Changes In CD4 Research Articles

Impact of lenalidomide and dexamethasone treatment on immune cell populations of the patients with refractory/relapsed multiple myeloma

Background: Lenalidomide combined with low-dose dexamethasone (Len-dex) is an effective treatment for the patients with refractory/relapsed multiple myeloma (RRMM). The anti-myeloma effect of lenalidomide is associated with activation of the immune system, but the exact immunomodulatory mechanisms in vivo and clinical impact of Len/dex in RRMM patients remains unclear. In this study, we analyzed immune cell populations in patients receiving Len-dex for the treatment of RRMM. Methods: Peripheral blood samples from 90 RRMM patients were taken on day 1 of cycles 1 (baseline), 2, 3, and 4 of Len/dex therapy. CD3+, CD4+, CD8+, CD161+ T cells, natural killer (NK) cell (CD16+/CD56+), NKT-like cell (CD3+/CD56+) and myeloid-derived suppressor cell (MDSC) including granulocytic (G-MDSC) and monocytic (M-MDSC) were analyzed by flow cytometry. In addition, response was assessed in 81 patients receiving more than 4 cycles of Len-dex and the comparison of cell populations according to an achievement of ≥very good partial response (VGPR) was performed. Results: Forty-eight men and 42 women were enrolled in this study. The median age was 61 years (range, 29-84 years). At baseline, peripheral blood CD3+ cell frequency was 51.65 ± 1.79% which was significantly decreased to 41.67 ± 2.44% (P=0.001) and 39.72 ± 2.90% (P Conclusion: Our data demonstrate that Len-dex therapy in patients with RRMM is associated with decreased frequency of T cells with a trend of increased NK cell frequency. Change in CD8+ cell and M-MDSC frequency can correlate with the quality of response to Len-dex. Baseline NKT-like cell frequency and change in CD3+ and CD8+ cells early after treatment may predict continuation of anti-myeloma effect of Len-dex therapy. Disclosures No relevant conflicts of interest to declare.

Abstract CT137: Combination of agonistic CD40 monoclonal antibody CP-870,893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma

Abstract Purpose: Combining immunostimulatory monoclonal (mAb) and checkpoint inhibition may improve response rates and overall survival in patients with metastatic melanoma. CP-870,893 is an agonistic fully human IgG2 mAb targeting the immune stimulatory molecule CD40 (αCD40). Tremelimumab (treme) is a fully human IgG2 mAb targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a negative co-stimulatory molecule found on T cells. Methods: Patients with metastatic melanoma were enrolled on a single-arm open-label phase I study. αCD40 and treme were dosed i.v. every 3 weeks and 12 weeks, respectively. Dose escalation followed a 3 + 3 strategy with alternating increases of αCD40 and treme in each cohort. Outcomes were scored using the Common Terminology Criteria for Adverse Events v3.0 and RECIST 1.0. Flow cytometric analysis of baseline blood samples was compared to samples obtained at least 3 weeks after the last dose of αCD40 and treme. Results: Toxicity: Twenty-four patients were enrolled and treated at 4 different dose levels. Two patients were replaced per protocol due to rapid, symptomatic progression of disease; these patients were not evaluated for response but were included in toxicity analysis. Dose-limiting toxicities (DLT), considered to be likely due to treatment, were colitis (n = 1) and hypophysitis (n = 1) and uveitis (n = 1). Cytokine release syndrome (CRS), grade 1-2, occurring within 24 hours of CP administration, was the most common treatment-related toxicity occurring in 19 (79.2%) patients. CRS symptoms were controlled with standard supportive care. All episodes of CRS resolved within 24 hours of αCD40 administration. The estimated maximum tolerated dose was 0.2 mg/kg of αCD40 and 10 mg/kg of treme. Outcomes: Overall objective response rate (ORR) was 27.3% (best response): two patients (9.1%) had complete responses to treatment and four (18.2%) patients had partial responses. The median follow-up was 22 months with a median progression free survival of 2.5 months and a median overall survival (OS) of 26.1 months. Correlative studies: Flow cytometric analysis of peripheral blood lymphocytes revealed changes in CD8+ T cell phenotypes. Double positive granzyme B+ and Ki-67+ cells increased from 0.56 ± 0.17% (mean ± standard error) for all CD8+ T cells at baseline to 1.01 ± 0.23% post-treatment (p = 0.03). CD8+ T cells expressing both programmed cell death 1 and eomesodermin were also higher after treatment, increasing from 7.7 ± 1.1% to 10.4 ±1.8% (p = 0.04) of all CD8+ T cells. Conclusion: Combination therapy with αCD40 and treme was well-tolerated in patients with metastatic melanoma, with rates of CRS and other toxicity similar to previously reported rates for αCD40 or treme treatments alone. Overall ORR was 27.3%, with median OS of 26.1 months. Given the tolerability, antitumor activity, and biomarker evidence of immune activation, expanded study of this combination should be pursued. Citation Format: David L. Bajor, Rosemarie Mick, Matthew J. Riese, Lee P. Richman, Xiaowei Xu, Drew A. Torigian, Erietta Stelekati, Martha Sweeney, Brendan Sullivan, Lynn M. Schuchter, Ravi Amaravadi, E. John Wherry, Robert H. Vonderheide. Combination of agonistic CD40 monoclonal antibody CP-870,893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT137. doi:10.1158/1538-7445.AM2015-CT137

Reconstitution of TCR/CD3 expression in B cell lymphoma to dissect the contribution of CD3 conformational change from downstream signaling events in the process of TCR/CD3 downregulation. (IRM7P.714)

Abstract T cell receptor (TCR) stimulation induces conformational change in CD3 (CD3Δc), followed by phosphorylation of ITAMs that leads to receptor downregulation among other T cell responses. To begin to dissect the contribution of CD3Δc from downstream signal transduction mechanisms associated with these events, we have transduced the mouse B cell lymphoma X63.Ag8.653, which lacks expression of the src-family kinase Lck required for proper ITAM phosphorylation, with the OT-I TCR chains Vα2 and Vβ5, together with all CD3 subunits and the coreceptor CD8αβ, generating the cell line X63.OT1.CD8. Stimulation of the OT-I TCR with anti-CD3 antibody or the cognate antigen SIINFEKL-H2Kb induces CD3Δc in this cell line. As expected by the absence of Lck, CD3 ITAM phosphorylation was undetectable in our cell line and no calcium mobilization was found upon antibody stimulation. Unexpectedly, stimulation with either SIINFEKL-H2Kb or anti-CD3 did induce TCR/CD3 downregulation in our cells. These data suggest CD3ε PRS exposure via CD3Δc might be sufficient to transduce some signals that contribute to TCR downregulation in the absence of CD3 ITAM phosphorylation.

Transcriptional Changes in CD8(+) T Cells During Antiretroviral Therapy Intensified With Raltegravir.

Background. Intensification of antiretroviral therapy with raltegravir does not affect levels of residual human immunodeficiency virus (HIV)-1 viremia, but it has led to increased levels of episomal HIV-1 DNA in some patients, suggesting antiviral activity against otherwise unresponsive components of the viral reservoir. Effects of raltegravir on host cells remain less well understood. Methods. We used comprehensive and unbiased microarray-based transcriptional profiling to analyze gene expression changes in CD8(+) T cells from participants in a randomized clinical trial (AIDS Clinical Trials Group [ACTG] A5244) comparing raltegravir-intensified to nonintensified antiretroviral therapy. Results. Although raltegravir intensification failed to induce statistically significant changes in HIV-1 DNA or residual plasma viremia, we observed significant increases in the expression intensity of 121 host gene transcripts. In functional annotations of these transcripts, we found that they were mainly involved in glucose and carbohydrate metabolism, immune regulation, control of cell proliferation, and tumor suppression. Two of the raltegravir-responsive gene transcripts were statistically correlated with levels of residual HIV-1 RNA, but none of the remaining 119 transcripts were associated with immunologic or virologic characteristics of the study patients. Conclusions. Together, these findings demonstrate that raltegravir intensification can induce previously unrecognized, statistically significant gene expression changes in host CD8(+) T lymphocytes.

Impact of exposure to intimate partner violence on CD4+ and CD8+ T cell decay in HIV infected women: longitudinal study.

Intimate partner violence (IPV) is a risk factor for HIV acquisition in many settings, but little is known about its impact on cellular immunity especially in HIV infected women, and if any impact differs according to the form of IPV. We tested hypotheses that exposure to IPV, non-partner rape, hunger, pregnancy, depression and substance abuse predicted change in CD4+ and CD8+ T-cell count in a dataset of 103 HIV infected young women aged 15-26 enrolled in a cluster randomised controlled trial. Multiple regression models were fitted to measure rate of change in CD4 and CD8 and including terms for age, person years of CD4+/CD8+ T-cell observation, HIV positivity at baseline, and stratum. Exposure variables included drug use, emotional, physical or sexual IPV exposure, non-partner rape, pregnancy and food insecurity. Mean CD4+ T cell count at baseline (or first HIV+ test) was 567.6 (range 1121-114). Participants were followed for an average of 1.3 years. The magnitude of change in CD4 T-cells was significantly associated with having ever experienced emotional abuse from a current partner at baseline or first HIV+ test (Coeff -132.9 95% CI -196.4, -69.4 p<0.0001) and drug use (Coeff -129.9 95% CI -238.7, -21.2 p=0.02). It was not associated with other measures. The change in CD8 T-cells was associated with having ever experienced emotional abuse at baseline or prior to the first HIV+ test (Coeff -178.4 95%CI -330.2, -26.5 p=0.02). In young ART-naive HIV positive women gender-based violence exposure in the form of emotional abuse is associated with a faster rate of decline in markers of cellular immunity. This highlights the importance of attending to emotional abuse when studying the physiological impact of IPV experience and the mechanisms of its impact on women’s health.

Characterization of CD8+ T cell differentiation following SIVΔnef vaccination by transcription factor expression profiling.

The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVΔnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from naïve animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVΔnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data support the hypothesis that ongoing stimulation by SIVΔnef promotes a distinct protective balance of CD8+ T cell differentiation and activation states.

Hypoglycemia affects the changes in endothelial progenitor cell levels during insulin therapy in type 2 diabetic patients.

Hypoglycemia is a barrier to the achievement of glycemic targets and limits the beneficial effects of improved glucose control on cardiovascular outcomes in type 2 diabetes (T2D). Circulating endothelial progenitor cells (EPCs) participate in cardiovascular homeostasis and predict future cardiovascular events. Therefore, we herein analyzed the association between occurrence of hypoglycemia and EPC changes in T2D patients after optimization of glucose control with basal insulin therapy. In the NCT00699686 trial, 42 T2D insulin-naïve patients received a 3 + 3-month cross-over therapy with glargine and detemir. There were 43 minor and 2 severe hypoglycemic episodes in 19 patients (45.2 %, 0.54 episodes/patient/year). Changes in EPCs were analyzed in relation to the occurrence of hypoglycemia during the trial. Patients with hypoglycemia had a higher final HbA1c at 6 months than patients without, although absolute HbA1c changes were not significantly different. Though PCs increased at study end, in patients experiencing at least 1 hypoglycemic episode, the changes in CD34(+), CD133(+) progenitor cells and CD34(+)KDR(+) EPCs were significantly lower than the respective changes in patients without incident hypoglycemia, even after correcting for confounders. During treatment with detemir, which induced >twofold less hypoglycemia than glargine, CD34(+)KDR(+) EPCs increased significantly more than during treatment with glargine. In naïve T2D patients initiating basal insulin, hypoglycemia prevents the increase in vasculoprotective PCs. Clinically, these data strengthen the importance of avoiding hypoglycemia to improve cardiovascular outcomes during the treatment of T2D.

The correlation between human chemokine type 1 chemokine ligand 3 and chemokine ligand 4 gene and the immune reconstitution of acquired immunodeficiency syndrome patients after antiretroviral therapy

Objective To investigate the correlation between the human chemokine type 1 chemokine ligand 3 (CCL3L1) and chemokine ligand 4 (CCL4L) gene expression and the immune reconstitution of acquired immunodeficiency syndrome (AIDS) patients after antiretroviral therapy. Methods The gene copy numbers of CCL3L1 and CCL4L were detected by real time polymerase chain reaction in 217 AIDS patients before antiretroviral therapy. And the correlation between CCL3L1 and CCL4L gene copy numbers and the level of CD4+ and CD8+ T lymphocytes were analyzed. The changes of CD4+ and CD8+ T lymphocytes were defined as mean change value per month after 48 months treatment. The change rates of CD4+ and CD8+ T lymphocytes were defined as the logarithm of the ratio of the value after 48 month to that at baseline. Comparison between groups was conducted using analysis of variance. Results The median of gene copy numbers of CCL3L1 and CCL4L were 2 (range: 0-8) and 3 (range: 0-7), respectively. After antiviral treatment, there were significantly different changes of CD8+ T lymphocyte level (F=3.054, P<0.05) and change rate of CD4+ /CD8+ (F=3.520, P<0.05) among groups of high (gene copy 4-8), median (gene copy 2-3) and low (gene copy 0-1) CCL3L1 gene copy numbers. The changes of CD8+ T lymphocyte levels (P=0.023) and change rates (P=0.038) in high and low CCL3L1 gene copy groups were both significantly different. There were significant differents changes rate of ratio of CD4+ /CD8+ T lymphocyte among high and median (P=0.010), high and low CCL3L1 gene copy numbers (F=4.397, P<0.05). The significant difference of the change rates of CD4+ /CD8+ were found between the gene copy 3 group and gene copy 4-7 group CCL4L (P=0.005) and between the gene copy 4-7 group and gene copy 0-2 group of CCL4L (P=0.030). The change ratio of CD4+ /CD8+ T lymphocytes increased with the increase of copy numbers of CCL4L gene. Conclusions The gene expressions of CCL3L1 and CCL4L might be associated with the ability of immune reconstitution of AIDS patients after antiretroviral therapy. Key words: Acquired immunodeficiency syndrome; Chemokine CCL3; Chemokine CCL4; Gene copy numbers; Immune reconstitution

Probiotic Effect of Lebanese Darfiyeh Cheese: A Randomized Case Control Prospective Study in the Elderly

A randomized case control prospective method was adopted in this study in order to assess the health benefit associated with the Darfiyeh cheese, a traditional Lebanese raw goat milk cheese ripened in goat skin. 21 participants were recruited and divided into 2 groups: test group (n = 10) and control group (n = 11). Venous blood and stools were collected at day 14, 21 and day 0 and 21 respectively. Flow cytometry was performed on blood sample. Fecal samples were plated on Plate Count Agar (PCA) and de Man, Rogosa and Sharpe Agar (MRS) agar in order to monitor any alteration in the gastrointestinal tract (GIT). At day 24, a significant increase by 27.67% (p = 0.043) in the bacterial count was seen on the MRS agar for the test group in comparison to no change in the total bacterial count. At day 14 a significant drop (p = 0.026) in CD3+/CD4+ population in the test group (35.67 vs 42.87), and a non-significant drop (p = 0.818) in CD8+ (24.76 vs 28.28) is seen when compared to control group. At day 24, no significant change (p = 0.20) in the CD3+/CD4+ population in comparison between both groups and no change in CD8+ or Geo means in both groups. In conclusion, an alteration in the GIT microflora could be hinted at by the lactobacilli bacterium, which was not reflected on the total bacterial count.

Treatment of Alopecia Areata in Mice by Stimulating the Hair Follicles Using Parathyroid Hormone Agonists Linked to a Collagen Binding Domain

Abstract Alopecia Areata is a patchy hair loss from autoimmune-mediated destruction of hair follicles, for which there is no adequate therapy. PTH-CBD is a fusion protein of parathyroid hormone and a bacterial collagen-binding domain, providing targeted delivery of a hair cycle stimulator to skin and promoting hair growth. Objectives: We tested the effects of PTH-CBD on hair growth in an established animal model for alopecia areata, the C3H/HeJ engrafted mouse. Methods: C3H/HeJ engrafted mice (Jackson Laboratories, Bar Harbor, ME) were treated subcutaneously for 8 weeks with either vehicle or different doses of PTH-CBD (320 mcg/kg x1, 1000 mcg/kg x1 or 1000 mcg/kg/wk). Results: Vehicle animals showed progressive hair loss, as expected. In PTH-CBD treated animals, grey scale quantification showed a greater proportion of PTH-CBD treated animals without significant hair loss at the end of the 2 month period (18/22 PTH-CBD vs. 4/11 vehicle), with no observed differences between the different PTH-CBD treatment regimens. Histological examination revealed no change in CD8+ cells, but there was a marked increases in the number of anagen VI hair follicles in PTH-CBD treated animals. There were also increased levels of betacatenin, a known initiator of the hair cycle, observed around the bulge region of hair follicles. Conclusions: C3H/HeJ engrafted animals treated with PTH-CBD showed rapid and persistent improvements in hair growth in the majority of tested animals. There were marked increases in anagen hair follicles despite an ongoing immune reaction. Increased beta catenin levels suggest that PTH-CBD stimulates hair growth by activating the Wnt pathway.

Transcriptional Changes in CD8+ T Cells During Antiretroviral Therapy Intensified With Raltegravir

Transcriptional Changes in CD8+ T Cells During Antiretroviral Therapy Intensified With Raltegravir

C-Myb Restrains Megakaryopoiesis through the Hsa-MiR-486-3p-Driven Down-Regulation of C-Maf

INTRODUCTIONThe transcription factor c-Myb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that c-Myb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which c-myb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs.METHODSRNA from CD34+ HPCs transfected with c-myb-targeting/non targeting control synthetic siRNAs was collected 24 hours post-Nucleofection for a set of 5 independent experiments.mRNA and miRNA expression for each sample were profiled by Affymetrix U219 GeneAtlas and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis.The effects of hsa-miR-486-3p overexpression and c-Maf silencing on CD34+ cells differentiation ability were studied by morphological and immunophenotypic analyses after liquid culture and by collagen-based clonogenic assay. Furthermore, gene expression changes in CD34+ cells upon hsa-miR-486-3p overexpression were profiled by Affymetrix U219.RESULTSThe integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs down-regulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p-overexpression in CD34+ cells.Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes down-regulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as up-regulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice.CONCLUSIONSIntegrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p up-regulation and subsequent c-maf down-regulation as a new molecular mechanism through which c-Myb favours erythropoiesis while restraining megakaryopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Defective antiviral CD8 T‐cell response and viral clearance in the absence of c‐Jun N‐terminal kinases

The c-Jun N-terminal kinase (JNK) signalling pathway appears to act as a critical intermediate in the regulation of lymphocyte activation and proliferation. The majority of studies on the importance of JNK are focused on its role in T helper responses, with very few reports addressing the mechanisms of JNK in governing CD8 T-cell-mediated immunity. By using a well-defined mousepox model, we demonstrate that JNK is involved in CD8(+) T-cell-mediated antiviral responses. Deficiency of either JNK1 or JNK2 impaired viral clearance, subsequently resulting in an increased susceptibility to ectromelia virus in resistant mice. The impairment of CD8 responses in JNK-deficient mice was not directly due to an inhibition of effector T-cell expansion, as both JNK1 and JNK2 had limited effect on the activation-induced cell death of CD8(+) T cells, and only JNK2-deficient mice exhibited a significant change in CD8(+) T-cell proliferation after acute ectromelia virus infection. However, optimal activation of CD8(+) T cells and their effector functions require signals from both JNK1 and JNK2. Our results suggest that the JNK pathway acts as a critical intermediate in antiviral immunity through regulation of the activation and effector function of CD8(+) T cells rather than by altering their expansion.

Nanostructural and Biochemical Changes in CD8+ T Cells Utilizing Atomic Force Microscopy and Raman Spectroscopy.

Nanostructural and Biochemical Changes in CD8+ T Cells Utilizing Atomic Force Microscopy and Raman Spectroscopy.

Issue highlights—July 2014

Issue highlights—July 2014

The effect of norepinephrine on CD8 T cell subsets in healthy human adults (CCR3P.215)

Abstract Chronic exposure to stress has detrimental effects on the adaptive immune response. Studies in vitro show that exposure of norepinephrine (NE), a stress hormone, decreases proliferation and functionality of T cells. T cells consist of several different types of cells and each of them serves distinct functions during an immune response. It is unknown how NE reduces functionality of T cells and whether NE has different impact on different types of T cells. Here, we assessed the effect of NE on CD8 T cell function in humans in vitro. We found that not all subsets of CD8 T cells respond the same to NE exposure; memory cells including central memory (Tcm) and effector memory (Tem) were more significantly affected by NE compared to naïve (Tn) CD8 T cells in terms of gene expression of cytokines (IL-2, IFNG, and TNF) important to proliferation and function in the adaptive immune response. Furthermore, expression of NE receptor, Beta-2 adrenergic receptor (B2AR), was higher in freshly isolated memory CD8 T cells (both Tcm and Tem) compared to Tn CD8 T cells. Together these findings show that memory CD8 T cells are more susceptible to NE induced reduction of cytokine production. Currently we are analyzing the effect of NE on general gene expression in Tcm and Tem cells as well as examining histone modifications in these cytokine loci to determine if NE effect via chromatin changes in CD8 T cells.

Contribution of Ang (1‐7) to Sex Differences in the Renal T cell Profile Following Chronic Ang II Infusion (LB714)

Angiotensin (Ang) II is pro‐inflammatory, and T cells contribute to Ang II hypertension in male animal models; little is known in females. Initial studies treated 12 wk old male and female Sprague Dawley rats (SD) with Ang II (200 ng/kg/min) and renal T cell profiles were measured via flow cytometry. Ang II increased renal T cell infiltration in both sexes; as well as immuno‐suppressive T regulatory cells (Tregs) only in kidneys of female SD (p=0.004), and male SD had greater increases in pro‐inflammatory Th17 cells (p&lt;0.001). We have previously shown that females also have greater renal Ang (1‐7) levels than males, and Ang (1‐7) has been suggested to be anti‐inflammatory. Therefore, the goal of the current study was to test the hypothesis that chronic Ang II infusion increases renal T cells in both sexes; however, greater Ang (1‐7) results in more Tregs in females than males. Additional male and female SD were treated with Ang II, or Ang II plus the Ang (1‐7) mas receptor antagonist A‐779 (48 µg/kg/hr). CD4+ T cell counts were greater in males compared to females following Ang II infusion (p&lt;0.01), inhibition of Ang (1‐7) did not alter counts in either sex. Ang II infusion increased Tregs in females and Th17 in males, inhibition of Ang (1‐7) did not alter these counts. Renal CD8+ T cell counts were reduced in males relative to females following Ang II infusion (p&lt;0.01), and inhibition of Ang (1‐7) caused a significant but comparable decrease in renal CD8+ T cell counts in both sexes (p=0.0003). In conclusion, our data implicates a role for Ang (1‐7) to modulate Ang II‐induced changes in CD8+ T cell counts; however, our results show that Ang (1‐7) does not mediate the sex difference in Tregs and Th17 in SD during Ang II infusion.Grant Funding Source: Supported by NIH grant 1R01 HL093271‐01A1 to J.C.S, and AHA Predoctoral Fellowship 12PRE11470003 to M.A.Z.

The dynamic microenvironment of pancreatic islet cell transplants precludes the use of common housekeeping genes

Background: Pancreatic islet graft survival is affected by early (<3 days) and late immune responses, displaying local CD3+ T cell predominance and loss of insulin production within a week. However, qRT–PCR gene expression profiles require normalisation to housekeeping genes that might be affected by metabolic, inflammatory, hypoxic, apoptotic, revascularisation–related and immune–cell infiltrate changes. Here, we examined eight housekeeping genes. Methods: Syngeneic and allogeneic mouse islet transplantations were performed; day 1 and 7 grafts were analysed for insulin and CD3 transcript levels, as normalised to each housekeeping gene. Results: Normalised qRT–PCR data was not uniform across transplant combinations and time from transplantation. 18s RNA was the superior housekeeping gene, as confirmed by valid changes in CD3 and insulin expression levels. Conclusions: While β–actin and GAPDH are the least–fitting housekeeping genes for the dynamic microenvironment of islet transplants, 18s RNA maintains authenticity across transplant combinations, time from transplantation and altered immune microenvironment.

Cutting Edge: Central Memory CD8 T Cells in Aged Mice Are Virtual Memory Cells

The number of memory phenotype CD8 T cells increases dramatically with aging in both humans and mice. However, the mechanism for this is unknown. The prevailing hypothesis is that memory T cells accumulate with aging as a result of lifelong antigenic stimulation. However, data supporting this supposition are lacking. In this study, we demonstrate that central memory CD8 T cells, which represent a large majority of memory CD8 T cells in aged mice, are not memory cells that develop in response to antigenic stimulation but are virtual memory cells that develop without antigenic stimulation. In addition to phenotypic evidence, we show that accumulation of central memory CD8 T cells is independent of CD4 T cells, CCR5, and CXCR3, all of which are known to be essential for Ag-driven development of central memory CD8 T cells. Thus, this study reveals a novel mechanism for aging-related changes in CD8 T cells.

DNA Methylation Changes In Aging Human CD34+ Cells Coincide With Hotspots Of Disordered Methylation In AML At Imprinted and Allelically Methylated Regions

Aging of the hematopoietic system in humans is associated with increased incidence of myeloid malignancies. Epigenetic machinery such as DNA methylation is known to change with age, and is disproportionately impacted by recurrent genetic mutations in acute myeloid leukemia (AML). We hypothesized that epigenetic changes in CD34+ hematopoietic stem and progenitor cells (HSPCs) may precede recurrent genetic changes in AML, and might be detected in normal aging HSPCs with increasing frequency. We also hypothesized that areas with increased variability in methylation may be hot-spots for the emergence of epigenetically distinct HSPC clones.In order to characterize these changes, we performed methylome-wide profiles of human HSPCs from different age groups (20-25 years (10), 40-45 years (10) and >60 years (9)).Adult HSPCs were obtained from Leukocyte Reduction System cones from healthy platelet donors. CD34+ cells were then isolated by Fluorescence Assisted Cell Sorting. 200 ng of DNA extracted from the CD34+ cells was processed using the Infinium Methylation 450k Beadchip (Illumina). Differentially methylated regions (DMRs) were identified using the bump hunting procedure of Jaffe (2011) to pool information across CpG loci into regions of consistent change and to quantify statistical significance.893 differentially methylated regions (DMRs) varied linearly with age in HSPCs; a set of 31 such regions yielded an accurate predictor of age in lineage-sorted cells (N=48, Reinius et al., 2012) and whole blood (N=656, Hannum et al., 2011), with a root-mean-squared error of 5.3 years. While age-related lymphopenia has previously been reported, DNA methylation marks for lineage commitment (Houseman et al., 2012) were nearly uniform within our subjects' CD34+ cells, and exhibited no relationship with age. However, regional summaries of methylation provided more accurate age predictions than specific CpG loci. We reasoned that differential variability at individual loci might be the cause.We thus investigated regions where methylation variability increased with age. Known imprinted clusters and allelically methylated regions (AMRs) identified by Fang (2012, PNAS) were disproportionately represented among these; 27% of known imprinting regions and 33.3% of allelically methylated regions in the genome coincided with at least one such region, while comprising only 0.3% of the genome and 0.7% of loci assayed. Among these, the H19 imprinting control region has been shown to crucially regulate long-term HSPC homeostasis in mice via IGF2, and the allelically methylated WT1/WT1-AS region on chromosome 11p is a highly recurrent hotspot for disordered methylation in AML, as well as sequential epigenetic defects in Wilms' tumor. The allelically methylated vault RNA VTRNA2-1 (recently shown to predict survival in AML) on chromosome 5q, and the monoallelically expressed TP73 and DIRAS3 genes on chromosome 1p, were also sites of greater methylation variability with age in normal HSPCs. Wu et al. (1997) showed that loss of imprinting at H19/IGF2 is common in AML, seemingly conferring a selective metabolic advantage, and global loss of imprinting in mice leads to widespread tumorigenesis (Holm et al., 2005). Recurrent methylation aberrations in induced pluripotent stem cells favor imprinted clusters (Nazor, 2012), and epigenetic polymorphisms arise in these regions over time in cultured cells (Tanay et al, 2012). However, to our knowledge, ours is the first report of this type of heterogeneity with age in normal human adult HSPCs.Clonal hematopoiesis has previously been documented in healthy elderly adults (Levine 2012), and the majority of patients in the Cancer Genome Atlas (TCGA) AML study exhibited mutations in one or more genes regulating epigenetic machinery. We propose that increased epigenetic heterogeneity in aging HSPCs, particularly at regions with allele-specific methylation (such as H19/IGF2), may precede malignant evolution in some leukemias, and warrants further investigation. Disclosures:No relevant conflicts of interest to declare.