Immune Profile Of Patients Research Articles

A combined genomic and immune profile in patients with head and neck squamous cell carcinoma treated with immunotherapy: A pilot study.

e18506 Background: In head and neck squamous cell carcinoma (HNSCC) only a small subset of patients (pts) really benefits from immunotherapy, suggesting the need of validated molecular and immunological predictive biomarkers. The aim of the study was to evaluate both the genomic and immune profile of HNSCC correlating it to early progression to immunotherapy. Methods: This is a pilot study evaluating immune and molecular profile in platinum-refractory HNSCC pts treated with Nivolumab. Blood samples were collected at baseline (T0) and at second cycle of therapy (T1). The immune profile was studied by multiplex assay, evaluating circulating: CD137, CTLA4, PD1, PDL1, PDL2, CD27, TIM3, LAG3, GITR, HVEM, BTLA, CD80, CD28 and IDO. The comprehensive genomic profile was evaluated at baseline on formalin-fixed paraffin-embedded samples of the tumor through Foundation One Cdx test. The results obtained were correlated with early progression (within 3 months from the start of therapy). Results: Ten pts were enrolled in the study. Early progression occurred in 6 pts (60%). Median progression free survival and overall survival were 3 months (1-11) and 6.5 months (1-12), respectively. In all patients ≥ 5 genetic mutations were detected; median number of mutation was 6 (5-14); 8/10 pts had a tumor mutational burden less than 10 Muts/mb. The more frequent genetic alterations involved the cycline-dependent-kynase pathway (9/10), the transcription factor associated gene TP53 (8/10) and the PI3K-Akt-PTEN signaling pathways (5/10). Less frequent alterations involved FGFR family (4/10), NOTCH signaling pathway (3/10) and TERT (4/10). Early progression was slightly associated with the number of mutations detected (p = 0.06). The rising or falling trend of circulating levels of biomarkers were detected in 9/10 pts and included modification in IDO (1/10), LAG3 (3/10), GITR (3/10), BTLA (3/10), CD137 (4/10), CTLA4 (4/10), PD1 (2/10), Tim3 (3/10), CD28 (3/10), HVEM (3/10), CD80 (2/10). No modifications were recorded in PDL1, PDL2 and CD27. Conclusions: Our results, although in a small cohort, highlighted the complexity and heterogeneity of landscape in HNSCC pts. A novel evaluation that combines molecular and immune profile is needed. In the context of a poor prognosis disease the combination of personalized molecular and immune approaches could represent a promising strategy to improve survival.

Comprehensive immune profiling of patients (pts) with metastatic urothelial cancer (mUC) or renal cell cancer (mRCC) receiving immune checkpoint inhibitors (CPIs).

532 Background: CPIs have had a major impact on pts with mUC and mRCC. Only a small subset of pts benefit from CPI, and predictive biomarkers are needed. The role of circulating immune cells is poorly understood, but early changes after CPI exposure may predict response. We aimed to study the changes in circulating immune cell populations of pts receiving CPIs. Methods: Whole blood was collected prior to, and 3 wks after initiation of CPI in CPI-naïve pts with mUC or mRCC. PBMCs were isolated and profiled using mass cytometry (CyTOF) to provide a comprehensive overview of immune cell populations, expression of immune checkpoints, proliferation, and viability. Expression of chemokine receptors and cytokines was measured by flow cytometry. Treatment-emergent changes were correlated with response. Effects of treatment were determined by Wilcoxon signed-rank test; interactions of treatment and other variables like cluster size were determined by repeated measures two-way ANOVA. Any effects described had a significance of p<0.05. Results: Ten pts enrolled in this pilot study (mRCC = 4, mUC = 6) received anti-PDL1 (n=5), anti-PDL1/anti-CTLA4 (n=3), and anti-PDL1/chemotherapy (n=2). Best response was: 1 CR, 3 PR, 2 SD, 4 PD. Treatment induced an increase in dendritic cells (DC) and a decrease in PD1+ CD4+ and CD8+ T-cells. Elevated Ki-67, CTLA-4, LAMP-1, granzyme B and perforin expression in PD1+ cells post CPI suggested re-invigoration of exhausted T-cells. PD1+ T-cells had increased expression of the chemokine receptors CCR4 and CCR5, and decreased expression of CCR7 and CXCR4, irrespective of treatment. Response was associated with fewer CCR4+ CD4+ T cells and fewer PD1+ CD8+ T cells. Conclusions: Deep profiling by CyTOF provides a means of immune monitoring, with potential applications in clinical trials involving CPIs. Immune responses to CPIs are heterogeneous, with pt subgroups segregated by shifts in both T cell and DCs, and patterns of chemokine receptors and cytokines. Therapy reinvigorates exhausted T cells, and may cause these cells to infiltrate tumors or tumor-draining lymph nodes via chemokine receptors.

Systemic Immune Profile in Patients With CRSwNP.

The immune pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) remains obscure. Our aim was to compare humoral immunity and white blood cell counts in patients with CRSwNP and controls. A prospective case-control study was carried out in 37 patients with CRSwNP and 34 controls without CRS. Clinical data were gathered through a systematic interview. Computed tomography scan, skin prick test, spirometry, and immunological parameters (leukocyte differential count, immunoglobulin classes, and immunoglobulin [Ig] G subclasses) in serum specimens were obtained. Statistical analysis was performed using SPSS v.23. The prevalence of chronic lower respiratory diseases was greater in the CRSwNP group (P < .001), but atopic disease had no significant difference. A significantly higher eosinophil (P < .001) and basophil relative count (P = .022) and a lower relative neutrophil count (P = .013) were found among CRSwNP group. Patients with CRSwNP had higher IgG1 (P = .022), but lower IgG2 (P = .014) and IgG3 (P = .018) serum levels compared to controls; IgG4, total IgG, IgA, IgM, and IgE serum levels did not differ between groups, as well as the prevalence of immunoglobulin classes or IgG subclasses deficiency. The variation observed in peripheral relative leukocyte count and the systemic IgG1 subclass shift are similar to what is known to happen in nasal polyp tissue. A unique systemic immune profile seems to be present in patients with CRSwNP.

Immune Profile of Patients With Sepsis

Immune Profile of Patients With Sepsis

Abstract 4063: Single cell immune profiling of patients with advanced biliary cancers treated with combination checkpoint inhibition and GM-CSF reveals diverse T cell and myeloid cell mechanisms of action

Abstract Background: Advanced biliary cancers (ABC) including cholangiocarcinoma and gallbladder adenocarcinoma are rising in incidence with limited standard treatment options. While checkpoint inhibition (CPI) achieves durable tumor responses in subsets of patients across many malignancies, less than 10% of ABC patients respond to single agent PD-1-targeted therapy. Combination immunotherapy aims to overcome pre-existing and adaptive resistance to immunotherapy. GM-CSF is a cytokine that activates and matures antigen-presenting cells, suggesting the potential to enhance immune responses. The exact mechanisms of action of this cytokine have not been defined in cancer patients. The combination of GM-CSF and anti-CTLA-4 CPI demonstrated safety along with inducing clinical responses in melanoma and prostate cancer. We conducted a phase II trial of the novel combination of GM-CSF and pembrolizumab (Pembro) in patients with ABC which has resulted in durable clinical responses in greater proportion than previously reported with anti-PD-1 monotherapy. Methods: We assessed peripheral blood mononuclear cells (PBMC) from patients on Pembro monotherapy and combined Pembro/GM-CSF by mass cytometry (CyTOF) and T cell receptor (TCR) sequencing. We explored for the differences between clinical responders versus non-responders. Results: We find that the addition of GM-CSF to Pembro leads to a higher frequency of myeloid cells overall; however, certain sub-populations of classical monocytes and conventional dendritic cells decreased in peripheral blood following upfront Pembro followed by GM-CSF. GM-CSF did not seem to change the phenotypes or relative frequencies of circulating T cell subsets. We also find that clinical responders possess specific circulating populations of classical monocyte and conventional dendritic cells prior to treatment. Responders also had higher percentages of CD8+ T cells expressing CD39 following Pembro treatment compared to non-responders. Combination therapy with Pembro/GM-CSF led to different effects on the T cell repertoire compared to Pembro alone. Conclusions: The addition of GM-CSF to Pembro leads to dynamic shifts in myeloid cell subsets in the peripheral blood of ABC patients treated with immunotherapy, whereas Pembro alone led to changes in the activation states of T cell subsets. The TCR repertoire shifts reflect distinct mechanisms of action for Pembro monotherapy versus Pembro/GM-CSF. Future studies will explore the mechanisms driving the increased response rate seen with combination immunotherapy in comparison to Pembro alone in ABC patients, both through the study of peripheral immune responses as well as via immune-profiling of tumors from sequential biopsies of patients on Pembro monotherapy versus combined Pembro/GM-CSF. Citation Format: Bridget P. Keenan, Whitney Tamaki, Eric Liu, Brandon Chen, Alexander Cheung, John D. Gordan, Brenna Sheldon, Li Zhang, Robin K. Kelley, Lawrence Fong. Single cell immune profiling of patients with advanced biliary cancers treated with combination checkpoint inhibition and GM-CSF reveals diverse T cell and myeloid cell mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4063.

The role of immune profile in patients with severe alcoholic hepatitis in predicting mortality and the development of infections: a prospective evaluation

Introduction: Acute alcoholic hepatitis (AAH) is characterized by high mortality rates mainly due to the development of infections.

Effects of ω-3 supplementation on the nutritional status, immune, and inflammatory profiles of gastric cancer patients: A randomized controlled trial.

The aim of this study was to study the effect of ω-3 supplementation on the nutritional status and the immune and inflammatory profiles of patients with gastric cancer during antineoplastic pretreatment. This was a randomized, open, controlled longitudinal study with intervention in outpatient patients with gastric cancer. Sixty-eight patients were randomized into two groups and received either a formula enriched with ω-3 (intervention group [IG]) or standard formula without ω-3 (control group) for 30 d consecutively. Nutritional status (based on patient-generated subjective global assessment, bioimpedance, and anthropometric measurements) and immune and inflammatory parameters were collected before and after supplementation. Results were expressed as frequency, median, and interquartile intervals and were compared by non-parametric test. P < 0.05 was considered statistically significant. Thirty-four patients were included in each group. Of the patients, 64.7% were men, 44.1% were older than 60 years, and 45.6% had stage III disease. There was an increase in C-reactive protein in the control group before and after supplementation, in addition to the worsening in some anthropometric parameters, such as arm muscle area and arm muscle circumference. There was maintenance of the immune profile in both groups. An increase in weight gain was observed in the IG but not in the control group (1.2 [0.9-9] versus 0.7 kg [0.4-1.3]; P = 0.03), as was a reduction of interleukin-6 (5.7 [4.1-6.4] versus 6.3 pg/mL [5.6-8.6]; P = 0.03) and a maintenance of nutritional status, after supplementation. Supplementation with ω-3 leads to weight gain, reduction in the inflammatory profile, and maintenance of the nutritional and immune profiles of these patients, but further studies are needed to examine changes in body composition.

Is inflammatory markers related with cognitive impairment in bipolar disorder?

Background Bipolar disorder (BD) diagnosis is based on specific set of mood and behavioural symptoms although they also exhibit cognitive dysfunction. Immune/inflammatory mechanisms have been implicated in the pathophysiology of BD. Objective To investigate whether cognitive functioning is associated with clinical parameters and immune profile in patients with BD in remission. Methods Twenty patients with BD type I in remission (15/20 female; age [mean ± SD] 43.80 ± 11.72 years; years of education [mean ± SD] 12.40 ± 3.39) and 25 healthy controls (16/25 female; age [mean ± SD] 43.52 ± 11.72 years; years of education [mean ± SD] 11.16 ± 2.66) matched for age, gender, and years of education were enrolled in this study. Subjects were evaluated with the Mini-Plus; Young Mania Rating Scale; Hamilton Depression Rating Scale; and with Brief Assessment of Cognition in Affective Disorders a comprehensive test battery to evaluate visuomotor, immediate affective and non-affective memory, verbal fluency, delayed affective and non-affective memory, inhibition, and problem solving domains. We assessed IL2, IL4, IL6, IL10, IL17, INF, TNF and its soluble receptors in plasma by flow cytometry. Results Patients with BD exhibited poorer performance on all cognitive domains in comparison with controls. Patients with BD also had high levels of soluble TNF receptors, and Th1/Th2/Th17 cytokines (p Conclusion Our data suggest that inflammatory dysfunction might contribute to the cognitive impairment in BD.

BCG immune activation reduces growth and angiogenesis in an in vitro model of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent cancers worldwide and is associated with poor survival and significant treatment morbidity. The immune profile in patients with HNSCC is immunosuppressive and presents cytokine-mediated adaptive immune responses, triggered apoptosis of T cells, and alterations in antigen processing machinery. Bacille Calmette-Guerin (BCG) immunotherapy has been used successfully as a treatment for several types of cancer. In the present study, we sought to determine the antitumor effect of soluble mediators from peripheral blood mononuclear immune cells (PBMCs) activated with BCG vaccine in a three-dimensional coculture model of HNSCC growth using FaDu hypopharynx carcinoma squamous cells. BCG activation of PBMCs led to an increase in CD4+ and CD8+ lymphocyte subsets concomitant with an elevation in the levels of the antitumor cytokines IL-6, TNF-α and IFN-γ, and a EGFR in FaDu cells. In addition, coculture with BCG-activated PBMCs reduced FaDu proliferation and increased cytotoxicity and apoptosis in parallel with an increase in caspase-3 activity and p53 expression. Finally, conditioned medium from BCG-activated PBMCs reduced the levels of the angiogenic factors vascular endothelial growth factor and angiopoietin-2 produced by human aortic endothelial cells (HAECs), and inhibited their proliferation and differentiation into capillary-like structures. Taken together, these results demonstrate that BCG vaccination induces antitumor responses in an HNSCC in vitro model and suggest that the BCG vaccine could be an effective alternative therapy for the treatment of HNSCC.

Comprehensive immune profiling of patients with urothelial carcinoma of the bladder identifies a novel immune target

Comprehensive immune profiling of patients with urothelial carcinoma of the bladder identifies a novel immune target

Immune dysregulation in patients with carpal tunnel syndrome

Peripheral immunity plays a key role in maintaining homeostasis and conferring crucial neuroprotective effects on the injured nervous system, while at the same time may contribute to increased vulnerability to neuropathic pain. Little is known about the reciprocal relationship between entrapment neuropathy and peripheral immunity. This study investigated immune profile in patients with carpal tunnel syndrome (CTS), the most prevalent entrapment neuropathy. All patients exhibited neurophysiological abnormalities in the median nerve, with the majority reporting neuropathic pain symptoms. We found a significant increase in serum CCL5, CXCL8, CXCL10 and VEGF, and in CD4+ central and effector memory T cells in CTS patients, as compared to healthy controls. CCL5 and VEGF were identified as having the highest power to discriminate between patients and controls. Interestingly, and contrary to the prevailing view of CCL5 as a pro-nociceptive factor, the level of circulating CCL5 was inversely correlated with neuropathic pain intensity and median nerve motor latency. In contrast, the level of central memory T cells was positively associated with abnormal neurophysiological findings. These results suggest that entrapment neuropathy is associated with adaptive changes in the homeostasis of memory T cells and an increase in systemic inflammatory modulating cytokines/chemokines, which potentially regulate neuropathic symptoms.

Healthcare-Associated Clostridium difficile Infections are Sustained by Disease from the Community.

Clostridium difficile infections (CDIs) are some of the most common hospital-associated infections worldwide. Approximately 5% of the general population is colonised with the pathogen, but most are protected from disease by normal intestinal flora or immune responses to toxins. We developed a stochastic compartmental model of CDI in hospitals that captures the condition of the host's gut flora and the role of adaptive immune responses. A novel, derivative-based method for sensitivity analysis of individual-level outcomes was developed and applied to the model. The model reproduced the observed incidence and recurrence rates for hospitals with high and moderate incidence of hospital-acquired CDI. In both scenarios, the reproduction number for within-hospital transmission was less than 1 (0.67 and 0.44, respectively), but the proportion colonised with C. difficile at discharge (7.3 and 6.1%, respectively) exceeded the proportion colonised at admission (5%). The transmission and prevalence of CDI were most sensitive to the average length of stay and the transmission rate of the pathogen. Recurrent infections were most strongly affected by the treatment success rate and the immune profile of patients. Transmission within hospitals is substantial and leads to a net export of colonised individuals to the broader community. However, within-hospital transmission alone is insufficient to sustain endemic conditions in hospitals without the constant importation of colonised individuals. Improved hygiene practices to reduce transmission from symptomatic and asymptomatic individuals and reduced length of stay are most likely to reduce within-hospital transmission and infections; however, these interventions are likely to have a smaller effect on the probability of recurrence. Immunising inpatients against the toxins produced by C. difficile will reduce the incidence of CDI but may increase transmission.

Alcohol abstinence ameliorates the dysregulated immune profiles in patients with alcoholic hepatitis: A prospective observational study.

AH patients were in a highly immune-dysregulated state, whereas HDC showed little evidence of immune activation; alcohol abstinence reversed most, but not all, of the immunological abnormalities. (Hepatology 2017;66:575-590).

Early Changes in Circulating T-Cell Immune Profiles in Patients with Relapsed Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: Data from the Phase 3, Double-Blind HELIOS Trial

Introduction Ibrutinib is a first-in-class, once-daily, oral, covalent inhibitor of Bruton9s tyrosine kinase (BTK) that as a single agent has significantly improved overall survival in patients (pts) with both treatment naive and relapsed/refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). In the phase 3 HELIOS trial, the addition of ibrutinib to bendamustine + rituximab (BR) resulted in an 80% reduction in disease progression or death (HR, 0.203, 95% CI, 0.150-0.276; p Methods HELIOS is a randomized, double-blind, placebo-controlled, phase 3 study. Pts with active CLL/SLL following ≥ 1 prior therapy were randomized 1:1 to receive BR (≤ 6 cycles) with either ibrutinib (420 mg daily; n = 289) or placebo (n = 289). Pts with del17p (> 20% of cells) were excluded. Peripheral blood was collected at the start of the study, at Cycle 1, Day 15 (C1D15) and at the end of treatment/time of progressive disease (EOT/PD). From these samples, peripheral blood mononuclear cells (PBMC) were separated and cryopreserved. PBMC were subjected to flow-cytometric analysis of T-cell subsets including CD4, CD8 and markers indicative of Treg cells (CD25+CD127low) and Th17 cells (CCR4+CCR6+). In addition, expression of T-cell checkpoints ICOS, PD-1 and OX-40 by CD4 and CD8 cells were evaluated as an indicator of cell activation or activation/exhaustion. The findings from the HELIOS study reported here are based on analysis of paired samples (C1D1 and C1D15) of 29 CLL cases from the I+BR arm and 22 cases from the P+BR arm. The Mann-Whitney U test was applied to determine the significance of the differences in each of T-cell subtype between the two arms. Results Overall, there was a net increase in the percentage of CD3+ cells with treatment in both the I+BR-treated pts and P+BR-treated pts, which was observed as early as Day 15 (+12.0% and +11.42% of total lymphocytes, respectively, at C1D15 compared with C1D1; Figure 1). The EOT/PD samples showed a continued increase in the percentage of CD3+ cells in the I+BR pts, the majority of whom (11/12) were responders, while the opposite trend was observed in the P+BR patients (8/21 responders). Focusing on the early changes in the analysis of T-cell subsets, a pronounced decrease in the percentage of Th17 CD4+ cells was noted in the I+BR pts (mean: -2.49%) but in the P+BR pts, an increase of this subset (mean: +2.66%) was observed (p = 0.011; Figure 2). When comparing other CD4+ subsets, decreases in Treg cells were seen in both pt groups, with a corresponding increase in the T-cell activation marker ICOS. Less significant changes were observed in the other T-cell markers studied, however, a trend to increased PD-1 was seen in the P+BR pts but not in the I+BR pts. The safety profile in these pts was consistent with previous reports for the study. Conclusions These results suggest that the addition of ibrutinib to BR helped restore T-cell proportions in this subset of cases and it is noteworthy that this effect was visible even within 15 days of initiation of therapy. Data from the I+BR pts showed a pronounced decrease in the percentage of Th17 CD4+ cells, which are primarily pro-tumorigenic but may sometimes have anti-tumor effects, and a concomitant decrease in the percentage of Treg cells, which may explain the overall increase in T-cell activation. These findings may also be related to the observed increase in PFS with the addition of ibrutinib to BR reported in the trial. Disclosures Damle:Janssen Research & Development: Employment. Schaffer:Janssen Research & Development: Employment. Chaturvedi:Janssen Research & Development: Employment. Phelps:Johnson & Johnson: Employment, Equity Ownership. Aquino:Janssen Research & Development: Employment. Mahler:Janssen Research & Development: Employment. Salman:Janssen Research & Development: Employment, Equity Ownership, Other: Travel, Accommodations, Expenses. Howes:Janssen Research & Development: Employment. Loscertales:Janssen Research & Development: Consultancy; Roche: Consultancy; Gilead: Consultancy; AbbVie: Consultancy. Trneny:Gilead: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Janssen Research & Development: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Balasubramanian:Janssen Research & Development: Employment.

Clinical heterogeneity of dominant chronic mucocutaneous candidiasis disease: presenting as treatment-resistant candidiasis and chronic lung disease

In gain-of-function STAT1 mutations, chronic mucocutaneous candidiasis disease (CMCD) represents the phenotypic manifestation of a complex immunodeficiency characterized by clinical and immunological heterogeneity. We aimed to study clinical manifestations, long-term complications, molecular basis, and immune profile of patients with dominant CMCD. We identified nine patients with heterozygous mutations in STAT1, including novel amino acid substitutions (L283M, L351F, L400V). High risk of azole-resistance was observed, particularly when intermittent regimens of antifungal treatment or use of suboptimal dosage occurs. We report a case of Cryptococcosis and various bacterial and viral infections. Risk of developing bronchiectasis in early childhood or gradually evolving to chronic lung disease in adolescent or adult ages emerges. Lymphopenia is variable, likely progressing by adulthood. We conclude that continuous antifungal prophylaxis associated to drug monitoring might prevent resistance to treatment; prompt diagnosis and therapy of lung disease might control long-term progression; careful monitoring of lymphopenia-related infections might improve prognosis.

B-cell–associated immune profiles in patients with decompensated cirrhosis

Objective. Previous observations on immune dysfunction in decompensated cirrhosis have raised the possibility of B-cell impairment. Methods. B-cell subsets in decompensated cirrhotic patients were investigated. Twenty-six decompensated cirrhotic patients and 26 healthy controls were included in this study. The percentages of B-cell subsets, such as mature, memory, immature B cells, and interleukin (IL)-10+-B-cell subpopulations, were measured using fluorescent activated cell sorting. B-cell–associated cytokines (IL-10, IL-21 and IL-4) were determined using an enzyme-linked immunosorbent assay. Results. The percentage of total B cells and mature B cells increased in patients with decompensated cirrhosis compared to healthy controls. The proportions of memory B cells were significantly lower in the decompensated cirrhosis group than the control group. However, the frequency of immature B cells and the percentage of IL-10–expressing cells that were CD19+, memory, mature, or immature B cells were not significantly different between the two groups. Serum levels of IL-10, IL-21, and IL-4 were significantly lower in the decompensated cirrhosis group compared to the control group. Conclusion. These results indicate significant alterations in peripheral blood B-cell subsets in patients with decompensated cirrhosis. Specifically, a profound reduction of memory B cells was observed in spite of an increase in total B-cell populations in decompensated cirrhotic patients. This implies the underlying mechanisms of impaired immune response in these patients.

Evaluation of Immune Profile in Patients with Multiple Myeloma Using Cytof Technology

Introduction: CyTOF (time-of-flight mass cytometry) is a novel high-dimensional technology which permits immunophenotyping and analysis of signaling in single cells. This approach enables simultaneous evaluation of up to 40 parameters using antibodies tagged with distinct elemental isotopes, by combining flow cytometry with atomic mass spectrometry. Since multiple myeloma (MM) is characterized by immune dysfunction, we used CyTOF technology to define the complex immune profile in MM patient bone marrow (BM) samples.Methods: We used 40 different markers to define various B, T, natural killer (NK) subsets, as well as cells of monocytic, granulocytic, erythroid and platelet lineages. Our preliminary data are results from 10 patients with MGUS/ smoldering MM (SMM); 10 newly diagnosed MM; 20 relapsed/refractory MM; and 15 WM patients (5 newly diagnosed and 10 receiving treatment) in comparison with age-matched healthy donors’ BM (HD). A significantly larger cohort of MM (N=150) and WM (N=50) patients is being similarly analyzed and will be presented. To evaluate phenotypic abnormalities in various B cell subsets, we used B lineage markers CD10, CD19, CD20, CD22, CD27, CD34, CD38, CD45, IgA, IgD, IgG and IgM to define B cells maturation stages from hematopoietic stem cells (HSC) to naïve to mature B lymphocytes (pro-B; pre-B-I; pre-B-II; immature B; and mature (naïve) B cells), as well as memory non-switched and memory switched B cells, plasmablasts, normal (CD138+CD38+CD19+CD45+) and clonal plasma cells (CD138+CD38+CD19-CD45-/low), which reside in the specific BM niche. Furthermore, natural killer (NK) subsets (such as NK and NKT cells) and T cells (such as memory CD4T, naive CD4T, memory CD8T, naive CD8T, T regulatory cells and Tg/d cells) were examined. High-dimensional data was obtained using CyTOF technology and analyzed by SPADE and viSNE software.Results: Our data showed significantly decreased HSC in patients with newly diagnosed and relapsed/refractory MM compared to HD (P<0.025). A significant increase in pre-B-I cells was detected in relapsed/refractory MM vs. MGUS/SMM (P<0.028), but the opposite trend was observed in the pre-B-II subpopulation (P<0.005). No differences in immature B cell populations were observed in different stages of MM. However, a significantly higher percentage of immature B cells was present in relapsed/refractory MM compared to HD (P=0.008), and transitional B cells were significantly decreased in newly diagnosed MM compared to HD (P<0.001). Moreover, memory B cells were significant decreased in all MM stages compared to HD (P<0.003). Non-switched memory B cells were significantly increased in MGUS and SMM compared to newly diagnosed MM, while a significant increase of switched memory B cells was present in newly diagnosed MM compared to relapsed/refractory MM. A significant increase in plasmablasts was seen in relapsed/refractory MM in comparison with other MM stages (P<0.011) by CyTOF analyses. Malignant plasma cells (PC) were defined as CD19-, CD38++, CD45-/dim, CD138+ and either cyk or cyl positive. Importantly, a significant increase in clonal PC was found in all MM stages vs. HD, as well as in newly diagnosed MM compared to relapsed/refractory MM (P<0.01). The percentage of PC from CyTOF analyses correlated with % of PC obtained using flow cytometry by Bland-Altman method comparison. We also observed significant differences in T cell subsets including naïve, central memory, effector, and effector memory CD4+ and CD8+T populations between MGUS and newly diagnosed MM, but no significant changes in T regulatory and Tg/d cells. Furthermore, plasmacytoid dendritic (pDC) cells were significantly increased in newly diagnosed MM, and PD-1 expressed on pDC was significantly decreased in newly diagnosed MM compared to MGUS (P=0.007). Interestingly, PD-1 and its ligand PD-L1 were variably expressed on B cells (2-9% and 3-27%) and PC (0.5-46% and 3-41%) from MM BM samples. Other surface molecules including CD269 (4-32%), CD289 (1-8%), CD362 (0.5-1%) and CD329 (1-4%), were variably expressed in PC.Conclusion: A better understanding of the neoplastic BM milieu will provide the framework for identifying and validating novel targeted therapies directed against MM. CyTOF technology represents a novel diagnostic tool to assess the status not only of MM, but also of host immunity, and may allow for the development of rational personalized therapies. DisclosuresNo relevant conflicts of interest to declare.

Изучение патогенетических механизмов депрессивных расстройств

The purpose of this research was to study the peculiarities of psychopathological symptoms, humoral and cellular immune responses in patients with depressive disorders and neurotic psychotic levels. Simple, randomized comparative study in parallel groups involving 157 patients suffering from neurotic and reactive depression was carried out. The control group consisted of 50 healthy donors. Psychiatric symptoms were assessed by the modified cards Avrutskaya-Zaitsev and Hamilton scale. The study of cellular and humoral immune reactions was made by conventional methods. Patients with depressive disorders were observed with the criteria of the signs of depression HAM medium and high severity and unidirectional, but depending on the severity of the disease of varying severity changes in the immune system: inhibition of the oxygen - dependent systems neutrophils, hypo-immune globulinemia of G class, raising a total complement activity of serum, while with reactive depression immune dis-orders wore maladaptive nature, which manifested imbalance in systemic immunity – the load index and leuco – T – index. The study of the immune profile of patients showed that dysfunctional disorders of cellular and humoral immunity depends on the depth of psychopathological disorders. The role of the immune system in the pathogenic mechanisms of depressive disorders was shown. It is necessary in the scheme of the survey include the study of their immune status.

Abstract CT411: Immune profiling of patients vaccinated with the survivin targeted therapeutic vaccine DPX-Survivac demonstrates durable polyfunctional CD4+ and CD8+ T cells in ovarian cancer patients

Abstract Survivin is a member of the inhibitor of apoptosis protein family and its over-expression has been reported in many cancer types such as melanoma, colon, brain, breast, and ovarian cancers making it a promising target for immunotherapy. DPX-Survivac is a Survivin multi-epitope vaccine that covers a broad range of HLA haplotypes (HLA-A1, A2, A3, A24, B7). The adjuvanted water free depot vaccine is designed to specifically induce CD8+ T cells. In a Phase I study in ovarian cancer patients (n=18), we aimed to evaluate one dose level of the vaccine (0.5 mL) without cyclophosphamide (Cohort A) and two dose levels (0.1 mL and 0.5 mL) combined with oral low dose cyclophosphamide as an immune modulator (Cohorts B and C). Using multiparametric flow cytometry, we measured the ex-vivo frequency, phenotype and function of Regulatory T cells (CD25+FoxP3+Ki67+) and Survivin-specific CD4 and CD8 T cells. Our data shows that cohort C preferentially mounted survivin-specific CD8 T cells which significantly peaked at one month following the third vaccination (study day 70) and persisted until at least day 126 post-vaccination. This polyfunctional T cell response was endowed with cytotoxic and proliferative functions as measured by the expression of IL-2, IFNγ, TNFα, Granzyme B and CD107a. The immune responses identified in this cohort functionally diversified over time and were distributed within the Effector memory (CD45RA-CD27-), Central Memory (CD45RA-CD27+) and Late Differentiated CD8 T cell pool (CD45RA+CD27-). Of note, polyfunctional CD4 helper responses directed against tetanus toxoid and Survivin were induced by DPX-Survivac and possibly contributed to the maintenance of the CD8 T cell response. Taken together, the data presented herein shows that DPX-Survivac induced durable polyfunctional CD4 helper and CD8 T cells providing a strong rationale for combining the targeted immune therapy with immune modulation using metronomic cyclophosphamide. Citation Format: Yoav Peretz, Dominike Sauvé, Dominic Gagnon, Salim Ahmed Khan, Geneviève Lévesque, Nathalie Saha, Gilbert Croteau, Valérie Hébert, Karyne Savard, Bader Yassine Diab, Jean-Francois Poulin, Claire Landry, Mohan Karkada, Marc Mansour. Immune profiling of patients vaccinated with the survivin targeted therapeutic vaccine DPX-Survivac demonstrates durable polyfunctional CD4+ and CD8+ T cells in ovarian cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT411. doi:10.1158/1538-7445.AM2014-CT411

B cell-associated immune profiles in patients with end-stage renal disease (ESRD)

Most of the previous studies on immune dysregulation in end-stage renal disease (ESRD) have focused on T cell immunity. We investigated B cell subpopulations in ESRD patients and the effect of hemodialysis (HD) on B cell-associated immune profiles in these patients. Forty-four ESRD [maintenance HD patients (n = 27) and pre-dialysis patients (n = 17)] and 27 healthy volunteers were included in this study. We determined the percentage of B cell subtypes, such as mature and immature B cells, memory B cells, and interleukin (IL)-10+ cells, as well as B cell-producing cytokines (IL-10, IL-4 and IL-21) by florescent activated cell sorting (FACS). B cell-associated gene expression was examined using real-time PCR and B cell producing cytokines (IL-10, IL-4 and IL-21) were determined using an enzyme-linked immunosorbent assay (ELISA). The percentage of total B cells and mature B cells did not differ significantly among the three groups. The percentages of memory B cells were significantly higher in the pre-dialysis group than in the HD group (P < 0.01), but the percentage of immature B cells was significantly lower in the pre-dialysis group than in the other groups. The percentages of IL-10-expressing cells that were CD19+ or immature B cells did not differ significantly (P > 0.05) between the two subgroups within the ESRD group, but the serum IL-10 concentration was significantly lower in the pre-dialysis group (P < 0.01). The results of this study demonstrate significantly altered B cell-associated immunity. Specifically, an imbalance of immature and memory B cells in ESRD patients was observed, with this finding predominating in pre-dialysis patients.