Influence of Race, Age and Sex on the Lymphocyte Subsets in Peripheral Blood of Healthy Malaysian Adults

The lymphocyte subsets in the peripheral blood of healthy Malaysian adults (212 subjects, age 18–71 years) were analysed using a flow cytometer FACScan in an effort to establish a reference range for the lymphocyte subsets. The lymphocyte subsets studied were T cells (CD3), B cells (CD19), natural killer (NK) cells (CD3−CD16+/CD56+), helper/inducer cells (CD4), cytotoxic/suppressor cells (CD8) and the helper/suppressor ratio (CD4/CD8). The distributions of T cells, CD4 cells and CD8 cells were symmetric about their means while B cells, NK cells and CD4/CD8 ratio followed a skewed distribution. Differences in race were observed for T cells, NK cells, CD4 cells and CD4/CD8 ratio where the Indians were significantly different from the Malays and the Chinese (higher T cells, CD4 cells and CD4/CD8 ratio and lower NK cells). The B cells were significantly lower in the Chinese than the Malays and the Indians. Age differences were seen only in the Chinese where increased CD4 cells and CD4/CD8 ratio, and decreased CD8 cells were observed. A sex difference was observed only in the Chinese where the CD4/CD8 ratio was significantly higher in females than males.

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Alterations of circulating lymphocyte subsets in patients with colorectal carcinoma

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-03127-8 ◽

2021 ◽

Author(s):

Johanna Waidhauser ◽

Pia Nerlinger ◽

Tim Tobias Arndt ◽

Stefan Schiele ◽

Florian Sommer ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Colorectal Carcinoma ◽

Healthy Volunteers ◽

Nk Cells ◽

Colon Carcinoma ◽

Lymphocyte Subsets ◽

Cd4 Cells ◽

Cd8 Cells ◽

Circulating Lymphocytes

Abstract Introduction Cellular immune response to cancer is known to be of great importance for tumor control. Moreover, solid tumors influence circulating lymphocytes, which has been shown for several types of cancer. In our prospective study we elucidate changes in lymphocyte subsets in patients with colorectal carcinoma compared to healthy volunteers. Methods Flow cytometry was performed at diagnosis of colon carcinoma to analyze B cells, T cells and NK cells including various subtypes of each group. Univariate and multivariate analyses including age, gender, tumor stage, sidedness and microsatellite instability status (MSI) were performed. Results Forty-seven patients and 50 healthy volunteers were included. Median age was 65 years in patients and 43 years in the control group. Univariate analysis revealed lower total lymphocyte counts, lower CD4 + cells, CD8 + cells, B cells and NKs including various of their subsets in patients. In multivariate analysis patients had inferior values of B cells, CD4 + cells and NK cells and various subsets, regardless of age and gender. Naïve, central memory and HLADR + CD8 + cells showed an increase in patients whereas all other altered subsets declined. MSI status had no influence on circulating lymphocytes except for higher effector memory CD8 + cells in MSI-high patients. Localization in the left hemicolon led to higher values of total cytotoxic T cells and various T cell subsets. Conclusion We found significant changes in circulating lymphocyte subsets in colon carcinoma patients, independent of physiological alterations due to gender or age. For some lymphocyte subsets significant differences according to tumor localization or MSI-status could be seen.

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Mezagitamab Induces Immunomodulatory Effect in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

Blood ◽

10.1182/blood-2020-140527 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-9

Author(s):

Michael Abadier ◽

Jose Estevam ◽

Deborah Berg ◽

Eric Robert Fedyk

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

Nk Cells ◽

Peripheral Blood ◽

Immune Cell ◽

Cell Populations ◽

Landscape Changes ◽

Ki 67 ◽

Immune Cell Subsets

Background Mezagitamab is a fully human immunoglobulin (Ig) G1 monoclonal antibody with high affinity to CD38 that depletes tumor cells expressing CD38 by antibody- and complement-dependent cytotoxicity. CD38 is a cell surface molecule that is highly expressed on myeloma cells, plasma cells, plasmablasts, and natural killer (NK) cells, and is induced on activated T cells and other suppressor cells including regulatory T (Tregs) and B (Bregs) cells. Data suggest that immune landscape changes in cancer patients and this may correlate with disease stage and clinical outcome. Monitoring specific immune cell subsets could predict treatment responses since certain cell populations either enhance or attenuate the anti-tumor immune response. Method To monitor the immune landscape changes in RRMM patients we developed a mass cytometry panel that measures 39-biomarkers to identify multiple immune cell subsets, including T cells (naïve, memory, effector, regulatory), B cells (naïve, memory, precursors, plasmablasts, regulatory), NK cells, NKT cells, gamma delta T cells, monocytes (classical, non-classical and intermediate), dendritic cells (mDC; myeloid and pDC; plasmacytoid) and basophils. After a robust analytical method validation, we tested cryopreserved peripheral blood and bone marrow mononuclear cells from 19 RRMM patients who received ≥ 3 prior lines of therapy. Patients were administered 300 or 600 mg SC mezagitamab on a QWx8, Q2Wx8 and then Q4Wx until disease progression schedule (NCT03439280). We compared the percent change in immune cell subsets at baseline versus week 4 and week 16. Results CD38 is expressed at different levels on immune cells and sensitivity to depletion by mezagitamab generally correlates positively with the density of expression. CD38 is expressed at high densities on plasmablasts, Bregs, NK-cells, pDC and basophils at baseline and this was associated with reductions in peripheral blood and bone marrow (plasmablasts, 95%, Bregs, 90%, NK-cells, 50%, pDC, 55% and basophils, 40%) at week 4 post treatment. In contrast, no changes occurred in the level of total T-cells and B-cells, which is consistent with low expression of CD38 on most cells of these large populations. Among the insensitive cell types, remaining NK-cells acquired an activated, proliferative and effector phenotype. We observed 60-150% increase in activation (CD69, HLA-DR), 110-200% increase in proliferation (Ki-67), and 40-375% increase in effector (IFN-γ) markers in peripheral blood and bone marrow. Importantly, NK-cells which did not express detectable CD38, also exhibited a similar phenotype possibly by a mechanism independent of CD38. Consistent with these data, the remaining CD4 and CD8 T-cell populations exhibited an activated effector phenotype as observed by 40-200% increase in activation, 60-200% increase in proliferation and 40-90% increase in effector markers in peripheral blood. A potential explanation for this acquisition of activated effector phenotypes could be a reduction in suppressive regulatory lymphocytes. Next, we measured levels of Tregs and Bregs, and observed that Bregs which are CD24hiCD38hi were reduced to 60-90% in peripheral blood and bone marrow. In contrast, total Tregs were reduced by only 5-25% because CD38 expression in Tregs appears as a spectrum where only ~10-20% are CD38+, and thus CD38+ Tregs were reduced more significantly (45-75%), reflecting the selectively of mezagitamab to cells expressing high levels of CD38. CD38+ Tregs are induced in RRMM patients, thus we looked at the phenotype of CD38-, CD38mid, and CD38high -expressing Tregs. We observed higher level of markers that correlate with highly suppressive Tregs such as Granzyme B, Ki-67, CTLA-4 and PD-1 in CD38high Tregs. Accordingly, the total Treg population exhibited a less active phenotype after exposure to mezagitamab, which selectively depleted the highly suppressive CD38+ Tregs. Conclusions Chronic treatment with mezagitamab is immunomodulatory in patients with RRMM, which is associated with reductions in tumor burden, subpopulations of B and T regulatory cells, and characterized by conventional NK and T cells exhibiting an activated, proliferative and effector phenotype. The immune landscape changes observed is consistent with the immunologic concept of converting the tumor microenvironment from cold-to-hot and highlights a key mechanistic effect of mezagitamab. Disclosures Berg: Takeda Pharmaceuticals Inc: Current Employment.

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Increased Expression of PD-1 on CD4+ T Cells from Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v112.11.4154.4154 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4154-4154

Author(s):

Mary M Sartor ◽

David J Gottlieb

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Nk Cells ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Effector Memory ◽

Cd4 Cells ◽

Normal Peripheral Blood

Abstract Although the predominant finding in patients with chronic lymphocytic leukemia (CLL) is an expansion of monoclonal B lymphocytes, a polyclonal expansion of T cells co-exists in CLL patients. Allogenic stem cell transplants for CLL suggest that a significant graft versus leukaemia effect mediated through recognition of minor MHC or leukaemia specific antigens can be achieved. Since it appears that the immune system and probably T cells recognise CLL cells, it is possible that one or more T cell defects might contribute to the initiation or maintenance of a clone of CLL lymphocytes. PD-1 is a coinhibitory molecule that is expressed on T cells in patients with chronic viral infections. It has been suggested that PD-1 expression might be a marker of cell exhaustion due to antigenic overstimulation. We examined the expression of PD-1 and its naturally occurring ligands PD-L1 and PD-L2 on both B and T cells in patients with CLL and compared this with expression on normal peripheral blood mononuclear cells. We found that PD-1 was expressed on over 10% of CD4+ T cells in 7 of 9 cases of CLL (mean 22±16%) but not on CD4+ T cells in any of 9 normal donors (mean 0±0%), p=0.0009. There was no difference in PD-1 expression on CD8+ or CD14+ PBMCs from CLL patients and normal donors (for CD8+ 24±21% and 19±16% for CLL and normals; for CD14+ 58±16% and 71±31% for CLL and normals). More than 10% of CD5+/19+ CLL cells expressed PD-1 in 7 of 10 cases (mean 18±18%) while more than 10% of normal B cells from 6 of 7 donors also expressed PD-1 (mean 49±30%). We examined the expression of PD-1 on naïve, central memory, effector memory and terminally differentiated subsets of CD4+ cells (CD62L+CD45RA+, CD62L+CD45RA−, CD62L−CD45RA− and CD62L−CD45RA+ respectively) from CLL patients and normal donors. The expression of PD-1 was higher on CD4+ cells from CLL patients in all subsets. The effect was most prominent in the effector memory subset (mean 54±4% for CLL patients versus 26±17% for normal donors, p=0.02). We looked for expression of PD-L1 and PD-L2 on T cells, B cells, monocytes and NK cells from CLL patients and normal donors. PD-L1 was only expressed on monocytes (mean 30±23%) and NK cells (mean 14±19%) from CLL patients and on monocytes from normal donors (mean 35±26%). There was no expression of PD-L2 on any cell type in either CLL patients or normal donors. We conclude that there is increased expression of the co-inhibitory molecule PD-1 on CD4+ T cells in patients with CLL. Ligation of PD-1 by PD-L1 expressed on monocytes or NK cells could inhibit immune responses to tumor and infectious antigens leading to persistence of clonally expanded cells and predisposition to opportunistic pathogens.

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Treatment and biology of lymphomatoid granulomatosis

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.8029 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 8029-8029 ◽

Cited By ~ 1

Author(s):

B. R. Healey Bird ◽

N. Grant ◽

K. Dunleavy ◽

J. Janik ◽

J. Cohen ◽

...

Keyword(s):

B Cells ◽

Lymphocyte Subsets ◽

High Dose ◽

Cd4 Cells ◽

Cd8 Cells ◽

Viral Loads ◽

The Mean ◽

Male Sex ◽

Age Range ◽

Grade Iii

8029 Background: LYG is a rare angiocentric-destructive process with EBV+ B-cells and reactive T-cells. LYG is graded with grades I-II showing rare-moderate large EBV+ B-cells (usually polyclonal or oligoclonal) and grade III showing numerous large EBV+ B-cells (usually monoclonal), likely reflecting progressive transformation. Historically, steroids and/or chemotherapy have a 14 mos median survival. Methods: We are investigating Interferon-a (I-a) for grade I/II and dose-adjusted EPOCH ±Rituximab (R) for grade III LYG. Results: Characteristics of 53 pts are: male sex 68%; median age (range) 46 (17–67) and median ECOG P.S. 1 (0–3). Disease sites include lung 98%, CNS 38%, kidney 15%, skin 17%, liver 19% and nodes 4%. On study LYG grades are I-30%, II-26% and III-44%. Prior treatment was none-28%, chemotherapy± R-34%, and steroids alone-40% of pts. For grades I/II, I-a is begun at 7.5 million IUs TIW and escalated as tolerated until disease regression and continued 1 yr after CR. Of 31 patients treated with I-a, PFS is 62% at the median f/u of 5.3 yrs. Of 25 evaluable pts (3 NE; 3 TE), 60% had sustained CR for a median of 60 mos (4–175). In 9 pts who progressed on I-a, grade III was found in 5. Thus, in 20 pts with only grade I/II, 75% had sustained CR with I-a. In 11 evaluable pts with CNS disease, 81% achieved remission with I-a alone. The median time to remission is 9 mos (3–40) and median I-a dose is 20 MIU (7–40). Among 24 pts receiving DA-EPOCH±R, PFS is 40% at the median f/u of 28 mos. Of 21 evaluable pts (2 NE, 1 TE), 66% achieved CR. OS of all 53 pts is 68% at the median f/u of 4 yrs. Median EBV viral loads in 29 pts at study entry were 18 copies/10e6 genome equivalents (0–22727) (normal<200). Lymphocyte subsets in 30 pts showed a median CD4–428 (24–2322) and CD8–165 cells/mm3 (42–1316). In 12 pts in CR and with serial values, the mean CD8 cells (131 ± 44) (p2= 0.013) but not CD4 cells (65 ± 75) increased with treatment. Conclusions: High dose I-a produces sustained remissions in grade I/II LYG and is effective in CNS LYG. DA-EPOCH±R can produce durable CRs in grade III LYG. We hypothesize LYG emerges in a compromised immune milieu and undergoes progressive transformation if not effectively treated. Historical results suggest steroids may allow transformation by compromising immune function. No significant financial relationships to disclose.

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Clinical Features and Lymphocyte Subsets in Recovered Covid-19 Patients With Prolonged Viral Rna Shedding Duration

10.21203/rs.3.rs-33073/v1 ◽

2020 ◽

Author(s):

Wei-yun Zhang ◽

Kai Wu ◽

Ying-ying Liu ◽

Chang-guo Wang ◽

Jian-an Huang ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Clinical Features ◽

Poor Prognosis ◽

Lymphocyte Subsets ◽

Viral Rna ◽

Viral Shedding ◽

Novel Coronavirus ◽

Immunological Dysfunction

Abstract Background:The 2019 novel coronavirus disease (COVID-19) spread in many countries.Data about viral shedding duration, particularly the prolonged ones of the pathogen SARS-Coronavirus-2 (SARS-CoV-2) is scarce. The longest viral RNA sheddingduration reported previously was 37 days. Herein, we report the clinical and immunologic features ofrecovered COVID-19cases with a medium viral RNA shedding duration of 44 days. Cases presentation: Nine laboratory-confirmed COVID-19 cases from Wuhan with viral RNA shedding duration more than 30 days were included in our study,5 of them were moderate.Althoughinflammatory markers were significantlyhigher, the medium duration in severepatients was similar to that in moderate patients (44.5days vs. 43.6days). Severepatients showed higher NK cells levels, although the T cells and B cells were lower as compared with moderate patients. Contrary to previous reports in influenza, prolonged viralshedding time did not cause poor prognosis in this study.Conclusions: There could be characteristic immunological dysfunction in COVID-19 patients with prolonged viral shedding durationand interestingly, prolonged viral shedding duration seemed not to be related with poor prognosis.

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Circulating cytokines and lymphocyte subsets in patients who have recovered from COVID-19

10.1101/2020.07.22.20160259 ◽

2020 ◽

Author(s):

Hasi Chaolu ◽

Xinri Zhang ◽

Xin Li ◽

Dongyan Li

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Lymphocyte Subsets ◽

Healthy Controls ◽

Tnf Α ◽

Ifn Γ ◽

Circulating Cytokines

To investigate the immune status of people who previously had COVID-19 infections, we recruited patients 2 weeks post-recovery and analyzed circulating cytokines and lymphocyte subsets. We measured levels of total lymphocytes, CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK cells, and the serum concentrations of interleukin (IL)-1, IL-4, IL-6, IL-8, IL-10, transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) by flow cytometry. We found that in most post-recovery patients, levels of total lymphocytes (66.67%), CD3+ T cells (54.55%), CD4+ T cells (54.55%), CD8 + T cells (81.82%), CD19+ B cells (69.70%), and CD56+ NK cells(51.52%) remained lower than normal, whereas most patients showed normal levels of IL-2 (100%), IL-4 (80.88%), IL-6 (79.41%), IL-10 (98.53%), TNF-α (89.71%), IFN-γ (100%) and IL-17 (97.06%). Compared to healthy controls, 2-week post-recovery patients had significantly lower absolute numbers of total lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells, along with significantly higher levels of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17. Among post-recovery patients, T cells, particularly CD4+ T cells, were positively correlated with CD19+ B cell counts. Additionally, CD8+ T cells positively correlated with CD4+ T cells and IL-2 levels, and IL-6 positively correlated with TNF-α and IFN-γ. These correlations were not observed in healthy controls. By ROC curve analysis, post-recovery decreases in lymphocyte subsets and increases in cytokines were identified as independent predictors of rehabilitation efficacy. These findings indicate that the immune system has gradually recovered following COVID-19 infection; however, the sustained hyper-inflammatory response for more than 14 days suggests a need to continue medical observation following discharge from the hospital. Longitudinal studies of a larger cohort of recovered patients are needed to fully understand the consequences of the infection.

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Circulating immune markers predict the therapeutic effect in primary lung cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e21203 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e21203-e21203

Author(s):

Liangliang Xu ◽

Jitian Zhang ◽

Li Yang ◽

Guangqiang Shao ◽

Taiyang Liuru ◽

...

Keyword(s):

Lung Cancer ◽

T Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Blood Lymphocyte ◽

Lymphocyte Subpopulations ◽

Significant Difference

e21203 Background: Radiotherapy (RT), surgical resection (SR), and immunotherapy (IT) as main therapies in lung cancer have either suppressive or stimulatory effects on the immune system. It’s still unclear the mechanism involved in the systemic changes of immune cells in the blood. Peripheral blood lymphocyte subpopulations were useful markers for evaluating immune response in tumor patients. Hence, we aimed to systematically investigate the alteration of lymphocyte subpopulations during the local therapies to evaluate antitumor treatment effects. Methods: Blood samples were obtained EDTA coated tubes and then centrifuged gently for white blood cell separation. The white blood cells in 10% DMSO and 90% FBS were frozen slowly in -80°C refrigerator. The following fluorochrome-conjugated surface and nuclear antibodies were used in the lymphocyte subtyping: CD11b, CD45, CD19, CD3, CD56, CD4, CD8a, CD25,CD127 and FOXP3. The staining cells were detected in the BD FACS machine and data were analyzed by the paired T-test. The percentage of Lymphocytes, Myeloid cells, B cells, T cells, Treg, CD8+ T cells, CD4+ T cells, NK cells, and NKT were examined. Results: Between July 2019 and January 2020, a total of 176 patients eligible, including 135 RT patients and 29 SR patients,12 IT patients, with both blood collection with both Pre, During and End therapies. Before local therapies, the percentage of total T cells in the RT group was significantly higher than SR (RT v.s SR mean:64.1 v.s 55.3, P = 0.02) while CD8+ T cells (RT v.s SR mean:28.2 v.s 34.5, P = 0.04)and Tregs (RT v.s SR mean:0.0 v.s 0.1, P = 0.055) were lower. The baseline level of T cells and their subtypes showed a significant difference in these two group patients. After local therapies, myeloid cells, lymphocytes, CD4+ T cells, CD8+ T cells, NK cells were significant different. There is no significant difference due to the smaller number of IT patients. In the RT group, lymphocytes (Pre-RT v.s End-RT mean:75.2 v.s 54.3, P = 0.004) and B cells (Pre-RT v.s End-RT mean:12.6 v.s 8.0, P = 0.03) were significantly decreased while other subpopulations didn’t show any significant difference after RT. Interestingly, in the SR group, there was a significant increase in CD4+ T cells (mean:59.0 v.s 62.1, p = 0.02) a trend of reduction in CD8+ T cells (mean:34.5 v.s 32.0, p = 0.055) after SR. In addition, there was an increased trend of Tregs after IT. Conclusions: There are some different patterns of distribution in subtypes of leukocytes in operable and inoperable patients and between different therapies. All RT, SR and IT changed the distribution of peripheral blood lymphocyte subpopulations. Further validation study is warranted to validate our findings particularly in circulating lymphocytes and B cells as a marker to evaluate immune status after RT, CD4+ T cells and CD8+ T cells after SR, Tregs after IT, as well as their relationship with tumor microenvironment and implication for personalized care.

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The Feature of δRec-ψJα sjTRECs Level and Frequency of 23 TCR Vβ-Dβ1 sjTRECs in Mononuclear Cells, CD4+ and CD8+ T Cells from Cord Blood and Peripheral Blood of Normal Individuals.

Blood ◽

10.1182/blood.v108.11.3873.3873 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3873-3873

Author(s):

Yangqiu Li ◽

Qingsong Yin ◽

Shaohua Chen ◽

Lijian Yang ◽

Grzegorz Przybylski ◽

...

Keyword(s):

T Cells ◽

Quantitative Analysis ◽

Cord Blood ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Output Function ◽

Cd4 Cells ◽

Cd8 Cells ◽

Normal Individuals

Abstract Thymic recent output function is characterized its importance of thymus to T-cell diversity in the periphery of both children and adults. The generation of TCR diversity occurs in the thymus through recombination of gene segments encoding the variable parts of the TCR α and β chains. During these processes, by-products of the rearrangements are generated in the form of signal joint T-cell receptor excision circles (sjTRECs), which is considered as a very valuable tool to estimate thymic function. Quantitative of δRec-ψJα sjTRECs can direct evaluate the recent thymic output function, but it is unable to analyze the particular thymic output function of different TCR Vβ subfamily naive T cells. The complexity of TCR Vβ repertoire is an important factor for immune reconstitution, quantitative analysis of series TCR Vβ-Dβ sjTRECs could be used to evaluate the levels of different Vβ subfamily naive T cells. In the present study, quantitative analysis of δRec-ψJα sjTRECs was performed in mononuclear cells, CD3+, CD4+ and CD8+T cells from peripheral blood of normal individuals and cord blood by real-time PCR(TaqMan). And the analysis of 23 TCR Vβ-Dβ1 sjTRECs was performed by semi-nested PCR. Different amounts of DNA (corresponding to 2*105, 5*104, 1*104 and 1*103 cells respectively) from all samples were amplified to estimate the frequency of TCR Vβ-Dβ sjTRECs. The mean value of δRec-ψJα sjTRECs was detected in 4.10±3.65/1000 PBMCs, 6.37±5.28/1000 CD3+cells, 3.28±1.24/1000 CD4+cells, 4.67±3.63/1000 CD8+cells from normal individuals (n=14) and 35.59±47.56/1000 CBMC, 71.48±86.42/1000 CD3+cells, 41.02±32.9/1000 CD4+ cells, 52.05±52.32/1000 CD8+cells from cord blood (n=9) (p=0.0208, p=0.0096, p=0.0003, p=0.0026, respectively). A part of Vβ subfamily sjTRECs could be detected in all samples from cord blood (Vβ2, 3, 4, 5, 10, 13, 14, 15, 19 and 22) and peripheral blood (Vβ10, 13 and 14) at 5*104 cells level, some of Vβ subfamily sjTRECs could be detected in 1*103 cells level. The frequencies of 23 Vβ-Dβ1 sjTRECs were different at the same cellular concentration. The number of detectable Vβ subfamily sjTRECs was 22.00±0.94/2×105, 18.8±1.87/5×104, 10.40±2.99/1×104 and 0.78±1.39/1×103 CBMCs, as compared with 18.70±2.45/2×105 (p=0.002), 13.7±2.67/5×104 (p<0.001), 5.5±2.07/1×104 (p=0.001) and 0.50±0.71/1×103 (p=0.739) in PBMCs from normal individuals. Similar results were found in CD4+ and CD8+ T cells which were sorted from both CBMCs and PBMCs, the number of detectable Vβ subfamily sjTRECs was 13.90±2.38/1×104 CD4+cells, 11.5±1.96/1×104CD8+cells from cord blood and 5.6±2.68/1×104 CD4+cells (p<0.001) and 8.2±2.57/1×104CD8+cells (p>0.005) from normal individuals. The results indicate that the number of detectable sjTRECs of Vβ subfamilies and the frequencies of most Vβ-Dβ1 sjTRECs in normal PBMCs, CD4+ and CD8+T cells were obviously lower than those in cord blood. In conclusions, the results provide the base data of naïve T cells levels and thymic recent output function in cord blood and peripheral blood of normail individuals in chinese.

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Number of PD-1+/CD8+ Cells in Peripheral Blood of Patients with Lymphoma Reflects Tumor Burden, Lymphoma Subtype, Disease Phase and Is Significantly Higher Compared to Healthy Volunteers.

Blood ◽

10.1182/blood.v120.21.2670.2670 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2670-2670

Author(s):

Vit Prochazka ◽

Martin Novák ◽

Zuzana Pikalova ◽

Tomas Papajik ◽

Karel Indrak ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Healthy Volunteers ◽

Peripheral Blood ◽

Healthy Subjects ◽

Cd4 Cells ◽

Cd8 Cells ◽

Programmed Death ◽

Programmed Death 1 ◽

Disease Stages

Abstract Abstract 2670 Background: Programmed death-1 (PD-1) and programmed death-1 ligand (PD-L) signaling pathways are involved in the functional impairment and “exhaustion” of cytotoxic CD8+ T cells in conditions such as chronic viral infection and in tumor immune evasion. The interaction of PD-1 with its ligand PD-L suppresses antitumor T cell function and indirectly stimulates Treg population. We investigated a hypothesis of whether examining PD-1 expression in peripheral T cells of patients with different lymphoma subtypes reflects tumor subtype or stage and compared results with healthy volunteers. Methods: Patients were assessed prior to their treatment or at the time of disease relapse or progression. We analyzed 5 patients with HL and 30 patients with NHL (T-cell n=6, diffuse large B-cell n=12, follicular lymphoma n=9, marginal zone lymphoma n=3). Twelve of the patients had relapsed or refractory diseases (B-NHL n=6, T-NHL n=2, HL n=4). Eleven patients (32%) had advanced (III/IV) disease stages. Data were compared with samples obtained from 12 healthy blood donors. Peripheral blood samples were stained with anti-CD3 FITC (Exbio), PD-1 (CD279) PE (BioLegend), anti-CD8 PerCP (Exbio), CD4 APC (Exbio), anti-CD25 FITC (BD), and anti-CD127 PE (BioLegend) using a lyse/no-wash protocol. Stained cells were acquired using the FACSCalibur cytometer (BD). Analysis of immunocompetent subpopulations was performed using the CellQuest Pro (BD) software. PD-1 (CD279) population was gated from CD3-positive T cells; minimal acquisition was designated as 10,000 CD3+ events. The percentage of PD-1+ cells within the live CD3+CD4+ and CD3+CD8+ populations was compared to isotype controls to establish baseline values. Absolute numbers were expressed as number of cells*10exp6 per liter. Population of Tregs was defined as CD4+/CD25int-hi / CD127low cells. Tregs were gated from CD4+ lymphocytes with minimal acquisition of 5,000 CD4+ cells. Results: Proportion of PD-1+/CD8+ of CD3+/CD8+ cells was significantly higher in patients with lymphoma than in healthy subjects: healthy volunteers (HV) 8.8%, B-NHL 16.0% (p=0.02), HL 21.8% (p<0.01), and T-NHL 30.8% (p<0.01). In absolute numbers of PD-1+/CD8+ cells, no significant difference was found when comparing healthy subjects and B-NHL: HV 0.23, B-NHL 0.56 (p=0.21), T-NHL 0.93 (p<0.01), and HL 1.51 (p<0.01). When analyzing the proportion of PD-1+/CD8+ cells according to disease phases, the highest numbers were found in patients with refractory/relapsed lymphoma as compared to patients with untreated disease and healthy subjects: HV 8.8%, untreated 14.6% (p=0.04), and relapsed 28.6% (p<0.01). Untreated patients had a significantly lower proportion of PD-1+/CD8+ cells than relapsed patients (p<0.01). Similar results were obtained with absolute numbers: HV 0.22, untreated 0.55 (p=0.03), and relapsed 1.24 (p=0.03). Untreated vs. relapsed patients p=0.05. Patients with limited disease stages had almost the same proportion of PD-1+/CD8+ lymphocytes compared to HV: HV 8.8%, limited stage 11% (p=0.21), and advanced stage 24.3% (p<0.01). In absolute numbers, HV had much less PD-1+/CD8+ cells in PB: HV 0.22, limited stage 0.49 (p<0.01), and advanced stage 0.97 (p<0.01). When analyzing the population of PD-1+/CD4+ cells, differences were only found in absolute numbers between HV (0.35) and HL (1.34; p<0.01), and between B-NHL (0.54) and HL (p=0.01). Regarding the population of Tregs, statistical differences were found between HV and B-NHL, HL or T-NHL in either relative or absolute numbers. On the other hand, there was a close correlation between absolute numbers of Tregs and PD-1+/CD4+ cells (p<0.01, correlation 0.73), and between Tregs and PD-1+/CD8+ cells (p<0.01, correlation 0.53). Conclusion: PD-1 expression in peripheral blood CD4+ and CD8+ cells is markedly different between lymphoma subtypes and compared with healthy subjects. The highest numbers of PD-1+/CD8+ are in patients with advanced lymphoma and at the time of disease relapse. This fact support the hypothesis that tumor clones actively switch effector CD8+ cells through the PD1L/PD-1 pathway into an immunotolerant state. PD-1 may be a potential marker of systemic immune dysregulation in lymphoma patients and further exploration of T cell subpopulations may define its role as a potential biomarker. Supported by grants: MSM 6198959205, LF-2012-007 and MZ ÈR IGA NT 11103. Disclosures: Prochazka: Roche: Travel grants Other.

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Higher HHV-6 Reactivation After Cord Blood Allo-SCT Is Not Related to HHV-6 Cell Receptor CD46 Expression

Blood ◽

10.1182/blood.v120.21.1931.1931 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1931-1931

Author(s):

Patrice Chevallier ◽

Nelly Robillard ◽

Marina Illiaquer ◽

Julie Esbelin ◽

Mohamad Mohty ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

B Cells ◽

Cord Blood ◽

Nk Cells ◽

B Lymphocytes ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Cell Receptor

Abstract Abstract 1931 Introduction: Cord Blood (CB) are increasingly used as an alternative stem cells source in adults for allogeneic Stem Cell Transplantation (allo-SCT). The risk of human herpes virus (HHV-6) reactivation is significantly higher after CB transplant vs unrelated peripheral blood stem cells (PBSC) allo-SCT (Chevallier et al, BMT 2010). Higher HHV-6 cell receptor CD46 expression on progenitor cells in CB may explain this difference (Thulke et al, Virol J 2006). Patients and Methods: We have prospectively compared the HHV-6 cell receptor CD46 expression on various cell subsets of three freshly harvested blood sources on one hand and of three graft sources on the other hand. 52 samples were used for the purpose of this study. They were issued from peripheral blood (PB, n=10), G-CSF mobilised PB (GCSF-PB, n=10), cord blood (CB, n=10), unmanipulated bone marrow (uBM, n=5), leukapheresis product (LP, n=10) and thawed CB graft (n=7). CD46 expression was assessed by FACS analysis using a FACS CANTO II (BD Biosciences, San Jose, CA, USA) on total lymphocytes, monocytes, NK cells, T and B cells subsets, plasmacytoid (pDCs) dendritic cells and stem cells. Results: As all cell subsets were found CD46 positive, CD46 mean fluorescence intensity (MFI) was then considered for comparison. When considering the three blood sources, CD46 MFI were found similar on T cells, CD4-/CD8+ and CD4-/CD8- T cells, NKT cells, Tregs, memory B lymphocytes, pDCs and CD34+ stem cells. CD46 MFI was significantly lower on CD4+/CD8- and CD4+/CD8+ T cells, transitional B cells, total and naïve B lymphocytes, and NK cells in CB while higher on monocytes. The highest CD46 MFI was observed on monocytes in CB and on CD4+/CD8+ T cells in GCSF-PB and PB. Also, highest CD46 MFI was detected on T cells compared to B lymphocytes and NK cells in all blood sources while CD46 MFI was higher on CD4+/CD8- T cells compared to CD8+/CD4- T cells. When considering the three graft sources, CD46 MFI was similar on CD4-/CD8- T cells and NKT cells. CD46 MFI was found significantly lower on all other sub-populations in thawed CB graft, except monocytes. The highest CD46 MFI was observed on monocytes in CB graft, on CD4+/CD8+ T cells in LP and on monocytes and on CD4+/CD8- T cells in uBM. Also, highest CD46 MFI was detected on T cells compared to B lymphocytes and NK cells in all graft sources while CD46 MFI was higher on CD4+/CD8- T cells compared to CD8+/CD4- T cells. Conclusion: This original study shows strong differences in term of quantitative CD46 expression between several blood and grafts samples. Our results suggest that other factors (such as another HHV-6 cell surface receptor) than the qualitative CD46 expression play a role in the higher HHV-6 reactivation observed after CB transplant in adults. Disclosures: No relevant conflicts of interest to declare.

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