The spatial anatomy of hematopoiesis in the bone marrow (BM) has been extensively studied in mice and other preclinical models, but technical challenges have precluded a commensurate exploration in humans. Institutional pathology archives contain thousands of paraffinized BM core biopsy tissue specimens, providing a rich resource for studying the intact human BM topography in a variety of physiologic states. Thus, we developed an end-to-end pipeline involving multiparameter whole tissue staining, in situ imaging at single-cell resolution, and artificial intelligence-based digital whole slide image analysis and then applied it to a cohort of disease-free samples to survey alterations in the hematopoietic topography associated with aging. Our data indicate heterogeneity in marrow adipose tissue (MAT) content within each age group and an inverse correlation between MAT content and proportions of early myeloid and erythroid precursors, irrespective of age. We identify consistent endosteal and peri vascular positioning of hematopoietic stem and progenitor cells (HSPCs) with medullary localization of more differentiated elements and, importantly, uncover new evidence of aging-associated changes in cellular and vascular morphologies, microarchitectural alterations suggestive of foci with increased lymphocytes, and diminution of a potentially active megakaryocytic niche. Overall, our findings suggest that there is topographic remodeling of human hematopoiesis associated with aging. More generally, we demonstrate the potential to deeply unravel the spatial biology of normal and pathologic human BM states using intact archival tissue specimens.

Objectives To examine В-cell subsets in peripheral blood of Sjogren`s syndrome (SjS) patients (pts) and to analyze the association between B-cell subsets and different clinical and immunological parameters. Methods 67 active SjS pts (65F/2M); mean age 51±14,4 years; disease duration 60 (24-120) months were included. SjS was diagnosed based on the ACR-EULAR2016 criteria. Twenty healthy donors composed the control group. CD19+B cells, memory B-cells (CD19+CD27+), non-switched memory B-cells (CD19+IgD+CD27+), switched memory B-cells (CD19+IgD-CD27+), naïve (CD19+IgD+CD27-), double negative (CD19+IgD-CD27-), transitional (CD19+IgD+CD10+CD38+) B-cells, plasmablasts (CD19+СD38+++IgD-CD27+) were analyzed by multicolor flow cytometry using cytometer Navios (Beckman Coulter, USA). The nonparametric Mann-Whitney test, the Pearson`s x2 criterion, Spearman rank correlation coefficient were used for statistical analysis. Data were shown as median (Me) with an interquartile range of 25 - 75 percentile. The differences were considered statistically significant when p<0.05. Statistica 10 for Windows (StatSoft Inc., USA) package was used for statistical data processing. Results The percentages/absolute numbers of plasmablasts, transitional B-cells, memory B-cells in SjS were significantly higher than in healthy donors, p<0.01 for all cases. At the same time percentages/absolute numbers of naïve cells in SjS were reduced compared to healthy donors (p=0.005, p=0.04). Shorter duration of SjS (less than 2 years) correlated with higher rates of naïve cells (p=0.02). ESSDAI score was ≥5 in 33 pts and <5 in 34 pts. We didn`t find any correlation between B cells subsets and disease activity. The presence of lymphoma in pts (n=5) had no impact on B-cells subsets as well. Significant changes were in B-cells subsets were found depending on immunological parameters. Pts with an isolated increase of antinuclear antibodies (ANA) (ANA positive, RF/aRo/aLa negative) had no plasmablasts in peripheral blood, level of plasmablasts in other pts was 1.5 (1-4.5)x10 3 /µL, р=0,004. ARo-positive pts had higher amount of plasmablasts and transitional B-cells than aRo-negative pts (2 (1-5) and 1 (0-1) x10 3 /µL, p= 0.004). Higher level of plasmablasts and transitional B-cells also correlated with decrease in C3 and/or C4 (p≤0.03) and increase in IgG (p<0.025). Conclusion According to our data elevated level of plasmablasts and transitional B-cells had positive correlation with some immunological diagnostic markers and immunological activity parameters of SjS but didn`t associate with ESSDAI. Disclosure of Interests None declared

Background: B-cells play a pivotal role in primary Sjogren’s syndrome (SS) pathogenesis. Recent studies have shown disturbances in the peripheral B cell populations in primary SS. Objectives: To examine В-cell subsets in peripheral blood of SS patients (pts) and to analyze the association between B-cell subsets and SS activity. Methods: Twenty active SS pts (19F/1M): median age 42 years (range) (32-54); disease duration 3 (2-10) years; ESSDAI score ≥5 in 6 pts, <5 in 14 pts), were included. SS was diagnosed based on the ACR-EULAR 2016 criteria. Twenty healthy donors composed the control group. CD19+B cells, memory B-cells (CD19+CD27+), non-switched memory B-cells (CD19+IgD+CD27+), switched memory B-cells (CD19+IgD-CD27+), naïve (CD19+IgD+CD27-), double negative (CD19+IgD-CD27-), transitional (CD19+IgD+CD10+CD38+) B-cells, plasmablasts (CD19+СD38+++IgD-CD27+), and plasma cells (CD19+СD38+) were analyzed by multicolor flow cytometry using cytometer Navios (Beckman Coulter, USA). The nonparametric Mann-Whitney test, the unpaired Student’s t test for group comparison, and the Pearson`s x2 criterion were used for statistical analysis. Data were shown as median (Me) with an interquartile range of 25 - 75 percentile. The differences were considered statistically significant when p <0.05. Statistica 10 for Windows (StatSoft Inc., USA) package was used for statistical data processing. Results: The percentages/absolute numbers of plasmablasts, plasma cells and transitional B-cells in SS were significantly higher than in healthy donors, р<0.01 for all cases (Table 1). Table 1. B-cell subsets in patients with SS, SS and MALT lymphoma and healthy donors. B-cell subsets SS HD Pt 1 FN % Abs. cells/μl % Abs. cells/μl % Abs. cells/μl B lymphocyte 12,8 (7,9 – 19,7) 151 (95-112) 8,5 (7,2-11,0) 200 (100-200) 4,5 31 ** plasma cells 6,9 (4,2-10) 12 (6-22) 0,1 (0,05-0,1) 0,1 (0-0,2) 5,7 13 * plasmablasts 0,88 (0.37-1.8) 2 (1-3) 0,1 (0,1-0,2) 0,2 (0,1-0,4) 26,6 12 */** transitional B-cells 5.7 (0.9-17) 8 (4-32) 0,1 (0-0,1) 0,1 (0-0,3) 0 0 * switched cells 9.1 (5.9-20.7) 18 (11-29) 12,8 (9,3-17,0) 20 (10-40) 52 16 ** non-switched memory B-cells 4.13 (2.7-5.4) 7 (3-15) 7,4 (3,7-11,1) 10 (5-20) 16 5 ** SS Sjogren’s patients; HD healthy donors, Pt 1 patient with SS and MALT lymphoma * SS and HD groups compared, р<0.01 ** Pt 1 and SS group compared One of the patients (Pt 1) was diagnosed with MALT lymphoma of the parotid salivary gland; she had a distinct B-lymphocyte subpopulations distribution, which was different from the rest of the SS group. She had the lowest percentage / absolute B lymphocyte count of all patients with SS, the highest percentage of switched cells and non-switched memory B-cells, and the highest percentages/absolute numbers of plasmablasts. For this reason, the patient was excluded from the subsequent analysis (Table 1). Patients with activity of SS by ESSDAI ≥ 5 had a significantly higher percentage of B cells (p=0.007), and significantly higher absolute B lymphocyte count of naïve cells (p=0.04) and plasmablasts (p=0.048). Conclusion: Immunophenotyping showed disturbed homeostasis of the B-cells subpopulations in our SS cohort. A significant increase in plasmablasts in SS, as well as a positive correlation of the level of plasmablasts with the SS activity and presence of lymphoma could suggest the important role of these cells in the pathogenesis of SS. Disclosure of Interests: None declared.

Background B-cells play a critical role in the regulation of systemic autoimmune diseases pathogenesis, that extends beyond antibodies production. Objectives To examine B-cells subsets in peripheral blood of patients (pts) with untreated systemic lupus erythematosus (SLE). To analyze the association between B-cell subsets and disease-specific autoantibodies. Methods Thirty six untreated SLE pts (31F/5M) were enrolled. SLE pts had median age of 37 years (range) (32–41), disease duration of 3.5 years (1–11), SLEDAI 2K of 7 (4–8), BILAG of 14,5 (10,2–23). The control group consisted of 29 volunteers (23F/6M, median age 38 [35; 48] years). CD19+B cells, memory B cells (CD19+CD27+), switched memory B cells (CD19+ CD27+IgD-), non-switched memory B cells (CD19+CD27+ IgD+), naive (CD19+CD27-IgD+), double-negative (CD19+CD27-IgD-), transitional (CD19+CD38++CD10+IgD+CD27-) B cells, and plasmablasts (CD19+CD38+++CD27+IgD-CD20-) were analyzed using multicolor flow cytometry. Results The absolute counts of memory B cells (CD19+CD27+), switched memory B cells (CD19+CD27+IgD-), transitional B cells (CD19+CD38++CD10+IgD+CD27-), and plasmablasts (CD19+CD38+++CD27+IgD-CD20-) were higher in untreated pts with SLE compared to healthy donors, p<0,01 for all cases. The absolute counts of double-negative B cells (CD19+CD27-IgD-) were lower in SLE pts than in donors, p=0,03 (Table 1). At significant correlation was found in SLE pts between anti-dsDNA levels and absolute counts of the following B-cell subtypes: CD19+B cells (r=0,72), memory B cells (CD19+CD27+) (r=0,76), switched memory B cells (CD19+ CD27+IgD-) (r=0,75) and naive B cells (CD19+CD27-IgD+) (r=0,73), p<0,01 for all cases. In addition, in SLE pts the a-Sm levels correlated with absolute counts of the switched memory B cells (CD19+ CD27+IgD-) (r=0,98; (r=0,51, p<0,01), antibodies to cardiolipin (aCL) IgG levels with absolute counts of CD19+B cells (r=0,64, p=0,02), and with naive B cells (CD19+CD27-IgD+) (r=0,59, p=0,01). Conclusions Immunophenotyping showed an increase in the absolute counts of memory B cells (CD19+CD27+), switched memory B cells (CD19+CD27+IgD-), transitional B cells (CD19+CD38++CD10+IgD+CD27-), and plasmablasts (CD19+CD38+++CD27+IgD-CD20-) in untreated SLE compared with healthy subjects. Positive correlation between the counts of B-cells subsets and values of disease-specific autoantibodies (anti-dsDNA, a-Sm, aCL IgG) suggests that B-lymphocytes may be involved in SLE pathogenesis. This work was supported by the Russian Science Foundation (Grant N 22–25-00358).

Background The level of circulating plasmablasts has been proposed as a good marker for diagnosing and monitoring IgG4-related disease (IgG4-RD) independent of serum IgG4 level [1]. However elevated plasmablasts can be seen in other rheumatologic conditions. Objectives To compare В-cell subsets in peripheral blood of IgG4-RD, Sjogren’s syndrome (SjS) patients (pts) and healthy controls. Methods Twelve pts with clinically active IgG4-RD (7F/5M, mean age 50,25 years (range 33-62), 20 active SjS pts (19F/1M, mean age 42 years (range 32-54 years); median disease duration 3 (2-10) years; ESSDAI score ≥5 in 6 pts,) and 20 healthy donors were included. SS was diagnosed based on the ACR-EULAR 2016 criteria. IgG4-RD was diagnosed according to comprehensive diagnostic criteria (H. Umehara, 2011). CD19+ B cells, memory B-cells (CD19+CD27+), non-switched memory B-cells (CD19+IgD+CD27+), switched memory B-cells (CD19+IgD-CD27+), naïve (CD19+IgD+CD27-), double negative (CD19+IgD-CD27-), transitional (CD19+IgD+CD10+CD38+) B-cells, plasmablasts (CD19+СD38+++IgD-CD27+), and plasma cells (CD19+СD38+) were analyzed by multicolor flow cytometry using cytometer Navios (Beckman Coulter, USA). The nonparametric Mann-Whitney test was used for statistical analysis. Data were shown as median (Me) with an interquartile range of 25 - 75 percentile. The differences were considered statistically significant when p <0.05. Statistica 10 for Windows (StatSoft Inc., USA) package was used for statistical data processing. Results There were no significant differences in the number of all studied B-cell subsets between pts with IgG4-RD and with SjS. But absolute numbers of plasmablasts, memory B-cells cells and transitional B-cells in IgG4-RD were significantly higher than in healthy donors: 4.5[1;9.8] х10 3 /μl vs. 0.2 [0.09;0.4] х10 3 /μl; 41 [33.5;57.5] х10 3 /μl vs. 2.5 [1;6.3]; 12 [6.5;21] х10 3 /μl vs. 0.1 [0;0.23] х10 3 /μl respectively, p<0.05 for all cases. Table 1. Peripheral blood B-cell subsets in patients with SS and healthy donors. B-cell subsets, IgG4-RD SjS HD P<0.05 Abs. х10 3 /μl B lymphocyte 166 [109.3;251.3] 164.5 [109.3;236.3] 150 [95;200] plasmablasts 4.5 [1;9.8] 2 [1;3.5] 0.2 [0.09;0.4] * transitional B-cells 12 [6.5;21] 8.5 [4;32.5] 0.1 [0;0.23] * switched cells 24.5 [20;41.8] 18.5 [11;29.8] 20 [9.75;40] non-switched cells 16.5 [10.5;21] 7 [3.75;15.5] 10 [4.75;20] memory B-cells 41 [33.5;57.5] 26 [14.8;40.8] 2.5 [1;6.3] * naïve B-cells 122 [63.3;176.5] 107.5 [49.5;193.8] 100 [57.5;100] double negative 13.5 [8.8;17] 10.5 [5.3;22.3] 20 [9.75;22.5] HD healthy donors * IgG4-RD and HD groups compared, р<0.05 Conclusion Immunophenotyping showed disturbed homeostasis of the B-cells subpopulations in IgG4-RD pts with a significant increase in plasmablasts compared to HD, but there were no differences between IgG4-RD and SjS pts. Further research is needed to evaluate the diagnostic utility of circulating plasmablasts in IgG4-RD. References [1]Wallace ZS, Mattoo H, Carruthers M, Mahajan VS, Della Torre E, Lee H, Kulikova M, Deshpande V, Pillai S, Stone JH. Plasmablasts as a biomarker for IgG4-related disease, independent of serum IgG4 concentrations. Ann Rheum Dis 2015 Jan;74(1):190-5. Disclosure of Interests None declared

BackgroundB-cells play a critical role in the regulation of systemic autoimmune diseases pathogenesis, that extends beyond antibodies production.ObjectivesTo examine В-cell subsets in peripheral blood of patients (pts) with untreated systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), to analyze of their associations with disease-specific autoantibodies.MethodsThirty six untreated SLE pts (31F/5M) and 32 untreated SSc pts (25F/7M) were enrolled. SLE pts had median age of 37(range)(32-41)years, disease duration of 3.5(1-11)years, SLEDAI 2K of 7(4-8), BILAG of 14,5(10,2-23). SSc pts had median age of 42(38-50)years, disease duration of 3.5(1-10)years, Disease Activity EScSG of 2.5 (1-3.2). The control group consisted of 29 volunteers (23F/6M, median age 38(35-48)years. CD19+B cells, memory B cells (CD19+CD27+), switched memory B cells (CD19+CD27+IgD-), non-switched memory B cells (CD19+CD27+IgD+), naive (CD19+CD27-IgD+), double-negative (CD19+CD27-IgD-), transitional (CD19+CD38++CD10+IgD+CD27-) B cells, and plasmablasts (CD19+СD38+++CD27+IgD-CD20-) were analyzed using multicolor flow cytometry.ResultsDifferences in the median percentage and of absolute B-cells subset levels in untreated pts with RA and SLE were not found. The absolute counts of memory B cells (CD19+CD27+), switched memory B cells (CD19+CD27+IgD-), transitional B cells (CD19+CD38++CD10+IgD+CD27-), and plasmablasts (CD19+СD38+++CD27+IgD-CD20-) were higher in SLE and SSc pts compared to healthy donors, p<0,01 for all cases. The absolute counts of double-negative B cells (CD19+CD27-IgD-) were lower in SLE pts than in donors, р=0,03 (Table 1). At significant correlation was found in SLE pts between anti-dsDNA levels and absolute counts of the following B-cell subtypes: CD19+B cells (r=0,72), memory B cells (CD19+CD27+) (r=0,76), switched memory B cells (CD19+CD27+IgD-) (r=0,75) and naive B cells (CD19+CD27-IgD+) (r=0,73); the a-Sm levels and absolute counts of the switched memory B cells (CD19+CD27+IgD-) (r=0,51); the antibodies to cardiolipin (aCL) IgG levels and absolute counts of CD19+B cells (r=0,64), and with naive B cells (CD19+CD27-IgD+) (r=0,59), p<0,01 for all cases. There was a correlation between the anti-topoisomerase-1 antibodies (a-Topo-1) and the absolute counts of double-negative B cells (CD19+CD27-IgD-) (R=0,62, p=0,01) in SSc pts.Table 1.Levels of the blood B-cell subsets in SLE pts, SSc pts and in control.Parameters, n (х109/l)SLESScControl groupCD19+B cells0,15 (0,12-0,25)0,14 (0,12-0,20)0,1 (0,08-0,2)memory B cells (CD19+CD27+)0,036(0,031-0,006) *0,042(0,027-0,053) *0,003(0,001-0,007) *switched memory B cells (CD19+ CD27+IgD-)0,02 (0,016-0,046) *0,026 (0,017-0,034) *0,01 (0,005-0,02) *non-switched memory B cells (CD19+CD27+IgD+)0,012(0,007-0,027)0,016(0,01-0,027)0,02(0,01-0,04)naive B cells (CD19+CD27-IgD+)0,09 (0,06-0,2)0,09 (0,06-0,16)0,1 (0,06-0,1)double-negative B cells (CD19+CD27-IgD-)0,011 (0,006-0,014) *0,02 (0,01-0,03)0,02 (0,01-0,02) *transitional B cells (CD19+CD38++CD10+IgD+CD27-)0,011 (0,004-0,026) *0,011 (0,005-0,012) *0,0001 (0-0,0003) *plasmablasts (CD19+СD38+++CD27+IgD-CD20-)0,001 (0,001-0,002) *0,001 (0-0,002) *0,0001 (0,00006-0,0003) *Note: * - Differences in B-cell subset of the control group with SLE and SSc groups.ConclusionImmunophenotyping showed similar levels of B-cell subset in untreated SLE and SSc pts, an increase in the absolute counts of memory B cells (CD19+CD27+), switched memory B cells (CD19+CD27+IgD-), transitional B cells (CD19+CD38++CD10+IgD+CD27-), and plasmablasts (CD19+СD38+++CD27+IgD-CD20-) in untreated SLE and SSc pts compared with healthy subjects. Positive correlation between the counts of В-cell subsets and values of disease-specific autoantibodies (anti-dsDNA, a-Sm, aCLIgG, a-Topo-1) suggests that B-lymphocytes may be involved in SLE and SSc pathogenesis.This work was supported by the Russian Science Foundation (Grant № 22-25-00358).Disclosure of InterestsNone declared

Diagnosis of low-grade myelodysplastic syndromes (LG-MDS) is one of the most challenging in hematopathology as it relies predominantly on morphologic assessment of dysplasia. Prior studies have demonstrated poor interobserver agreement among pathologists. Histomorphological evaluation of bone marrow core biopsy samples remains the gold standard for diagnostic workup of LG-MDS, including myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, this approach may be subjective, and cannot quantitatively assess subtle differences in marrow topography and the cellular microenvironment. Multiparametric in situ imaging (MISI) through various techniques enables multiple biomarker detection in a single tissue. BostonGene has developed an AI-based image analysis platform to reveal spatial information and subtle histomorphologic features in an objective, quantitative fashion. Here, we demonstrate the potential for automated AI-based imaging analysis of MISI to assist in the differentiation of LG-MDS samples from normal marrow tissues (NBM).

Взаимосвязь маркеров ранней и поздней активации лимфоцитов с эффективностью неоадъювантной химиотерапии больных трижды негативным раком молочной железыФедеральное государственное бюджетное учреждение «Национальный медицинский

Background:Pleiotropic effects of IL-6 receptor antagonist tocilizumab (TCZ) in rheumatoid arthritis (RA) involve circulating T-cells. Preliminary reports have suggested that TCZ therapy can modulate T cell function and humoral immune responses in RA [1,2]. According to other studies, TCZ had no influence on T-lymphocyte subdifferentiation [3].Objectives:To assess the effect of 12 months (m) TCZ therapy on T-cell phenotype, and to analyze the association between T-cell subsets and RA activity.Methods:36 active RA pts (29F/7M); median age 55[46; 64] years; disease duration 120[72; 1928]m; DAS28 score 6,3[5,7;6,7]; RF+100%, ACCP+ 86% were treated with TCZ (8 mg/kg every 4 weeks) in an open-label study. Immunophenotyping was performed at baseline, 6m and 12m. T-cell subpopulations and key laboratory parameters (CRP, RF, ACCP, MCV, MMP-3) were assessed.Results:At baseline, the absolute counts of T cell subpopulations in RA pts - (CD3+), Th (CD3+CD4+), Tc (CD3+CD8+), NK cells (CD3-CD56+) - were comparable with counts in healthy donors (table1). At baseline, the absolute NK cells (CD3-CD56+) count correlated with MCV (r=0,35, p<0,05) and with MMP-3 (r=0,57, p<0,05) in RA pts. After 6m of TCZ therapy, 34% of pts were classified as good responders, 66% - as moderate responders; after 12m of TCZ therapy 68% of pts - as good responders, 32% - as moderate responders, according to the response criteria of the EULAR. Tc (CD3+CD8+) absolute count reductions were documented after 6m of TCZ therapy, absolute of T cells (CD3+) count and Th (CD3+CD4+) count - after 12m of TCZ therapy (table1). A positive correlation between Tc (CD3+CD8+) absolute counts and RF (r=0,50, p<0,05) was found after 6m of TCZ therapy. A negative correlation of Δ CRP was established with Δ T cells (CD3+) (r=-0,45], Δ Th (CD3+CD4+) (r=-0.47), and Δ Tc (CD3+CD8+) (r=-0,59, p<0,05 in all cases) after 12m of TCZ. The median absolute counts of T cells (CD3+), Th (CD3+CD4+) was lower than on healthy donors after 12m of TCZ therapy. NK cells (CD3-CD56+) counts and Th/Tc index did not change after 12 m of TCZ therapy vs healthy donors and baseline values.Conclusion:Immunophenotyping in pts with active RA showed no difference in the absolute counts of T-cell subpopulations (T cells (CD3+), Th (CD3+CD4+), Tc (CD3+CD8+), NK cells (CD3-CD56+)) compared to healthy donors. T cells (CD3+), Th (CD3+CD4+) and Tc (CD3+CD8+) T cell counts dropped after 6 or 12m of TCZ therapy. Significant correlation between the T-cell subpopulations counts and values of RA laboratory indicators (CRP, MCV, RF, MMP-3) suggest potential T-lymphocytes involvement in RA pathogenesis.References:[1]Kasama T, Isozaki T, Takahashi R, Miwa Y.Clinical effects of tocilizumab on cytokines and immunological factors in patients with rheumatoid arthritis. Int Immunopharmacol. 2016 Jun;35:301-306. doi: 10.1016/j.intimp.2016.03.016.[2]Schinnerling K, Aguillón JC, Catalán D, Soto L. The role of interleukin-6 signalling and its therapeutic blockage in skewing the T cell balance in rheumatoid arthritis. Clin Exp Immunol. 2017 Jul;189(1):12-20. doi: 10.1111/cei.12966.[3]Schwaneck EC, Renner R, Junker L. et al. T cells, natural killer cells, and gammadeltaT cells in a large patient cohort with rheumatoid arthritis: influence of age and anti-rheumatic therapy. Scand J Rheumatol. 2020 Jan;49(1):8-12. doi: 10.1080/03009742.2019.1634755.Table 1.The effect of tapering tocilizumab on achieving outcomesDonorsBaseline6m12mT cell (CD3+)1,3 (1,2-1,8)∧1,2 (0,9; 1,7)*1,1 (0,7; 1,4)1,0 (0,7; 1,5)* ∧Th (CD3+CD4+)0,9 (0,7-1,2) ∧0,8 (0,6; 1,0)*0,7 (0,5; 1,0)0,6 (0,4; 1,1) * ∧Tc (CD3+CD8+)0,4 (0,3-0,5)0,4 (0,2; 0,5)**0,3 (0,2; 0,04)**0,3 (0,3; 0,5)Тh/Тc2,5 (1,9-3,1)2,3 (1,4; 3,0)2,3 (1,5; 3,6)2,3 (1,4; 4,0)NK (CD3-CD56+)0,2 (0,2-0,5)0,2 (0,1; 0,2)0,2 (0,1; 0,3)0,2 (0,1; 0,3)Note: * - p<0,05 between pts at baseline and after 12 m of TCZ therapy;**- p= 0,02 between pts at baseline and after 6 m of TCZ therapy;∧ - - p<0,05 between donors and pts after 12 m of TCZ therapy.Disclosure of Interests:None declared

Background. To enhance the antitumor immune response, new promising methods of immunotherapy are being developed. They consist in the blockade and activation of immune check-point molecules, in particular, the blockade of the Lag‐3 molecule (lymphocyte-activation gene 3) and the activation of the GITR receptor (Glucocorticoid induced TNF receptor). In the studies of combined use with PD-1 blockers, encouraging results were obtained, which makes the assessment of the expression of Lag-3 and GITR on immunocompetent cells of peripheral blood (PB) and tumor tissue necessary for the personalization of such treatment and understanding of the mechanisms of the antitumor immune response. Materials and methods. The study included peripheral blood samples and surgical material from 39 breast cancer patients being treated at the Blokhin National Medical Research Center of Oncology. The subpopulation composition and expression of PD-1, Lag-3, and GITR molecules were evaluated by flow cytometry. Results. The analysis of the main populations of PB lymphocytes showed that in patients with breast cancer, the content of NKT-lymphocytes was increased, and the proportions of lymphocytes expressing CD11b and CD25 markers were increased compared to the donor group. It was revealed that the tumor tissue is dominated by T-cells, an increase in the proportion of which occurs due to a reduced content of NK-lymphocytes and B-lymphocytes. The structure of Tumor-infiltrating lymphocytes (TILs) is dominated by subpopulations with immunosuppressive activity, which is indicated by a decrease in the content of CD11b+, CD25+ and perforin-positive cells, increased expression of Lag-3 and PD-1. For PB and tumor tissue, the average degree of dependence of Lag-3 expression on the content of PD-1+ lymphocytes was shown. There is an increase in the content of immunosuppressive subpopulations with high PD-1 values in PB and TILs. The direct dependence of the number of perforin-containing lymphocytes and CD11b expression on the GITR content in the PB was established, but it is not typical for breast cancer tissue. Conclusion. Since the blockade of the Lag-3 molecule by monoclonal antibodies can enhance the effect of anti-PD-1 therapy in cancer patients, it is necessary to evaluate the expression and co-expression of these two markers. A high content of GITR-positive lymphocytes in the tumor tissue, on the one hand, and a decrease in the proportion of effector subpopulations of lymphocytes, on the other, indicates the influence of the tumor microenvironment on the functioning of GITR-mediated activation of the immune response. Further investigation of GITR expression and functional activity is required to understand the nature of this contradiction.

Introduction.Long-term monitoring of immune system parameters in cancer patient in FSBI “N.N. Blokhin Medical Research Center of Oncology” of the Ministry of Health of Russia, as well as the absence of an immunological research algorithm and a spectrum of significant markers, served as the basis for this study.Purpose.To present reference values and determine the normal range for subpopulations of systemic immunity lymphocytes.Materials and methods.The phenotype of peripheral blood lymphocytes was studied in 186 healthy donors (86 men and 100 women), mean age 41,9 ± 12,5 years. To assess the multivariate phenotype by flow cytometry, monoclonal antibodies to CD45, CD3, CD4, CD8, CD16, CD56, CD19, CD25, CD28, CD11b, CD127, HLA-Dr, TCR-γ / δ, Perforin and Granzime B labeled various fluorochromes.Results.A panel of markers of immunocompetent cells of systemic immunity was developed as a basis for assessing the state of the immune system of cancer patients. Reference values and normal boundaries are given for characterizing the linearity of cells with detailing of the phenotypic and functional heterogeneity of the population of CD8+-lymphocytes, activation markers and the pool of naive cells; characteristics of various types of regulatory cells, functional activity of cells of the effector link of immunity. A comparative analysis of immunoregulatory indices was carried out and the vulnerability of the formula CD3+CD4+/CD3+CD8+to the CD4+/CD8+index was proved.Conclusion.The spectrum of the proposed indicators is the product of many years of research carried out in the laboratory of clinical immunology of the Federal State Budgetary Institution “N.N. Blokhin National Medical Research Center of Oncology” оf the Ministry of Health of the Russian Federation, in order to search for significant criteria for assessing the state of the immune system in cancer.

Introduction. Age is considered as an important clinical and pathological factor in cancer patients. Malignant tumors are more likely to develop in older people, but the disease is less aggressive than in young patients. According to various authors, the influence of age on the development of tumors largely depends on the age-related features of the immune system.The aim of the present study was to determine the relationship of indicators of systemic antitumor immune response with the age of patients with primary operable breast cancer and cancer of the oral mucosa.Materials and methods. The study included patients with all subtypes of primary-operable breast cancer (n = 145) and patients with cancer of the oral mucosa (n = 29). Immunophenotyping of peripheral blood lymphocytes was performed using a wide panel of monoclonal antibodies to markers of adaptive and innate immunity cells.Results. In elder patients (40 years and older) with primary-operable breast cancer, the percentage of activated CD25+ lymphocytes and CD4+CD25+ and CD3+CD4+ T cells, NKT cells, activated HLA-DR+ lymphocytes, including activated CD3+HLA-DR+ T cells before treatment, was statistically significantly higher than in patients younger than 40 years. Patients of this group showed increase of CD8+CD - 11b+CD28– CTLs and a decrease in the number of naive lymphocytes (CD4 – CD62L+ and CD8+CD11b – CD28+) in comparison with control percentage, and the downward trend in CD4+CD25+CD127– Treg, with increased numbers of CD4+CD25+ T cells. In patients with cancer of the oral mucosa, an increase in the number of cells of some populations of the immune effector link and a decrease in the number of suppressor lymphocytes were revealed with age.Conclusion. The results suggest that age-related differences in the state of systemic antitumor immune response contribute to a more favorable course of breast cancer and some other malignancies in older persons. It is obvious that the features of age differences in the immune response to the tumor should be taken into account when prescribing systemic therapy, including immunotherapy.All patients gave written informed consent to participate in the study

Aim. To study the state of local immunity in patients with gastric adenocarcinoma. Materials and methods. From 2017 to 2018, 45 previously untreated patients with gastric adenocarcinoma (25 with stage IIII, 20 with stage IV) received surgical/combined treatment or chemotherapy, respectively, at the Blokhin Scientific Research Center of Oncology. Tumor tissue was taken before treatment. By using flow cytometry there were evaluated the percentage of tumor tissue infiltration by lymphocytes (CD45+CD14-TIL); T cells (CD3+CD19-TIL); B cells (CD3-CD19+TIL); NK cell (CD3-CD16+CD56+TIL); effector cells CD16 (CD16+Perforin+TIL) and CD8 (CD8+Perforin+TIL) with their cytotoxic potential active CD16TIL and active CD8TIL; subpopulations of regulatory T cells NKT cells (CD3+CD16+CD56+TIL), regulatory cells CD4 (CD4+CD25+CD127-TIL) and CD8 (CD8+CD11b-CD28-TIL). The prognostic value of immune cells for overall survival (OS) and progression-free survival (PFS) was assessed. Results. A favorable prognosis factor for progression-free survival in patients with local and locally advanced forms of gastric cancer was an increase in the number of CD3-CD19+TIL (HR 0.862, 95% CI 0.7820.957, p=0.005), and an unfavorable prognosis was an increase in NK cells (CD3-CD16+CD56+TIL); HR 1.382, 95% CI 1.0871.758, p=0.008. The negative effect of the relative content of NK cells (CD3-CD16+CD56+TIL) and NKT cells (CD3+CD16+CD56+TIL) on OS of patients with metastatic gastric cancer noted (HR 1.249, 95% CI 0.9971.564, p=0.053; HR 1.127, 95% CI 1.0251.239, p=0.013). At the same time, an increase in the percentage of tumor tissue infiltration by lymphocytes (CD45+CD14-TIL) and an increase in the age of patients (HR 1.005, 95% CI 1.0021.008, p=0.003; HR 1.098, 95% CI 1.0311.170, p=0.004) reduce the incidence of PFS in patients with metastatic gastric carcinoma. Conclusion. Indices of local immunity can serve as additional prognostic factors for gastric carcinoma.

Objective: to study the state of cellular immunity in patients with gastric adenocarcinoma.Materials and methods. From 2017 to 2018, 45 previously untreated patients with gastric adenocarcinoma (25 with stage I–III, 20 with stage IV) received surgical / combined treatment or independent chemotherapy, respectively, at the N. N. Blokhin National Medical ResearchCenter of Oncology. Peripheral blood sampling was carried out before starting treatment. We studied the cellular composition of peripheral blood, as well as systemic immunity parameters determined by flow cytometry (CD3+CD4+; CD3+CD8+; CD4+CD8+; CD4+/CD8+; CD3–CD16+CD56+; CD3–CD19+), and their prognostic significance in relation to overall survival and progression-free survival.Results. The prognostic value of the relative indicator of platelet lymphocytic index was determined: progression-free survival in patients with a high level of platelet-lymphocytic index (>208.7) was higher: 8.1 months versus 4.5 months (p = 0.0027). A favorable prognosis for overall survival was an increase in the number of CD3–CD19+ lymphocytes (hazard ratio (HR) 0.91; 95 % confidence interval (CI) 0.85–0.97; p = 0.007), and an unfavorable prognosis was an increase in the number of neutrophils (HR 1.26; 95 % CI 1.05–1.50; p = 0.012), platelet count (HR 1.01; 95 % CI 1.0–1.01; p = 0.043), as well as an increase in the number of NK cells (HR 1.04, 95 % CI 1.0–1.09; p = 0.029).Conclusion. Indicators of the cellular composition of peripheral blood, characterizing a systemic inflammatory reaction, as well as indicators of systemic immunity, can serve as additional prognostic factors for gastric cancer.

Introduction. Natural killer T lymphocytes (NKT) take place between the innate and acquired immune response. The ability of these cells to activate the antitumor immune response and inhibit immunological activity makes them the target of research in cancer patients. The radicality of surgical treatment of patients with metastatic melanoma (stage III–IV) is relatively conventional. In this regard, the possibility of adjuvant effective therapy of melanoma is actively investigated worldwide.The aim of the study is investigation of the importance of increasing the number of NKT cells in the peripheral blood of patients with metastatic melanoma after radical surgical removal of the tumor. Patients were treated with adjuvant regimen antitumor autologous dendritic cell therapy in form of vaccination.Materials and methods. The study included 39 patients with stage III and IV metastatic melanoma with regional and / or distant metastases after radical surgery. From the peripheral blood monocytes of each patient, an autologous vaccine was created from mature dendritic cells loaded with tumor lysate. The therapy continued until objective progression. The study included patients who received from 5 to 120 injections. The follow-up period ranged from 5 to 168 months.Results. It was shown that 14 (36 %) of patients had the number of NKT cells exceeding the norm (0–10 %) and in the course of vaccine therapy they had the progression of the disease in the period up to 2 years. In patients with relapse-free course of the disease in vaccine therapy (n = 13), the number of NKT lymphocytes did not exceed the norm both before and during therapy. Significantly shorter time to progression was revealed in patients with high initial content of NKT lymphocytes compared with patients with normal indices of NKT cells (6.5 months) – 95 % confidence limit 2,4–10,7 % vs 96,2 months (95 % confidence limit 63.8–128.6 %). Conclusion. An increased number of NKT cells in patients with stage III–IV metastatic melanoma after radical surgical treatment is a marker of early progression.

In patients with primary resectable breast cancer, a positive correlation between the age and the count of CD16 + lymphocytes and a negative correlation of this parameter with the number of regulatory CD4 + CD25 + CD127 — cells and proliferative activity of Ki-67 tumor cells were revealed. Higher level of Ki-67 was associated with reduced number of effector lymphocytes (CD8 + and CD16 + ) and elevated content of regulatory CD8 + CD11b — CD28 — T cells. The absence of expression of estrogen receptors was associated with reduced cytotoxic potential of CD8 + T cell in comparison with ER + breast cancer. The percentage of CD8 + lymphocytes (CD3 + CD8 + and CD8 + CD11b + CD28 + ) among lymphocytes infiltrating the tumor was higher in PR + breast cancer than in PR — tumors. With increasing the tumor load, the number of lymphocytes expressing CD16 marker and their cytotoxic potential decreased.