Immune Function Gene Expression Research Articles

Flow cytometry of DNMT1 as a biomarker of hypomethylating therapies.

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

Immunologic Signatures of Peripheral Blood T Cells Reveal the Outcome of p53MVA Vaccine and Pembrolizumab Treatment in Patients with Advanced Ovarian Cancer.

Our previous studies indicated that p53-reactive T cells were associated with clinical benefit in patients with advanced ovarian cancer who were treated with p53-expressing modified vaccinia Ankara (p53MVA) vaccine and gemcitabine chemotherapy. To replace chemotherapy with an approach that will enhance vaccine efficacy and antitumor immunity, we treated patients with p53MVA in combination with PD-1 checkpoint blocker, pembrolizumab. We also attempted to further characterize the activation status of T cells prior to vaccination and during treatment. Patients received up to three triweekly vaccinations concurrent with pembrolizumab, followed by pembrolizumab monotherapy at 3-week intervals. Correlative studies analyzed peripheral blood T-cell phenotypes and profiles of immune function gene expression. We observed 6/28 (21%) patients with a clinical benefit to therapy, including 3 partial responses (PR) and 3 patients with stable disease (SD) for 6+ months. The median progression-free survival was 1.8 months (95% confidence interval: 1.7-3.8) and median overall survival was 15.1 months (9.4-30.4). Two patients remain progression-free at 28 and 33 months. Of the 18 patients evaluable in correlative studies, 6 were immunologic responders of whom 5 had clinical benefit (3 PR, 2 SD). Immunologic non-responders expressed in pretreatment peripheral blood mononuclear cell samples high levels of mRNA for multiple molecules associated with terminally differentiated T cells. p53MVA/pembrolizumab immunotherapy showed promising antitumor activity in patients who demonstrated functionally competent peripheral blood T cells. Detection of markers of terminally differentiated T cells before treatment may identify patients unlikely to respond to p53MVA/pembrolizumab. The activity of a combination immunotherapy of p53 vaccine and PD-1 checkpoint blockade in patients with platinum-resistant ovarian cancer was evaluated in a phase II trial. Clinical benefit was correlated with the responsive immune status of patients before and during the treatment, defining potential predictive markers for immune therapy.

Chicken adaptive response to nutrient density: immune function change revealed by transcriptomic analysis of spleen.

Feed accounts for the largest portion (65-70%) of poultry production costs. The feed formulation is generally improved to efficiently meet the nutritional needs of chickens by reducing the proportion of crude protein (CP) and metabolizable energy (ME) levels in the diet. Although many studies have investigated the production performance during dietary restriction, there is a lack of research on the mechanisms by which immune cell function is altered. This study examined the effects of ME and CP restriction in the chicken diet on serum immunoglobulins and expression of immune function genes in spleen. Changes in serum immunoglobulins and immune-related gene expression were analyzed in 216 YS-909 broilers fed with 9 different dietary treatments, including experimental treatment diets containing low, standard, and high levels of ME or CP in the diet. At 42 days of age, serum immunoglobulins and expression of spleen immune genes in 6 female chickens selected randomly from each dietary treatment (3×3 factorial arrangement) group were measured by enzyme-linked immunosorbent assay (ELISA) and transcriptomic analysis using RNA sequencing, respectively. The results showed that the IgM level in the low ME group chickens was significantly (p < 0.05) lower than that in other groups. In addition, immune-related genes, such as MX1, USP18, TLR4, IFNG and IL18 were significantly upregulated when the dietary nutrient density was reduced, which may put the body in an inflammatory state. This study provided general information on the molecular mechanism of the spleen immune response to variable nutrient density.

Evaluating beluga (Delphinapterus leucas) blow samples as a potential diagnostic for immune function gene expression within the respiratory system.

Evaluating respiratory health is important in the management of cetaceans, which are vulnerable to respiratory diseases. Quantifying the expression of genes related to immune function within the respiratory tract could be a valuable tool for directly assessing respiratory health. Blow (exhale) samples allow DNA analysis, and we hypothesized that RNA could also be isolated from blow samples for gene expression studies of immune function. We evaluated the potential to extract RNA from beluga blow samples and tested whether transcripts associated with immune function could be detected with endpoint polymerase chain reaction. A total of 54 blow samples were collected from clinically healthy aquarium belugas (n = 3), and 15 were collected from wild belugas temporarily restrained for health assessment in Bristol Bay, Alaska (n = 9). Although RNA yield varied widely (range, 0-265.2ng; mean = 85.8; SD = 71.3), measurable RNA was extracted from 97% of the samples. Extracted RNA was assessed in 1-6 PCR reactions targeting housekeeping genes (Rpl8, Gapdh or ActB) or genes associated with immune function (TNFα, IL-12p40 or Cox-2). Fifty of the aquarium samples (93%) amplified at least one transcript; overall PCR success for housekeeping genes (96/110, 87%) and genes associated with immune function (90/104, 87%) were similarly high. Both RNA yield and overall PCR success (27%) were lower for wild beluga samples, which is most likely due to the reduced forcefulness of the exhale when compared with trained or free-swimming belugas. Overall, the high detection rate with PCR suggests measuring gene expression in blow samples could provide diagnostic information about immune responses within the respiratory tract. While further study is required to determine if quantitative gene expression data from blow samples is associated with disease states, the non-invasive nature of this approach may prove valuable for belugas, which face increasing anthropogenic disturbances.

RNA-Sequencing of Umbilical Cord Blood to Investigate Spontaneous Preterm Birth: A Pilot Study.

Objective To analyze the transcriptomic gene expression of umbilical cord blood leukocytes using RNA-sequencing from preterm birth (PTB) and term birth (TB). Study Design Eight women with spontaneous PTB (sPTB) and eight women with unlabored TB were enrolled prospectively. The sPTB and TB cohorts were matched for maternal age, race, mode of delivery, and fetal sex. Cord blood RNA was extracted and a globin depletion protocol was applied, then sequenced on the Illumina HiSeq 4000. Raw read counts were analyzed with DESeq2 to test for gene expression differences between sPTB and TB. Results 148 genes had significant differential expression ( q < 0.01). Cell cycle/metabolism gene expression was significantly higher and immune/inflammatory signaling gene expression significantly lower in the sPTB cohort compared with term. In African American (AA) infants, 18 genes specific to cell signaling, neutrophil activity, and major histocompatibility complex type 1 had lower expression in preterm compared with term cohort; the opposite pattern was seen in non-Hispanic Whites (NHWs). Conclusion Compared with term, preterm fetuses have higher cell cycle/metabolism gene expression, suggesting metabolic focus on growth and development. Immune function gene expression in this pilot study is lower in the sPTB group compared with term and differs in AA compared with NHW infants.

Abstract 1020: Characterization of the immune landscape and analysis of tumor response after anti-PD-1 blockade in a 3D ex vivo system of non-small lung cancer

Abstract Background: Immuno-oncology drugs face the challenge that cell lines and animal models cannot recapitulate the full intimacies of the tumor microenvironment. Although these models have aided in the discovery of new medical modalities, only 8% of these models have had a successful translation. Furthermore, the phenotype of the microenvironment of various tumor types has not been assessed in depth. Nilogen Oncosystems' proprietary 3D-EX vivo drug screening platform analyzes fresh patient tumor tissue that remains embedded in its natural environment. With our model, the characteristics of the tumor microenvironment can be accurately revealed in response to checkpoint inhibitors in non-small cell lung cancer (NSCLC) and correlates can be found among different platforms. Methods: For the 3D-EX vivo platform, microspheres were produced from procured fresh tumor tissue from NSCLC cancer patients. They were then treated with Keytruda ex vivo and treatment-mediated changes in TIL subpopulations were analyzed using flow cytometric analysis, cytokine release by Bio-Rad's 17-plex cytokine assay as well as gene expression by NanoString's 770 gene Immune Panel. Results: Ex vivo treatment of the 3D microspheres with Keytruda, showed significant changes in T-cell activation and immune cell populations in 26% of NSCLC tumors. This was observed by the simultaneous increase in IFN-γ and TNF-α upon cytokine analysis. Furthermore, we found a differential expression of signature genes such as CD8, CXCL10, CXCL9, EOMES, Granzyme A/B, IFN-γ, related to T-cell subpopulations via Nanostring analysis, which was accompanied by an increase in Granzyme B via flow cytometry. Conclusion: The positive and negative associations between expression of immune function genes, TIL activation, and cytokine production by ex vivo treatment shows that Nilogen Oncosystems' 3D-Ex vivo platform can accurately recapitulate the tumor microenvironment and exhibits the importance of a comprehensive analysis of the tumor immune microenvironment for a better understanding of the mechanism of action of immuno-oncology drugs that may aid in developing biomarkers that can be used for patient selection. Citation Format: Melanie Mediavilla-Varela, Melba Marie Page, Jenny Kreahling, Soner Altiok. Characterization of the immune landscape and analysis of tumor response after anti-PD-1 blockade in a 3D ex vivo system of non-small lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1020.

262: Fetal immune function gene expression varies by gestational age and race

262: Fetal immune function gene expression varies by gestational age and race

Anti-PD1 treatment to induce M1 polarization of tumor infiltrating macrophages in a 3D ex vivo system of lung cancer patients.

e23090 Background: Nivolumab and pembrolizumab treatment targeting the PD1/PD-L1 axis has demonstrated increased survival benefit in subpopulations of patients with advanced non-small cell lung cancer (NSCLC). The impact of these therapeutics on tumor immune microenvironment is not fully understood. Classical M1 macrophages are critical components involved in the inflammatory response and antitumor immunity. In this study, we evaluated the effect of PD1/PD-L1 blockade on M1 polarization of tumor-associated macrophages (TAMs) and activation of cytotoxic T-cells in a 3D ex vivo system of NSCLC. Methods: Fresh tumor tissues obtained from consented patients with NSCLC at the time of surgical resection were utilized in a 3D ex vivo tumor miscrosphere assay. 3D tumor microspheres were treated with nivolumab or pembrolizumab at 10 mg/ml for 36 hours within an intact tumor microenvironment. Flow cytometry analysis was performed to evaluate treatment-mediated TAM polarization, activation of T-cells and changes in CD4 and CD8 subpopulations. A multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines. Additionally, a NanoString platform containing probes to quantitate 770 immune function genes was used to determine potential positive or negative associations between expression of immune function genes and TIL activation by ex vivo treatment. Results: Both nivolumab and pembrolizumab treatment increased population of M1 macrophages (CD68+, CD80+, CD163-) and simultaneous release of MIP1b, IFN-ɣ, TNF-a, and GM-CSF cytokines as well as expression of genes related to the M1 phenotype that was accompanied by activation of CD8 cells assessed by Ki67 and CD107a expression and increased expression of genes involved in the IFNg pathway. Conclusions: Our studies showed that anti PD1/PD-L1 treatment leads to M1 macrophage polarization and T-cell activation in subgroups of NSCLC patients emphasizing the importance of comprehensive analysis of tumor immune microenvironment for a better understanding of the mechanism of action of immuno-oncology drugs that may help developing rationale combination treatments in NSCLC.

Characterization of basic immune function parameters in the fathead minnow (Pimephales promelas), a common model in environmental toxicity testing

The fathead minnow (Pimephales promelas) is an environmental sentinel species, commonly used in toxicity testing. However, there is a lack of data regarding basic immune function in this species. To improve the usefulness of the fathead minnow as a model for basic immune function and immunotoxicity, this study sought to 1) compare the differential expression of immune function genes in naïve fathead minnows and 2) determine the effects of pathogen exposure on immune gene expression and spleen index. To accomplish this, kidney, spleen and liver tissue were collected three days post injection (dpi) from adult male fathead minnows from each of the following groups: 1) uninjected control 2) sham-injected (Hank's balanced salt solution) and 3) pathogen-injected (Yersinia ruckeri). Spleen tissue was also collected at seven and 14 dpi. Differential tissue expression of immune function genes was evaluated in naïve minnows and expression patterns were similar to those found in other fish species, with liver tissue generally having the highest amount of expression. Following pathogen injection, the expression of complement component 3 (c3) (4.4-fold, kidney; 2.5-fold, liver), interleukin 11 (il11) (4.8-fold, kidney; 15.2-fold, liver) and interleukin 1β (il1β) (8.2-fold, kidney; 17.2-fold, spleen; 2.6-fold, liver) were significantly upregulated. Elastase 2 (elas2) was significantly downregulated (5.8-fold) in liver tissue. A significant increase in spleen index at seven dpi was also observed in pathogen-injected minnows. This study has identified endpoints that are part of the normal response to pathogen in fathead minnows, an essential step toward the development of the fathead minnow as a model for immunotoxicity evaluations.

The transcriptome-wide effects of exposure to a pyrethroid pesticide on the Critically Endangered delta smelt Hypomesus transpacificus

Research into the effects of contaminants on fishes is often conducted on well- studied model test species, whose responses may be different than those of endangered species. We developed an oligonucleotide microarray consisting of 12 595 genes to examine the effects of the pesticide permethrin on an endangered fish in California, USA, the delta smelt Hypomesus transpacificus. The microarray was used in combination with quantitative PCR (qPCR) assess- ments. We exposed larval delta smelt to permethrin for 96 h at concentrations of 0.69, 1.37, 2.56, 4.84, 12.88 and 24.94 µg l �1 , and compared the responses to a control group. The 96 h 50% lethal concentration (LC50) was 4.07 µg l �1 , which is lower than those reported for model test species and similar to other endangered species. With increasing exposure concentration, we detected the dif- ferential expression of 3342 microarray features with elevated expression of genes involved in protein degradation and apoptosis, and decreased expression of immune function genes. Func- tional analysis indicated that genes involved in protein degradation, immune function, an unfolded protein response, meta bolism and cell signaling cascades were affected by exposure to permethrin. Many of the gene responses in the 0.69 µg l �1 treatment group differed in their direc- tional change in expression from those at higher exposure concentrations, suggesting a potential mechanistic threshold of sub-lethal toxicity at concentrations below the lowest observed effect concentration (LOEC) of 2.56 µg l �1 . These results are consistent with non-monotonic response patterns to contaminants, and demonstrate the effects on a sensitive fish species of a widely used pyrethroid pesticide at concentrations below those that affect model test species.

Genomic analysis reveals that immune function genes are strongly linked to clinical outcome in the North Central Cancer Treatment Group n9831 Adjuvant Trastuzumab Trial.

To develop a genomic signature that predicts benefit from trastuzumab in human epidermal growth factor receptor 2-positive breast cancer. DASL technology was used to quantify mRNA in samples from 1,282 patients enrolled onto the Combination Chemotherapy With or Without Trastuzumab in Treating Women With Breast Cancer (North Central Cancer Treatment Group N9831 [NCCTG-N9831]) adjuvant trastuzumab trial. Cox proportional hazard ratios (HRs), adjusted for significant clinicopathologic risk factors, were used to determine the association of each gene with relapse-free survival (RFS) for 433 patients who received chemotherapy alone (arm A) and 849 patients who received chemotherapy plus trastuzumab (arms B and C). Network and pathway analyses were used to identify key biologic processes linked to RFS. The signature was built by using a voting scheme. Network and functional ontology analyses suggested that increased RFS was linked to a subset of immune function genes. A voting scheme model was used to define immune gene enrichment based on the expression of any nine or more of 14 immune function genes at or above the 0.40 quantile for the population. This model was used to identify immune gene-enriched tumors in arm A and arms B and C. Immune gene enrichment was linked to increased RFS in arms B and C (HR, 0.35; 95% CI, 0.22 to 0.55; P < .001), whereas arm B and C patients who did not exhibit immune gene enrichment did not benefit from trastuzumab (HR, 0.89; 95% CI, 0.62 to 1.28; P = .53). Enriched immune function gene expression as defined by our predictive signature was not associated with increased RFS in arm A (HR, 0.90; 95% CI, 0.60 to 1.37; P = .64). Increased expression of a subset of immune function genes may provide a means of predicting benefit from adjuvant trastuzumab.

The Contraceptive MPA, Unlike NET, Modulates Expression of Immune Function Genes and Increases HIV-1 Infection in Cervical Tissue Explants and PBMCs

Background: The synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET-EN) are widely used in developing countries as injectable contraceptives where disease burden is high. Some studies suggest that MPA unlike NET increases HIV-1 acquisition in women. Whether MPA and NET differentially affect HIV-1 infection and the expression of key genes relevant to HIV-1 acquisition via differential molecular mechanisms is key to understanding choice of progestin contraceptive for HIV-1 prevention. Methods: Regulation of selected genes was investigated in cervical tissue explants and peripheral blood mononuclear cells (PBMCs) by qRT-PCR western blotting and Luminex assays in response to physiologically relevant doses of progestogens. Infection assays were performed in the absence and presence of HIV-1 using HIV-1BAL-RENILLA or HIVpNL4.3 IMCs. The GR specific antagonist RU486 or GR siRNA knockdown were used to determine the role of the GR in modulating ligand-specific effects. Results: In PBMCs MPA like dexamethasone (DEX a GR specific agonist) showed anti-inflammatory effects decreasing pro-inflammatory IL6 IL8 and RANTES levels and increasing anti-inflammatory GILZ gene expression levels while NET and progesterone (P4) did not. In primary cervical tissue explants DEX and MPA repressed IL6 and IL8 and increased GILZ gene expression levels. Differential gene expression by MPA versus NET and P4 were mediated via the GR in PBMCs. Similarly MPA and DEX unlike NET and P4 increased HIV-1 replication in viable PBMCs. In primary cervical explants MPA but not NET increased HIV-1 replication. Conclusions: Collectively the data suggest that NET unlike MPA would be a safer choice of injectable progestin contraceptive in young women in high risk areas for HIV-1 infection. The molecular basis for this choice most likely involves differential effects of MPA as compared to NET and P4 on transcription of immunomodulatory genes due to their differential actions via the ubiquitous GR.

Divergent gene expression responses to Complicated Grief and Non-complicated Grief

The "widowhood effect" (i.e., morbidity/mortality in recently bereaved spouses) may be related to changes in immune function, but little is known about the impact of bereavement on gene transcription in immune cells. This study examined how Complicated Grief and Non-complicated Grief responses to bereavement differentially affect leukocyte gene expression. Genome-wide transcriptional profiling and bioinformatic analyses were completed on 63 older adults. Thirty-six of them had lost their spouse/partner on average 2years ago, and 27 were nonbereaved, married controls. Twelve of the bereaved participants met criteria for Complicated Grief. Compared to nonbereaved controls, bereavement (both Complicated Grief and Non-complicated Grief) was associated with upregulated expression of genes involved in general immunologic activation and a selective downregulation of genes involved in B lymphocyte responses. However, Complicated Grief and Non-complicated Grief differed markedly in their expression of Type I interferon-related transcripts, with Non-complicated Grief subjects showing substantial upregulation relative to nonbereaved controls and Complicated Grief subjects showing substantial downregulation. Bereavement significantly modulates immune function gene expression. The magnitude of bereavement-related distress (i.e., Complicated Grief vs. Non-complicated Grief) is linked to differential patterns of transcription factor activation and gene expression involved in innate antiviral responses. These findings provide a molecular framework for understanding the health effects of bereavement, as well as new insights into the particular gene modules that are most sensitive to the individual's psychological response to loss.

RNA Sequencing Identifies Multiple Fusion Transcripts, Differentially Expressed Genes, and Reduced Expression of Immune Function Genes in BRAF (V600E) Mutant vs BRAF Wild-Type Papillary Thyroid Carcinoma

The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2-3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.

Widespread Decreased Expression of Immune Function Genes in Human Peripheral Blood Following Radiation Exposure

We report a large-scale reduced expression of genes in pathways related to cell-type specific immunity functions that emerges from microarray analysis 48 h after ex vivo γ-ray irradiation (0, 0.5, 2, 5, 8 Gy) of human peripheral blood from five donors. This response is similar to that seen in patients at 24 h after the start of total-body irradiation and strengthens the rationale for the ex vivo model as an adjunct to human in vivo studies. The most marked response was in genes associated with natural killer (NK) cell immune functions, reflecting a relative loss of NK cells from the population. T- and B-cell mediated immunity genes were also significantly represented in the radiation response. Combined with our previous studies, a single gene expression signature was able to predict radiation dose range with 97% accuracy at times from 6-48 h after exposure. Gene expression signatures that may report on the loss or functional deactivation of blood cell subpopulations after radiation exposure may be particularly useful both for triage biodosimetry and for monitoring the effect of radiation mitigating treatments.

146. Does gene regulation make it complicated? Differences in gene expression responses to Complicated Grief and Non-Complicated Grief

Increased morbidity and mortality in recently bereaved spouses (e.g. “widowhood effect”) may be due to alterations in immune function. However, little is known about the impact of bereavement on gene regulation of immune cells. Complicated Grief (CG), a syndrome characterized by persistent separation distress with feelings of overwhelming yearning and preoccupation with the deceased is distinct to Non-complicated Grief (Non-CG) in various psychoneuroimmunologic aspects. In this study we examined whether the type of grief (CG vs. Non-CG) differentially affects leukocyte gene expression. Sixty-three older adults were recruited for genome-wide transcriptional profiling and bioinformatic analyses; 36 had lost their spouse/partner, with 12 of them meeting criteria for CG. The remaining 27 were nonbereaved married controls. Bereavement (both CG and Non-CG, compared to nonbereaved controls) was linked to an increased expression of genes involved in general immunologic activation and a decreased expression of genes involved in B lymphocyte responses. In contrast, CG and non-CG showed significantly different gene expression of Type I interferon-related transcripts: compared to nonbereaved controls there was substantial upregulation in Non-CG subjects and substantial downregulation in CG subjects. Bereavement significantly modulates immune function gene expression. Furthermore, innate antiviral genes are differentially expressed in response to the magnitude of bereavement-related distress (i.e., CG vs. non-CG). These molecular insights might help to understand the health effects of bereavement.

Unique transcriptional profile of liver-resident memory CD8+ T cells induced by immunization with malaria sporozoites

Sterile immunity against live Plasmodium infection can be achieved by immunization with radiation attenuated sporozoites. This protection is known to be mediated in part by antigen-specific memory CD8+ T cells, presumably those residing in the liver. We characterized and compared the transcriptional profile of parasite-specific memory CD8+ T cells residing in the liver and spleen after immunization of mice with irradiated sporozoites. Microarray-based expression analysis of these memory CD8+ T cells indicated that liver resident memory cells display a distinct gene expression profile. We found major differences in the expression of immune function genes as well as genes involved in the cell cycle, cell trafficking, transcription and intracellular signaling. Importantly, the malaria parasite-induced liver resident CD8+ T cells display a transcriptional profile different to that described for CD8+ T cells following other microbial challenges.

Differential gene expression of γ-actin, Toll-like receptor 2 (TLR-2) and interleukin-1 receptor-associated kinase 4 (IRAK-4) in Mya arenaria haemocytes induced by in vivo infections with two Vibrio splendidus strains

Immune function gene expression in Mya arenaria haemocytes was evaluated following in vivo infection with Vibrio splendidus LGP32-GFP and 7SHRW. Elongation factor 1α (EF-1α) with 2 (EF-2), after challenge with LGP32-GFP, and EF-1α with the ribosomal protein S-18, after challenge with 7SHRW, were found to be the most stable housekeeping genes. Using these internal controls and comparing the regulation induced by both strains, up-regulation of γ-actin, down-regulation of TLR-2 and up-regulation of IRAK-4 was significantly higher after challenge with LGP32-GFP ( p < 0.001, p = 0.001 and p < 0.05, respectively). These results suggest specific responses at a molecular level modulated by the bacterial strains. LGP32-GFP induced marked responses which coincide with a similar trend previously found on phenotypic responses under our experimental model.

Phenotypic and functional characterization of a mouse model of targeted Pig-a deletion in hematopoietic cells

Somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) causes glycosyl phosphatidylinositol anchor deficiency in human patients with paroxysmal nocturnal hemoglobinuria. We produced an animal model of paroxysmal nocturnal hemoglobinuria by conditional Pig-a gene inactivation (Pig-a(-/-)) in hematopoietic cells; mice carrying two lox sites flanking exon 6 of the Pig-a gene were bred with mice carrying the transgene Cre-recombinase under the human c-fes promoter. We characterized the phenotypic and functional properties of glycosyl phosphatidylinositol-deficient and glycosyl phosphatidylinositol-normal hematopoietic cells from these Pig-a(-/-) mice using gene expression microarray, flow cytometry, bone marrow transplantation, spectratyping, and immunoblotting. In comparison to glycosyl phosphatidylinositol-normal bone marrow cells, glycosyl phosphatidylinositol-deficient bone marrow cells from the same Pig-a(-/-) animals showed up-regulation of the expression of immune function genes and contained a significantly higher proportion of CD8 T cells. Both characteristics were maintained when glycosyl phosphatidylinositol-deficient cells were transplanted into lethally-irradiated recipients. Glycosyl phosphatidylinositol-deficient T cells were inactive, showed pronounced Vbeta5.1/5.2 skewing, had fewer gamma-interferon-producing cells after lectin stimulation, and contained fewer CD4(+)CD25(+)FoxP3(+) regulatory T cells. However, the levels of T-cell receptor signaling proteins from glycosyl phosphatidylinositol-deficient cells were normal relative to glycosyl phosphatidylinositol-normal cells from wild type animals, and cells were capable of inducing target cell apoptosis in vitro. Deletion of the Pig-a gene in hematopoietic cells does not cause frank marrow failure but leads to the appearance of clonally-restricted, inactive yet functionally competent CD8 T cells.

Clonally-Restricted T-Lymphocytes in PigA Mutant Mice.

Somatic mutation in the X-linked phosphatydylinositol glycan class A (PIG-A) gene causes glycosyl phosphatidylinositol (GPI) anchor deficiency in hematopoietic stem and progenitor cells, in humans, a requirement for the development of the disease paroxysmal nocturnal hemoglobinuria (PNH). While progress has been made in understanding PNH and especially in treatment of intravascular hemolysis secondary to cell surface deficiency of CD59, why PIG-A mutant stem cells expand in the setting of immune-mediated bone marrow failure remains obscure. We produced a conditional PigA knock-out animal model (PigA−/−) by cross-breeding mice carrying germline insertion of two lox sites flanking exon 6 of PigA gene with mice carrying the transgene Cre-recombinase driven by the human c-fes promoter. The resultant B6 Fes-cre PigAflox (PigA−/−) mice had PigA gene inactivation specifically in hematopoietic cells. We observed that GPI-deficient (GPI−) bone marrow (BM) and spleen cells from PigA−/− mice contained much larger proportions of lymphocytes, especially CD8+ T cells, in comparison to GPI+ cells. The expansion of GPI−CD8+ T cells was not associated with any obvious hematological phenotype, and blood and BM cell counts were relatively normal in PigA−/− mice. In comparison to GPI+ cells analyzed by microarray, GPI− BM cells showed up-regulation in expression of genes important for immune function responses. Pathway analysis revealed that differentially-expressed genes were clustered in several groups related to immunological function, such as lymphocyte markers (CD8b1, CD8a, CD3e, CD3d, CD7, CD2, CD5, CD6, CD28, CD96, CD27), proteins related to T cell activation (Lck, Zap70, Fyn, Zeta, Lat, Traf1, Tcf7, Ctla2a/Ctla2b), TCR components (Tcr-beta-V13, Tcr-beta-V8.2, Tcr-alpha, Tcr-beta- J, Tcr-gamma), chemokines and C-C motifs (Ccl5, CXCR6, Ccr7), and molecules of the killer lectin-like receptor subfamily (Klrc1, Klrc2, Klra7, Klra8). We transplanted into lethally-irradiated recipients BM cells from PigA−/− mice (pre-incubated with aerolysin to lyse GPI+ cells) or BM cells from normal PigA+/+ donors. By microarray, transplanted GPI− cells retained the phenotype of untransplanted GPI− cells, with a much increased CD8+ T cell proportion and up-regulated immune function gene expression in comparison to transplanted normal BM cells. The enlarged GPI−CD8+ T cell pool had a significantly lower proportion of CD11a+ cells than did GPI+CD8+ T cells, suggesting that GPI−CD8+ T cells were generally less active. There was no difference in the proportion of CD44− naive T cells between GPI−CD8+ and GPI+CD8+ T cells; GPI−CD8+ T cells were not NK cells as they lacked surface NK1.1 staining. The percentage of CD4+CD25+FoxP3+ regulatory T cells in GPI− cells was only 10% of that in GPI+ cells in peripheral blood in both untransplanted and transplanted animals, indicating that the expanded T cell population in the GPI− cell fraction contained few cells with immunosuppressive property. We further investigated T cell clonality by usage of T cell receptor beta variable region (Vbeta); approximately 5-6 Vbeta subfamilies were over represented in the GPI− CD8+ T cells. In particular, Vbeta 5.1/5.2 was prominent in GPI−CD8+ T cells, constituting 22-23 ± 5% GPI− T cells from untransplanted and transplanted animals; a significant increase in comparison to 8-9.1 ± 0.5% Vbeta 5.1/5.2 clonal representation in GPI+CD8+ T cells. Our results are consistent with an antigen-driven T cell response in the GPI− lymphocyte population, independent of pancytopenia. Functionally, GPI−CD8 T cells showed no response to lectin stimulation as measured by gamma interferon production, but they were capable of effecting target cell apoptosis when co-incubated with minor-H antigen mismatched BM cells in vitro. Our data agrees with observations in humans, in which an immune process driven by a restricted set of (unknown) antigens appears active in the pathogenesis of PNH (Gargiulo et al., Blood 2007). We conclude that deletion of PigA gene in hematopoietic cells, independent of frank hematopoietic failure, leads to enrichment of lymphocytes, especially CD8 T cells, in the GPI− cell fraction that have an inactive and naive phenotype. These expanded, clonally-restricted, T cells may provide an initial pool of immune effectors, which in the proper immune activated environment, contribute to bone marrow failure in PNH.