Changes In Lymphocyte Subsets Research Articles

Treatment-induced changes of lymphocyte subsets in patients with adenoid cystic carcinoma of the head and neck.

Adenoid cystic carcinoma (ACC) of the head and neck is a rare and highly malignant tumor, characterized by perineural growth and early distant metastases. The composition of immune cells in the peripheral blood and the tumor microenvironment is critical to tumor growth and control. However, little is known about the frequency and function of the relevant immune cell subsets in this entity. In ACC patients (n = 11) and matched healthy donors (n = 11), the frequency of peripheral blood T and B cells was measured by flow cytometry at different treatment stages of disease (24 samples). Cells were further characterized by their expression of CCR7, PD-1, CD39 and CD73. Tumor-infiltrating lymphocytes (TIL) were analyzed by immunohistochemistry for ten patients and for three patients by flow cytometry. CD4+ T cells had significantly lower frequency after radiotherapy (RT). All other cell frequencies, including Treg, were stable through course of the disease. In B cells, CD73 was reduced after RT. CCR7 expression on T and B cells in patients with relapse/metastases (R/M) differed significantly from patients with active disease. PD-1 remained stable. Treg were more present in TIL compared to peripheral blood. Composition of lymphocyte subgroups behaves similar to squamous cell carcinoma in the head and neck, except for Treg, which remained stable. Nevertheless, the CD4+/Treg ratio was lower after RT, which could stand for an immunosuppressive effect in these patients. Therefore, it could be beneficial treating ACC with combined RT and immunomodulatory drugs.

B and NK Cells Closely Correlate with the Condition of Patients with Acute Pancreatitis.

Purpose Pancreatitis can lead to systemic inflammatory response, but the relationship between lymphocyte changes and patients with pancreatitis remains unclear. In this study, we evaluated the feedback function of changes in peripheral lymphocyte subsets on the condition of patients with pancreatitis. Materials and Methods 131 acute pancreatitis (AP) patients and 11 chronic pancreatitis (CP) patients constituted the patients' group; 20 healthy individuals were enrolled as healthy controls (HC). Serum concentration of C-reactive protein (CRP), amylase, and lipase and the frequency and absolute number of many types of peripheral lymphocytes (including T, B, NK, CD16+/CD56+ T, CD4+ T, CD8+ T, CD4+CD8+ T, and CD4−CD8− T cells) were detected on admission and the seventh day of standard treatment. Besides, the length of hospital stay was recorded. Results The absolute number of all lymphocytes we studied decreased in patients with CP and in patients with almost all types of AP. The frequency change of lymphocytes varies among the different types of AP. During disease onset, B cell frequency correlated positively with CRP concentration and NK cell frequency correlated positively with amylase and lipase concentration. B cell frequency and CD4+ T cell absolute number were recovering towards normal after short-term treatment. The frequency of B cells and NK cells correlated positively with the length of hospital stay. Conclusions B cells and NK cells closely correlate with patients' condition and may help to diagnose AP more accurately and reflect treatment effect of AP in time, affecting the recovery speed of patients with M-AP, which may help physicians to better understand the pathophysiology of pancreatitis.

Age-related changes in the BACH2 and PRDM1 genes in lymphocytes from healthy donors and chronic lymphocytic leukemia patients

BackgroundAge-related genetic changes in lymphocyte subsets are not currently well documented. BACH2 is a transcription factor that plays an important role in immune-mediated homeostasis by tightly regulating PRDM1 expression in both B-cells and T-cells. BACH2 gene expression is highly sensitive to DNA damage in aged mice. This concept led us to investigate the variation in BACH2 and also PRDM1 expression in major lymphocyte subsets with age.MethodsLymphocyte subsets from 60 healthy donors, aged from 20 to 90 years, and 41 untreated chronic lymphocytic leukemia patients were studied. BACH2 and PRDM1 gene expression was analyzed by real-time quantitative PCR. BACH2 gene expression was correlated with its protein expression. Lymphocyte apoptosis was evaluated after intracellular oxidative stress-inducing etoposide treatment of T and B cells.ResultsOur analysis shows BACH2 mRNA downregulation with age in healthy donor CD4+, CD8+ T-cells and CD19+ B-cells. Decreased BACH2 expression was also correlated with an age-related reduction in CD8 + CD28+ T-cells. We found a strong correlation between age-related BACH2 downregulation and decreased CD4+ T-cell and CD19+ B-cell apoptosis. PRDM1, as expected, was significantly upregulated in CD4+ T-cells, CD8+ T-cells and CD19+ B-cells, and inversely correlated with BACH2. A comparison of untreated chronic lymphocytic leukemia patients with age-matched healthy donors reveals that BACH2 mRNA expression was further reduced in CD4+ T-cells, CD8+ T-cells and leukemic-B cells. PRDM1 gene expression was consequently significantly upregulated in CD4+ and CD8+ T-cells in chronic lymphocytic leukemia patients but not in their leukemic B-cells.ConclusionOverall, our data suggest that BACH2 and PRDM1 genes are significantly correlated with age in human immune cells and may be involved in immunosenescence.

Factors associated with dimethyl fumarate-induced lymphopenia

BackgroundLymphopenia is a major concern in MS patients treated with dimethyl-fumarate (DMF) as it increases the risk of progressive multifocal leukoencephalopathy. ObjectiveTo identify factors associated with lymphopenia in DMF-treated patients and explore changes in blood lymphocyte subsets associated with DMF-induced lymphopenia. MethodsProspective longitudinal study including 106 patients initiating DMF treatment followed for a median time of 24.67 months. Blood lymphocyte subsets were studied in 64 patients by flow cytometry at baseline and 6 months after. ResultsMean absolute lymphocyte counts (ALCs) decreased by 29% during the first year of DMF-treatment. Patients developing lymphopenia showed a faster decline within the three first months. A reduction of ALCs higher than 38% at this time was associated to subsequent development of grade 2–3 lymphopenia (OR = 5.93, 95% CI: 1.9–18.6, p = 0.002). All patients showed a significant decrease in different T and B lymphocyte subsets upon DMF therapy. In addition, lymphopenic patients experienced a selective decrease in natural killer T (NKT) cell percentages (p = 0.01), and a high drop in NKT total counts (p < 0.0001). ConclusionsPatients who experience a drop in ALCs by >38% at three months of DMF-treatment are about 6-times more likely to develop significant lymphopenia. This decrease is clearly associated with a considerable loss of NKT cells.

CD38 Expression Levels on Myeloma Cells and the Frequency of Circulating CD38-Positive Treg Cells Are Associated with the Response to Daratumumab in Multiple Myeloma

Background: CD38 is highly expressed on plasma cells and is a good target for multiple myeloma (MM) therapies. Daratumumab (DARA), a humanized antibody to CD38, has emerged as a promising treatment for MM. DARA targets CD38-expressing myeloma and non-plasma cells, including CD38-positive (CD38+) regulatory T (Treg) cells. To investigate the mechanism of action of DARA, we analyzed baseline CD38 expression levels and lymphocyte subsets, including CD38+ Treg cells, before and after DARA treatment.Methods: This study comprised 32 patients with relapsed/refractory MM, who were treated with DARA at our Center and were followed up for more than one cycle of the drug. Peripheral blood and bone marrow samples were analyzed before and during treatment. Using flow cytometric analysis, CD38 expression (mean fluorescence intensity (MFI)) and lymphocyte subsets (CD4/CD8 T cells, natural killer (NK) cells, and Treg cells) were evaluated. Treg cells were identified as a fraction of the CD4+CD25highCD127dim population.Results: The median age of the patients was 78 years (range, 53-92 years). Twenty-two patients (69%) had received more than three prior therapies. Patients received a median of four prior lines of therapy (range, 2-11 prior therapies). All patients were refractory to their last line of therapy, and 89% were refractory to both proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs). Eight patients (25%) received a PI-based DARA-containing regimen, 21 (66%) received an IMiD-based DARA-containing regimen, and 3 (9%) received other DARA-containing regimens. Twenty patients (63%) had a partial response (PR) or better (responders), whereas 12 patients (37%) did not respond to the drug (non-responders).The pretreatment levels of CD38 MFI were significantly higher in responders than in non-responders (p < 0.0005). Before treatment, the absolute number of CD38+ Treg cells was significantly higher in responders (median, 12.3 cells/µL; range, 5.1-37.3 cells/µL) than in non-responders (median, 4.5 cells/μL; range, 1.1-12.8 cells/µL, p = 0.001), but absolute Treg cell numbers were not associated with DARA response. CD38+ Treg cells were depleted in all treated patients, but CD38-negative Treg cell numbers remained relatively stable after DARA treatment. The absolute CD8+ T-cell numbers were significantly increased after DARA treatment (236.1 ± 146.2 vs. 388.6 ± 183.4/μL, p = 0.002). In addition, the absolute HLA-DR+ activated T-cell numbers were also significantly increased (p = 0.002). However, the increased numbers of CD8+ and HLA-DR+ T cells were not associated with clinical responses. The numbers of CD56+ NK cells were immediately depleted in all treated patients. As a control, we also examined the changes in lymphocyte subsets in patients treated with elotuzumab, the first approved monoclonal antibody for MM treatment. However, there was no change in CD38+ Treg and total Treg cell numbers.Next, to investigate the role of CD38+ Treg cells in disease progression, we analyzed the frequencies of Treg cells in healthy volunteers and in patients with monoclonal gammopathy of undetermined significance, smoldering MM, newly diagnosed MM, and relapsed MM. The CD38+ Treg cells were significantly higher in the relapsed MM group than in the other disease or control groups, suggesting that CD38+ Treg cells may play an important role in the progression of MM.Finally, we investigated the CD38 expression and frequency of CD38+ Treg cells in durable responders. Among 20 responders, 13 patients showed a long-lasting response (>6 months) or deepening response (defined as durable responders). The absolute number of CD38+ Treg cells was significantly higher in the durable responders (p = 0.009). On the other hand, the CD38 MFI of myeloma cells was not associated with a durable response.Conclusions: Pretreatment levels of the CD38 MFI could be a predictive marker for the early response to DARA treatment. Moreover, the frequency of CD38+ Treg cells present before treatment could also serve as a durable-response marker. These results suggest that the depletion of CD38+ Treg cells could contribute to some immunological mechanism of DARA. This study provides evidence to support multiple mechanisms of action for DARA, including antibody-dependent cell-mediated cytotoxicity and immunomodulatory effects. DisclosuresNo relevant conflicts of interest to declare.

Effectiveness of Haemophilus influenzae type b vaccination after splenectomy - impact on selected immunological parameters

ABSTRACTSplenectomy is a surgery indicated in case of splenic rupture after injury, when there are tumors in the spleen, or as a treatment for certain diseases, such as idiopathic thrombocytopenic purpura and spherocytosis. The aims of the study were to assess the immunological response to the Haemophilus influenzae type b (Hib) vaccine and the post-vaccination changes in lymphocyte subsets and cell activation markers in splenectomized patients and healthy volunteers. Blood samples were collected from 25 patients that had undergone splenectomy and from 15 healthy, non-splenectomized volunteers. All participants received a single dose of Hib vaccine. The concentration of specific Hib antibodies was assessed by an enzyme-linked immunosorbent assay. Selected immune cell populations were evaluated using flow cytometry. The analysis of the antibody titers against Hib showed statistically significant differences in both groups. There was a significantly higher percentage (p = 0.0012) and absolute value (p = 0.0003) of natural killer T (NKT)-like cells (CD3+/CD16+ CD56+) in the study group, compared to the control group. The levels of natural killer (NK) and NKT cells did not change relative to the cause and age of splenectomy. The quantity and percentage of regulatory T (Treg) cells were higher in the study group compared to the control group (p < 0.0001). No significant correlations were found between the time elapsed since splenectomy, the age of the patients, and the Treg levels. Our study showed that spleen resection results in an important deterioration of Treg cells and Th17 cell balance which may contribute to an incomplete immunological response.

Characterization and Changes of Lymphocyte Subsets in Baricitinib-Treated Patients With Rheumatoid Arthritis: An Integrated Analysis.

ObjectiveBaricitinib is an orally administered inhibitor of JAK1 and JAK2 that has been shown to be effective in treating rheumatoid arthritis (RA). This study was undertaken to analyze changes in lymphocyte cell subsets during baricitinib treatment and to correlate these changes with clinical outcomes.MethodsAn integrated analysis was conducted by pooling data from 3 completed phase III trials comparing placebo with baricitinib treatment (RA‐BEAM, RA‐BUILD, and RA‐BEACON) and 1 ongoing long‐term extension study (RA‐BEYOND) in patients with active RA (n = 2,186).ResultsBaricitinib treatment was associated with an early transient increase in total lymphocyte count at week 4, which returned to baseline by week 12. Transient changes within normal reference ranges in T cells and subsets were observed with baricitinib treatment, up to week 104. B cells and relevant subpopulations increased after 4 weeks of baricitinib treatment, with no further increases noted through 104 weeks of treatment. Natural killer (NK) cells temporarily increased after 4 weeks of baricitinib treatment, before decreasing below baseline levels and then stabilizing over time. With baricitinib treatment, few correlations were observed between changes in lymphocyte subsets and clinical end points, and most correlations were also observed within the placebo group. A modest potential association between low NK cell numbers and treatment‐emergent infections was observed in the baricitinib 4 mg/day treatment group, but not for serious infections or herpes zoster.ConclusionOverall, these findings demonstrate that changes in lymphocyte subsets were largely within normal reference ranges across the baricitinib phase III RA clinical program and were not associated with increased risk of serious infections.

Effect of Stereotactic Ablative Radiation Therapy (SABR) on Peripheral Lymphocyte Subsets in 20 Cancer Patients

Abscopal effect reveals that local radiotherapy enhances systemic antitumor immune response, especially for stereotactic ablative radiotherapy (SABR). And the mechanism remains unclear. In this research, we retrospectively investigated the dynamic change of lymphocyte subsets in the patients treated with SABR. We analyzed the data from 20 cancer patients with I-IV clinic stage who underwent SABR. The tumor types covered urinary system tumor (n=9), non-small cell lung cancer (n=5), hepatobiliary cancer (n=2), head and neck cancer (n=1), soft tissue sarcoma (n=1), ovarian cancer (n=1) and thymic carcinoma (n=1). Twenty-five percent (4 in 20) of the gross tumors were in I-III clinic stage and 75% (16 in 20) were metastatic lesions or relapse lesions. The radiation treatment (RT) delivered SABR combined or not combined with convention fraction radiotherapy(CFRT). All of the 20 patients had peripheral lymphocyte subsets tests at the start of RT (pretreatment or within 1 week after the first radiation). Seventeen of them had the same tests at the end of RT (within 1 week after the last radiation) and 13 did it after RT (in 1-3 months after the last radiation). Paired t test was used to evaluate statistical significances. The median age of 20 patients was 67 (range 40-92). The patients received SABR with 12-70Gy/2-10 fractions (median 24Gy, median 4 fractions) and following convention fraction radiotherapy (CFRT) with 0-52Gy/0-26 fractions (median 45.45Gy, median 20.5 fractions). The biological effective dose was median 100.85 Gy (range 79.20-186.08Gy) in the center of the tumors. In the comparison of the peripheral lymphocyte subsets at the start of RT and the end of RT, we found peripheral T cells, B cells and NK cells were significantly decreased at the end of RT (P =0.000, P =0.000, P =0.002). In contrast, the proportions of CD3+/CD8+ T cells, CD3+/CD4+ T cells and CD4+/CD8+ ratio did not differ at the start of RT and at the end of RT. The results showed that the proportion of CD3+/CD8+ T cells was significantly increased after RT compared with the start of RT (mean 50.60% vs.41.37%, P =0.044). In addition, compared with the start of RT, the proportion of CD3+/CD4+ T cells was significantly decreased (mean 46.15% vs.57.21%, P =0.041) and also the CD4+/CD8+ ratio (mean 1.03 vs.1.53, P =0.045). There were no significant differences in peripheral T cells, B cells and NK cells after RT compared with the start of RT. Increased T cells (P =0.031), increased proportion of CD3+/CD8+ T cells (P =0.043) and decreased proportion of CD3+/CD4+ T cells (P =0.049) were found after RT compared with the end of RT. Stereotactic ablative radiotherapy (SABR) has dynamic impact on the proportion of T cells subsets, particularly CD3+/CD8+ T cells involved. SABR may have a prolonged and effective impact on systemic antitumor immune response.

The clinical importance of changes in Treg and Th17 lymphocyte subsets in splenectomized patients after spleen injury.

Splenectomized patients are more prone to bacterial infections due to their immunocompromised status. Little is known about the role of T helper 17 (Th17) and T regulatory cells (Treg) in the immune system of patients after the removal of the spleen. The aim of the present study was to analyze possible changes in CD4+ lymphocyte T subsets, especially Treg and Th17, in patients who had undergone splenectomy. The study included a group of 67 male patients (41.74 ±16.22 years). All patients had undergone splenectomy because of spleen injury. Mean time elapsed from splenectomy to analysis was 9.1 ±4.6 years. Control samples were obtained from 20 male healthy volunteers. The percentages and absolute counts of Th17 and Treg were measured using the flow cytometry method. The analysis of the antibody titer against 23 serotypes of Streptococcus pneumoniae (S. pneumoniae) in the splenectomized patients revealed its elevated values compared to controls (p = 0.0016). Higher percentages and absolute counts of Treg cells were found in the splenectomized group vs controls (p < 0.000007). Lower percentages and absolute counts of the Th17 subset were found in the study group vs controls (p < 0.000002 and p < 0.00006, respectively). The Treg cell percentage was positively correlated with the antibody titer against S. pneumoniae (p < 0.02). Th17 cells were reversely correlated with the antibody titer (p < 0.004 and p < 0.001 for absolute counts and percentage values, respectively). The Th17 subset values were significantly lower in the splenectomized patients who reported a higher frequency of upper respiratory tract infections (URTI) (p < 0.0001). No correlations were found between the time elapsed since splenectomy and the Treg or Th17 cell values in the study group. Splenectomy results in an important deterioration of the Treg/Th17 cell balance with a predominance of immunoregulatory Tregs, which can contribute to insufficient immune response to infection.

Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs

While dogs are increasingly being utilized as large-animal models of disease, important features of age-related immunosenescence in the dog have yet to be evaluated due to the lack of defined naïve vs. memory T lymphocyte phenotypes. We therefore performed multi-color flow cytometry on peripheral blood mononuclear cells from young and aged beagles, and determined the differential cytokine production by proposed memory subsets. CD4+ and CD8+ T lymphocytes in aged dogs displayed increased cytokine production, and decreased proliferative capacity. Antibodies targeting CD45RA and CD62L, but less so CD28 or CD44, defined canine cells that consistently exhibited properties of naïve-, central memory-, effector memory-, and terminal effector-like CD4+ and CD8+ T lymphocyte subsets. Older dogs demonstrated decreased frequencies of naïve-like CD4+ and CD8+ T lymphocytes, and an increased frequency of terminal effector-like CD8+ T lymphocytes. Overall findings revealed that aged dogs displayed features of immunosenescence similar to those reported in other species.

Effect of resection of adenoids and tonsillectomy on immune function in children with obstructive sleep apnea hypopnea syndrome

Objective:To remove the immune function of children with obstructive sleep apnea hypopnea syndrome by resection of adenoids and tonsillar. Method: The fasting peripheral venous blood of children with OSAHS and healthy children were collected. The lymphocyte subsets, NK cells and immunoglobulin levels were compared between the two groups. The fasting blood samples of OSAHS children were collected again after 2d, 30d, 60d and 90d after operation. The changes of lymphocyte subsets, NK cells and immunoglobulin levels were observed after operation. Result: Before operation, at the 0.05 test level, it was not yet clear that the levels of IgG, IgM, IgA, NK, CD3+, CD4+ and CD8+ in OSAHS children were significantly different from those in the control group. The levels of IgA, CD3+ and CD4+ decreased significantly in children at 2d and OSAHS after operation, and IgG, IgM, IgA, NK, CD3+ and IgA continued to rise after 60d until ninetieth day after operation. Conclusion: There is no statistical disorder of immune level in children with OSAHS compared with healthy children. The implementation of adenoidectomy and tonsillectomy in children with OSAHS, the level of immunity in 90 days can be restored to the preoperative level.

Pregnancy Outcome, Changes in Lymphocyte Subsets in Peripheral Blood, and Plasma Osteopontin in Japanese Patients with Multiple Sclerosis after Assisted Reproductive Technology (P4.369)

Pregnancy Outcome, Changes in Lymphocyte Subsets in Peripheral Blood, and Plasma Osteopontin in Japanese Patients with Multiple Sclerosis after Assisted Reproductive Technology (P4.369)

Differential Dynamics of HIV Infection in Humanized MISTRG versus MITRG Mice

Abstract Humanized mice are a powerful tool to study HIV in vivo. The recently generated mouse strains MITRG and MISTRG, which differ in human SIRPα expression, support an improved human myeloid lineage development from human hematopoietic stem and progenitor cells. The rationale of the study was the characterization of the two mouse strains during an HIV infection with CCR5- and CXCR4-tropic viruses. Upon HIV infection, we observed HIV dissemination and sustained viral load over 20 wk in peripheral blood in both reconstituted mouse strains. However, HIV RNA levels were significantly lower in MITRG mice compared with MISTRG mice during the first 8 wk postinfection. HIV-infected MISTRG mice showed lymphocyte activation and changes in lymphocyte subsets in blood and spleen, recapitulating hallmarks of HIV infection in humans. Depletion of murine tissue-resident macrophages in MITRG mice led to significantly elevated viral loads, and lymphocyte levels were similar to those in HIV-infected MISTRG mice. Depletion of CD8+ T cells in MISTRG mice before HIV infection resulted in substantially decreased CD4+ T cell levels, indicating functionality of human CD8+ T cells; depletion of CD4+CD8+ thymocytes may have contributed, in part, to the latter finding. In summary, MITRG and MISTRG mice represent novel HIV mouse models, despite differential HIV dynamics.

Serial changes in lymphocyte subsets in patients with newly diagnosed high grade astrocytomas treated with standard radiation and temozolomide.

The immune system plays a significant role in cancer prevention and outcome. In high grade astrocytomas (HGA), severe lymphopenia is associated with shortened survival due to tumor progression. This study was performed to quantify serial changes in lymphocyte subsets in HGA following standard radiation (RT) and temozolomide (TMZ). Adults (KPS >60, HIV negative) with newly diagnosed HGA scheduled to receive concurrent RT and TMZ and adjuvant TMZ were eligible. Blood was collected before beginning concurrent RT/TMZ and at weeks 6, 10, 18, and 26, and 3 months after completing adjuvant TMZ. Lymphocyte subsets were analyzed by flow cytometry. Twenty patients (70% glioblastoma, median age 53, 50% male, 80% Caucasian) who enrolled from January 2014 to August 2014 were followed until April 2016. Baseline dexamethasone dose was 0.5mg/day and 15% had absolute lymphocyte counts (ALC) <1000 cells/mm3 before starting RT/TMZ. However, 75% developed lymphopenia with ALC <1000 cells/mm3 after completion of RT/TMZ. NK cells, B cells and all T lymphocytes subsets dropped significantly after concurrent RT/TMZ and remained depressed for the 48 weeks of observation. The CD4+/CD8+ ratio was not affected significantly during follow-up. Severe lymphopenia involving all subsets occurred early in treatment and remained present for nearly 1 year. To our knowledge, this is the first report of serial trends in lymphocyte subsets following standard RT and TMZ for HGA.

Abstract 1687: Systemic immunological changes during first line chemotherapy in patients with high-grade serous ovarian cancer

Abstract Ovarian cancer remains the most lethal gynecological malignancy and new therapeutic strategies are urgently needed. High-grade serous ovarian cancer (HGSC), in particular, is associated with a five-year survival of only 40%. A promising new approach for treating HGSC is immunotherapy, which has resulted in complete and durable responses, albeit in a minority of patients. One potential approach for improving these response rates is by combining chemo- and immunotherapy. Indeed, carboplatin/taxol chemotherapy was shown to augment immune responses in cervical cancer patients by depleting circulating myeloid suppressor cells. As patients with HGSC are treated in first line with similar carboplatin/taxol chemotherapy, we explored the effects of chemotherapy on systemic immunity in HGSC patients. Within this prospective observational study, 75 patients with suspected ovarian cancer were included. Blood was collected at three different time points during first line chemotherapy treatment, namely: prior to chemotherapy, between the third and fourth cycle of chemotherapy and 4-6 weeks after the sixth cycle of chemotherapy. All patients received 6 cycles of carboplatin/taxol chemotherapy and cytoreductive surgery (either as primary debulking, or after 3 cycles of neo-adjuvant chemotherapy). Peripheral blood mononuclear cells were isolated and analyzed for a total of 36 immune markers using 9 flow cytometry panels, in total analyzing 49 immune cell subsets. Results were compared to an age-matched cohort consisting of women surgically treated for a benign disease, and a cohort of healthy young volunteers. 75 patients have been included so far from which 22 were diagnosed with benign disease, 3 were diagnosed with another malignancy, and 50 were diagnosed with OC. Of the 50 OC patients, 18 were diagnosed with HGSC and from 9 HGSC patients, multiple time points were available for analysis of chemotherapy-dependent changes in the immune cell subsets. All patients developed leukopenia as a result of chemotherapeutic treatment. Age-related changes in lymphocyte and myeloid cell subsets were observed in all HGSC patients and patients with benign disease. Chemotherapy-dependent depletion of myeloid cells was observed in a subset of patients. Depletion of myeloid subsets was equally distributed among monocytes, macrophages and dendritic cells. No HGSC- or chemotherapy-dependent changes in T cell subsets or migration and activation markers were observed. Taken together, we observed no major systemic changes in immune cell subsets during carboplatin/taxol chemotherapy treatment, suggesting that combined chemo-immunotherapeutic strategies could be feasible during first line treatment of HGSC. Citation Format: Fenne L. Komdeur, Florine A. Eggink, Ninke Leffers, Hagma H. Workel, Kim L. Brunekreeft, Annechien Plat, Marco de Bruyn, Hans W. Nijman. Systemic immunological changes during first line chemotherapy in patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1687. doi:10.1158/1538-7445.AM2017-1687

Changes of peripheral lymphocyte subsets and cytokine environment during aging and deteriorating gastrointestinal tract health status.

Human immune senescence accompanies with the physical and physiological frailty. The functional change and shift of NK, NKT and T cell subsets by aging have been widely studied. However, it remains largely unclear how the aging and disease conditions affect the distribution of lymphocytes. In the present study, 233 subjects with age range from 20 to 87 year old, including healthy people, people with chronic gastrointestinal tract disease or cancers were investigated. We found that the proportion of NK cells, CD8+ T cells and NKT cells remained relatively unchanged with aging. However, NKG2D and CD16 expression level on NK cells decreased with aging indicating impaired NK cell function. Surprisingly, the proportion of NK, NKT and T cells all declined with deteriorating health status from health to chronic gastrointestinal tract disease and cancer. Furthermore, cytokine and chemokine profiles changed with aging, but did not vary with different health status. Our results highlight new evidence for a continuum of change during immunologic aging and show unique data for variations of NK cells, CD8+ T cells, NKT cells, and cytokine microenvironment with human aging and health status transformation.

Evidence of bone marrow downregulation in brain-dead rats.

Experimental findings support the evidence of a persistent leucopenia triggered by brain death (BD). This study aimed to investigate leucocyte behaviour in bone marrow and blood after BD in rats. BD was induced using intracranial balloon catheter inflation. Sham-operated (SH) rats were trepanned only. Thereafter bone marrow cells were harvested every six hours from the femoral cavity and used for total and differential counts. They were analysed further by flow cytometry to characterize lymphocyte subsets, granulocyte adhesion molecules expression and apoptosis/necrosis [annexin V/propidium iodide (PI) protocol]. BD rats exhibited a reduction in bone marrow cells due to a reduction in lymphocytes (40%) and segmented cells (45%). Bone marrow lymphocyte subsets were similar in BD and SH rats (CD3, P=0.1; CD4, P=0.4; CD3/CD4, P=0.4; CD5, P=0.4, CD3/CD5, P=0.2; CD8, P=0.8). Expression of L-selectin and beta2 -integrins on granulocytes did not differ (CD11a, P=0.9; CD11b/c, P=0.7; CD62L, P=0.1). There were no differences in the percentage of apoptosis and necrosis (Annexin V, P=0.73; PI, P=0.21; Annexin V/PI, P=0.29). In conclusion, data presented suggest that the downregulation of the bone marrow is triggered by brain death itself, and it is not related to changes in lymphocyte subsets, granulocyte adhesion molecules expression or apoptosis and necrosis.

Circulating immune-profile as predictor of outcome in advanced NSCLC patients treated with nivolumab.

e14514 Background: Detection of predictive markers of anti-PD-1/PD-L1 antibodies activity is of pivotal interest in non-small cell lung cancer (NSCLC). This study aimed to identify a circulating immuno-profile as predictor of outcome in NSCLC patients treated with nivolumab Methods: A peripheral blood immuno-profile evaluation was performed at baseline (T0), after 2 (T1) and 4 cycles (T2) of bi-weekly nivolumab in advanced pre-treated NSCLC patients from two Italian Institutions. First tumor assessment was performed after 4 cycles and then every 2 months. FACS analysis of lymphocyte subpopulations [CD3, CD4, CD8, NK (CD56), Treg (FOXP3) and MDSC] was performed. Absolute and % changes of lymphocyte subsets together with their functional and proliferative activity were assessed. Quali-quantitative leucocyte composition at baseline and its variation during therapy were correlated with tumor response and survival. Results: In the overall population of 54 treated patients, baseline Neutrophil-to-Lymphocyte ratio and NK count, lymphocyte count and CD4 variations during therapy showed a statistically significant prognostic role (p &lt; 0.001; p = 0.012; p &lt; 0.001; p = 0.010, respectively). Among 31 patients (squamous carcinoma, n = 17; adenocarcinoma, n = 14) in which all 3 time-points samples were available, 19 were responders (response and stable disease) and 12 non-responders. In responders, absolute numbers of total NK and NKCD56dim subset were higher at baseline and their increase between T0 and T1 was statistically significant (p &lt; 0.05). Responders also displayed increased cytotoxic capability as shown by a higher baseline expression of CD3ζ, perforin and granzyme in NKCD56dim subset. No significant variation was documented in absolute number and functional activity of CD4+ and CD8+ lymphocytes. A higher percentage of CD8+PD-1+ cells at baseline was observed in responders, while non-responders showed a statistically significant increase in the absolute number of MDSC during therapy (p &lt; 0.05). Conclusions: The number and function of NKs and the frequency of PD-1 expression in CD8+ cells could represent predictive peripheral immuno-biomarkers for nivolumab treatment in advanced NSCLC.

Dynamic changes of laboratory parameters and peripheral blood lymphocyte subsets in severe fever with thrombocytopenia syndrome patients

ObjectivesThe aim of this study was to dynamically investigate laboratory parameters and peripheral blood lymphocyte subsets in severe fever with thrombocytopenia syndrome (SFTS) patients at different stages, to evaluate the significance of these changes in the infection process and its influence on prognosis. MethodsCase-control study was used in the research. Sixty-nine confirmed thrombocytopenia syndrome virus(SFTSV) infected patients were enrolled. They were divided into two groups, recovery group and poor prognosis group, according to the clinical prognosis of the diseases. The laboratory parameters were measured by matched fully-automatic detector. The dynamic lymphocyte subsets of each group were tested by flow cytometry. Independent-group Student’s t-test, Bonferroni test and Nemenyi test were used to compare the mean value of every group. ResultsThe clinical manifestations typically became worse on about the 7th day. Most of them had multi organ dysfunction, and part of them had hemophagocytic lymphohistiocytosis histiocytosis (HLH). The characteristic laboratory findings in the early stage were the drop of platelets (PLT), while the increase of alanine aminotransferase (ALT), aspartate amino transferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH). SFTSV viral loads reached the highest on Days 7–10 after onset of fever in SFTS patients. CD3+, CD3+CD4+ T cell counts were significantly reduced in poor prognosis group, more so on Days 7–10 after onset of fever. CD3–CD19+ (B cell) counts in SFTS patients were significantly higher than that of healthy controls. 11 days after illness onset, symptoms were improved, accompanied by resolution of laboratory abnormalities. ConclusionsThese results indicated that SFTS had an acute onset and self-limited course. It was a systemic infection. The host immune response caused tissues and organs injury. The improvement of symptoms and laboratory tests was consistent with the elimination of the virus and recover of immune response. Further investigation should be done in order to reveal the mechanisms of SFTSV pathogenesis and guide the clinical treatment.

Changes in hematological indices and lymphocyte subsets in response to whole blood donation in healthy male donors

Whole blood donation has immunomodulatory effects, and most of these have been observed at short intervals following blood donation. This study aimed to investigate the impact of whole blood donation on lymphocyte subsets over a typical inter-donation interval. Healthy male subjects were recruited to study changes in complete blood count (CBC) (n = 42) and lymphocyte subsets (n = 16) before and at four intervals up to 106 days following blood donation. Repeated measures ANOVA were used to compare quantitative variables between different visits. Following blood donation, changes in CBC and erythropoietin were as expected. The neutrophil count increased by 11.3% at 8 days (p < .001). Novel changes were observed in lymphocyte subsets as the CD4/CD8 ratio increased by 9.2% (p < .05) at 8 days and 13.7% (p < .05) at 22 days. CD16-56 cells decreased by 16.2% (p < .05) at 8 days. All the subsets had returned to baseline by 106 days. Regression analysis showed that the changes in CD16-56 cells and CD4/CD8 ratio were not significant (Wilk’s lambda = 0.15 and 0.94, respectively) when adjusted for BMI. In conclusion, following whole blood donation, there are transient changes in lymphocyte subsets. The effect of BMI on lymphocyte subsets and the effect of this immunomodulation on the immune response merit further investigation.