGVHD) is a major limiting factor for the success of allo-HSCT. In this prospective study we aimed to evaluate the association between the kinetics of Regulatory T cells (TREG) and Conventional CD4+T cells (TCON) reconstitution and the emergence of cGVHD. We performed a detailed phenotypic analysis by multiparametric flow cytometry using fresh blood from 39 patients undergoing unrelated donor HSCT after a reduced intensity conditioning regimen containing ATG over a 2 year period, representing a total of 213 samples analyzed. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil. 11 patients were excluded due to disease relapse and/or death due to infection or acute GVHD in the first 9 months post-HSCT.We observed indiscriminately low numbers of TREG (CD4+CD127lowFoxp3+CD25bright) until mo 6 after HSCT and reduced TREG numbers in patients developing cGVHD (GVHD+) versus those who did not (GVHD-) (p=0.02 at mo 9).We further studied the dynamics of TREG subset reconstitution. CM TREG (CD45RA-CD62L+) was the predominant population in both patient groups. EM (CD45RA-CD62L-) was the second most abundant TREG subset. CM and EM TREG numbers were similar between patient groups. EMRA (CD45RA+CD62L-) TREG remained very low throughout the follow-up but were significantly increased at mo 9 in GVHD+ (p= 0.03). Interestingly, Naïve TREG (CD45RA+CD62L+CD95-) started to emerge at mo 9 and were significantly reduced in GVHD+ patients at mo 12 (0.71 vs 0.14 cells/µl; p=0.02)The Stem Cell Memory subset (SCM), identified as CD45RA+CD62L+CD95+, is thought to be self renewing and multipotent, being able to differentiate into CM, EM and TEMRA memory subsets. While in GVHD- SCM TREG started to emerge at mo 9, this subset remained low in GVHD+. Statistically significant differences were observed at mo 18 (0.91 vs 0.15 cells/µl; p=0.02).To ascertain the role of thymic output in TREG reconstitution we quantified CD31+ naïve TREG. Recent Thymic Emigrant cells (RTE) TREG were significantly reduced in GVHD+ at mo 12 (0.8 vs 0.16cells/µl; p=0.02) and at mo 18 (1.93 vs 0.28 cells/µl; p=0.02).In order to clarify whether both thymic output and peripheral expansion were contributing factors to decreased Naïve TREG, we quantified proliferation using Ki-67. TREG from GVHD+ patients proliferated less from months 2 to 18 reaching statistical significance at mo 9 and 12 (p= 0.003 and 0.02, respectively), suggesting that decreased TREG numbers are at least partly due to reduced peripheral expansion.Taken together, these observations suggest a compromised peripheral expansion and de novo generation of TREG through thymic output in cGVHD patients. Of note, susceptibility to apoptosis was not increased in TREG from GVHD+ patients, as Bcl-2 levels tended to be higher in relation to GVHD- patients.In the CD4+Foxp3- TCON population, we observed a clear predominance of EM in all patients. These cells, together with CM, are the first to appear at similar levels in both patient groups. EMRA emerged at higher numbers in GVHD+, although the results did not reach statistical significance.Naïve and RTE TCON started to emerge after mo 6 in GVHD-. Interestingly, these cells remained much lower in GVHD+ vs GVHD- patients throughout the follow-up, reaching statistical significance at mo 12 (p=0.03 for naïve; p=0.03 for RTE TCON).SCM TCON remained very low in all patients showing a tendency to be reduced in GVHD+ when compared to GVHD-. This difference reached statistical significance at mo 18 (6.85 vs 2.13 cells/µl; p=0.02).The small number of TREG and TCON subsets in GVHD+ patients were unlikely due to increased susceptibility to apoptosis, as assessed by Bcl-2 expression. The low numbers of cells within naïve and SCM TREG gates precluded the analysis of Bcl-2 expression in these subsets. Bcl-2 tended to be increased in total TREG CM and EM in GVHD+. In TCONs, Bcl-2 levels were similar in both patient groups.In summary, our data in patients developing cGVHD suggest that decreased thymic output and increased differentiation into terminally differentiated effector cells may have a negative impact on the number of naïve and SCM TCON and TREG. We speculate that the inability to generate and maintain the more immature TREG subsets may lead to decreased TREG numbers after 6 months post transplant, potentially resulting in decreased control of effector T cells contributing to the development of cGVHD. DisclosuresRitz:Kiadis: Membership on an entity's Board of Directors or advisory committees.

Premature aging and immune senescence in HIV-infected children.

Several pieces of evidence indicate that HIV-infected adults undergo premature aging. The effect of HIV and antiretroviral therapy (ART) exposure on the aging process of HIV-infected children may be more deleterious since their immune system coevolves from birth with HIV. Seventy-one HIV-infected (HIV+), 65 HIV-exposed-uninfected (HEU), and 56 HIV-unexposed-uninfected (HUU) children, all aged 0-5 years, were studied for biological aging and immune senescence. Telomere length and T-cell receptor rearrangement excision circle levels were quantified in peripheral blood cells by real-time PCR. CD4 and CD8 cells were analysed for differentiation, senescence, and activation/exhaustion markers by flow cytometry. Telomere lengths were significantly shorter in HIV+ than in HEU and HUU children (overall, P < 0.001 adjusted for age); HIV+ ART-naive (42%) children had shorter telomere length compared with children on ART (P = 0.003 adjusted for age). T-cell receptor rearrangement excision circle levels and CD8 recent thymic emigrant cells (CD45RACD31) were significantly lower in the HIV+ than in control groups (overall, P = 0.025 and P = 0.005, respectively). Percentages of senescent (CD28CD57), activated (CD38HLA-DR), and exhausted (PD1) CD8 cells were significantly higher in HIV+ than in HEU and HUU children (P = 0.004, P < 0.001, and P < 0.001, respectively). Within the CD4 cell subset, the percentage of senescent cells did not differ between HIV+ and controls, but programmed cell death receptor-1 expression was upregulated in the former. HIV-infected children exhibit premature biological aging with accelerated immune senescence, which particularly affects the CD8 cell subset. HIV infection per se seems to influence the aging process, rather than exposure to ART for prophylaxis or treatment.

Thymic Activity and T Cell Repertoire Recovery after Autologous Hematopoietic Stem Cell Transplantation Preceded by Myeloablative Radiotherapy or Chemotherapy

It was previously postulated that pretransplant myeloablative treatment may impair thymopoiesis, contributing in this way to delayed reconstitution of T cells after hematopoietic stem cell transplantation (HSCT). On the other hand, de novo generation of T cells after HSCT requires a competent thymus. Various myeloablative conditioning regimens (total body irradiation [TBI] or high-dose chemotherapy) routinely used in clinical practice may have potentially different impacts on the thymus. However, no comparative study on thymic output and T cell repertoire in autologous (auto)HSCT model has been presented so far. Here we evaluated thymic output and TCR diversity in 45 lymphoma patients submitted to autoHSCT differing in respect to conditioning regimen: high-dose chemotherapy as monotherapy (BEAM, n = 22) or combination of total body irradiation with cyclophosphamide chemotherapy: Cy/TBI (n = 23). Thymic output was assessed before and on days +100, +180, and +365 after autoHSCT by flow cytometric counts of recent thymic emigrant (RTE) cells (CD31+ CD62L+ CD45RA+ CD4+) and quantification of signal joint TCR receptor excision circles (sjTRECs) by quantitative PCR. T cell repertoire diversity was analyzed on day +365 after autoHSCT by spectra-typing of the CDR3 region in the TCRVβ chain. The BEAM group, in contrast to the Cy/TBI group, manifested significantly higher proportions of RTE cells and sjTREC copy numbers on days +100 and +180. Analysis of TCRVβ spectra-types on day +365 revealed more restricted (monoclonal or oligoclonal) T cell repertoires in the Cy/TBI versus BEAM group (48.8% versus 18.2%, P = .0002). In conclusion, the conditioning scheme based on BEAM chemotherapy may be performed with lower risk of thymic destruction and T cell repertoire distortion than Cy/TBI scheme. This finding may help to potentially improve conditioning schemes to efficiently perform myeloablation and maintain active thymopoiesis.

Preliminary Evidence for a Compromised T-Cell Compartment in Maltreated Children with Depression and Posttraumatic Stress Disorder

Objective: Adverse childhood experiences, such as maltreatment, and affective disorders are associated with a proinflammatory state and/or variably compromised counts in lymphocyte subsets in adults. Animal models of social stress indicate that recent thymic emigrant cells (RTE), which maintain the T-cell compartment, are affected. Methods: In this study, we examined the association between lymphocyte subsets, and depression and posttraumatic stress disorder (PTSD) among 16 maltreated children (aged 6-17 years) 1-3 years after the intervention by the Child Protection Team and among 14 healthy age-matched controls. The participants completed psychological assessment and had blood drawn for fluorescent-activated cell sorting analysis. Results: Among maltreated children and adolescents, depression was associated with lower counts of RTEs and T-helper cells after controlling for age. We found additional trends and large effect sizes with regard to the percentages of these cells, as well as for related lymphocyte subsets. Similar effects were found for PTSD, i.e. lower counts of naïve T cells, which was also supported by a trend for their percentage. Compared to controls, maltreated participants with a clinical level of depression had decreased percentages of RTEs, with a similar trend for PTSD. Conclusion: Limited by the nature of a pilot study and the small sample size, these preliminary findings of a compromised T-cell compartment related to psychiatric symptoms in maltreated children and adolescents need to be further studied; particularly the role of RTEs needs further evaluation.

Changes in the frequencies of human hematopoietic stem and progenitor cells with age and site

This study enumerated CD45(hi)/CD34(+) and CD45(hi)/CD133(+) human hematopoietic stem cells (HSCs) and progenitor granulocyte-macrophage colony forming cells (GM-CFCs) in blood and trochanteric and femoral bone marrow in 233 individuals. Stem cell frequencies were determined with multiparameter flow cytometry and using an internal control to determine the intrinsic variance of the assays. Progenitor cell frequency was determined using a standard colony assay technique. The frequency of outliers from undetermined methodological causes was highest for blood, but less than 5% for all values. The frequency of CD45(hi)/CD133(+) cells correlated highly with the frequency of CD45(hi)/CD34(+) cells in trochanteric and femoral bone marrow. The frequency of these HSC populations in trochanteric and femoral bone marrow rose significantly with age. In contrast, there was no significant trend of either of these cell populations with age in the blood. Trochanteric marrow progenitor GM-CFCs showed no significant trends with age, but femoral marrow GM-CFCs trended downward with age, potentially because of the reported conversion of red marrow at this site to fat with age. Hematopoietic stem and progenitor cells exhibited changes in frequencies with age that differed between blood and bone marrow. We previously reported that side population (SP) multipotential HSC, which includes the precursors of CD45(hi)/CD133(+) and CD45(hi)/CD34(+), decline with age. Potentially the increases in stem cell frequencies in the intermediate compartment between SP and GM progenitor cells observed in this study represent a compensatory increase for the loss of more potent members of the HSC hierarchy.

Angiotensin II Receptor Type 2 Activation Is Required for Cutaneous Sensory Hyperinnervation and Hypersensitivity in a Rat Hind Paw Model of Inflammatory Pain

Many pain syndromes are associated with abnormal proliferation of peripheral sensory fibers. We showed previously that angiotensin II, acting through its type 2 receptor (AT2), stimulates axon outgrowth by cultured dorsal root ganglion neurons. In this study, we assessed whether AT2 mediates nociceptor hyperinnervation in the rodent hind paw model of inflammatory pain. Plantar injection of complete Freund's adjuvant (CFA), but not saline, produced marked thermal and mechanical hypersensitivity through 7 days. This was accompanied by proliferation of dermal and epidermal PGP9.5-immunoreactive (ir) and calcitonin gene–related peptide-immunoreactive (CGRP-ir) axons, and dermal axons immunoreactive for GFRα2 but not tyrosine hydroxylase or neurofilament H. Continuous infusion of the AT2 antagonist PD123319 beginning with CFA injection completely prevented hyperinnervation as well as hypersensitivity over a 7-day period. A single PD123319 injection 7 days after CFA also reversed thermal hypersensitivity and partially reversed mechanical hypersensitivity 3 hours later, without affecting cutaneous innervation. Angiotensin II–synthesizing proteins renin and angiotensinogen were largely absent after saline but abundant in T cells and macrophages in CFA-injected paws with or without PD123319. Thus, emigrant cells at the site of inflammation apparently establish a renin-angiotensin system, and AT2 activation elicits nociceptor sprouting and heightened thermal and mechanical sensitivity. PerspectiveShort-term AT2 activation is a potent contributor to thermal hypersensitivity, whereas long-term effects (such as hyperinnervation) also contribute to mechanical hypersensitivity. Pharmacologic blockade of AT2 signaling represents a potential therapeutic strategy aimed at biologic mechanisms underlying chronic inflammatory pain.

Angiotensin receptor type 2 mediates hypersensitivity and hyperinnervation in an inflammatory pain model

Abnormal sensory axon proliferation occurs in many pain syndromes. The objective of this study was to determine if the angiotensin type 2 receptor (AT2), a mediator of dorsal root ganglion axon sprouting, is required for increased innervation density and hypersensitivity in a rat hind paw model of inflammatory pain. Injection of complete Freund's adjuvant (CFA), but not saline, produced marked thermal and mechanical hypersensitivity through 7 days, accompanied by proliferation of cutaneous axons immunoreactive for the pan‐neuronal marker PGP9.5, calcitonin gene‐related peptide (a marker of heat‐sensitive nociceptors) and GFRα2 (a marker of mechanosensitive nociceptors). Continuous infusion of the AT2 antagonist PD123319 beginning with CFA injection completely prevented hyperinnervation and hypersensitivity. A single PD123319 injection 7 days after CFA also reversed thermal hypersensitivity and partially reversed mechanical hypersensitivity 3 hours later, without affecting cutaneous innervation. Angiotensin II‐synthesizing proteins renin and angiotensinogen were largely absent after saline but abundant in T‐cells and macrophages in CFA‐injected paws. We conclude that emigrant inflammatory cells establish a renin‐angiotensin system, which leads to AT2 activation required for nociceptor sprouting and heightened thermal and mechanical sensitivity. Supported by HD049615.

Establishment of an In Vitro System Representing the Chicken Gut-Associated Lymphoid Tissue

The bursa of Fabricius is critical for B cell development and differentiation in chick embryos. This study describes the production in vitro, from dissociated cell suspensions, of cellular agglomerates with functional similarities to the chicken bursa. Co-cultivation of epithelial and lymphoid cells obtained from embryos at the appropriate developmental stage regularly led to agglomerate formation within 48 hours. These agglomerates resembled bursal tissue in having lymphoid clusters overlaid by well organized epithelium. Whereas lymphocytes within agglomerates were predominantly Bu-1a+, a majority of those emigrating onto the supporting membrane were Bu-1a− and IgM+. Both agglomerates and emigrant cells expressed activation-induced deaminase with levels increasing after 24 hours. Emigrating cells were actively proliferating at a rate in excess of both the starting cell population and the population of cells remaining in agglomerates. The potential usefulness of this system for investigating the response of bursal tissue to avian Newcastle disease virus (strain AF2240) was examined.

Molecular Assessment of Thymus Capabilities in the Evaluation of T-Cell Immunodeficiency

T-cell immunodeficiency may pose a diagnostic challenge to clinicians, especially when the basic T-cell immune workup is not sufficiently informative. An intensive assessment of thymus capabilities that involves either measuring the recent thymic emigrant cells or analyzing the T-cell receptor (TCR) repertoire is often required to estimate the severity and nature of the immune disorder. A comprehensive T-cell immune workup, including TCR excision circles (TRECs) and TCR repertoire analyses, was performed in three patients with various degrees of severity of T-cell immunodeficiency. All three patients had normal peripheral CD3+ T lymphocytes. TCR repertoire analysis revealed oligoclonal (patient 1), restricted (patient 2), and near-normal (patient 3) patterns. TREC quantification was significantly reduced in patients 1 and 2 but normal in patient 3. Based on clinical features at presentation and at follow-up, and supported by the results of immunologic studies, patients 1 and 2 were diagnosed as having significant T-cell immunodeficiency and patient 3 as having T-cell immunocompetence. Assessment of thymus capabilities by TRECs and TCR repertoire analyses is helpful in diagnosing patients with T-cell immunodeficiency and should be part of the evaluation of every patient suspected of having that condition.