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An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Hepatitis C virus (HCV)-specific CD8 T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8 T cells (pentamer-positive [pentamer]/CD8 T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer/CD8 cells. In PI, pentamer/CD8 cells had impaired antigen-specific reactivity that worsened when these cells were not detectable Short/midduration PI was characterized by detectable peripheral PD-1 CD127 TRAF1 cells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1 to exhaustion. treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatment enhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1 HCV-specific CD8 T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8 T cells are rarely detectable , but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivity in slow fibrosers. Hepatitis C virus (HCV) infects 71 million people worldwide. Two-thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection, but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8 T cells. During chronic infection, these cells are functionally impaired, which could be due to the failure of costimulation. This study describes exhausted specific T cells, characterized by low levels of expression of the signal transducer TRAF1 of the positive costimulatory pathway 4-1BB/4-1BBL. IL-7 upregulated TRAF1 expression and improved T cell reactivity in patients with short/midduration disease, while in patients with long-lasting infection, it was also necessary to block the negative PD-1/PD-L1 checkpoint. W...

Chronic myelomonocytic leukaemia (CMML) is an entity with a heterogeneous clinical evolution, initially included among myelodysplastic syndromes (MDS) according to the FAB classification. The recent WHO classification brings CMML closer to myeloproliferative disorders, and moved it into a new category termed myelodysplastic/myeloproliferative diseases (MDS/MPD). Aim: To describe the immunophenotypic profile of CMML and to compare it with the phenotype described in MDS and MPD. Patients: 76 patients: 20 CMML (15 CMML I, 5 CMML II), 38 MDS (refractory cytopenia with multilineage dysplasia), and 18 MPD (11 essential thrombocytemia, 2 chronic idiopathic myelofibrosis, and 5 polycythemia vera). Material and Methods: Flow cytometry immunophenotyping (FCI) in bone marrow samples. Data studied: 1 In the myeloid lineage: abnormalities in granularity and CD45 distribution, abnormalities in the phenotypic pattern of myeloid development (CD16/CD11b/CD13), and the absence of CD10 expression on mature granulocytes (CD10−). 2 In the monocytic lineage: CD2 and CD56 expression. 3 In the red cells: abnormalities in CD71 and glycophorin A distribution. 4 In B-cells, detection of a low percentage of CD10+ B-cell precursors (less than 1% of bone marrow B-cells). 5 In myeloblasts, identification of more than >5% CD34+ cells, and evaluation of aberrant expression of CD7 and TdT in these cells (positive expression was described when >10% of CD34+ cells were positive for any of these antigens). Results: Abnormal cases among those studied for each parameter analysed is specified in the table. Patients with CMML and MDS showed statistically significant differences (p<0.05) in their B-cell development and CD56 monocyte expression. Significant differences between CMML and MPD were found in CD45 myeloid distribution, myeloid antigenic profile, CD56 and CD2 monocyte expression and B-cell development. Immunophenotypic data in patients with MDS (row1), CMML (row2), and MPD (row3). Abnormal SSC/CD45 Abnormal myeloid CD16/CD11b/CD13 CD10− neutrophils CD2+ monocytes CD56+ monocytes Abnormal erythroid CD71/GlyA <1% CD10+ B cells >5% CD34+ MDS: myelodysplastic syndromes; CMML: chronic myelomonocytic leukemia; MPD: myeloproliferative disorders. 33/38 35/38 15/30 5/27 5/31 27/38 16/31 2/38 20/20 20/20 4/17 8/17 9/16 16/19 14/17 0/20 4/18 5/18 1/18 1/18 0/16 12/17 4/18 0/18 Conclusions: With the number of cases studied, higher similarities can be found between CMML and MDS immunophenotypic profiles. The antibody panel selected, including several abnormalities previously described in patients with MDS, might justify the differences found between CMML and MPD. However, it does not explain the relationship between the immunophenotypic profile of CMML and MDS. Abnormalities in B-cell development and aberrant expression of CD56 on monocytes were the most useful data to distinguish CMML and MPD. Although the number of patients studied is still low, quantification of CD34+ cells did not help to identify CMML with an aggressive clinical course.

Introduction It was proposed that peripheral blood (PB) monocyte subset analysis evaluated by flow cytometry, hereafter referred to as "monocyte assay", could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay required a large multicenter validation. Methods PB and/or bone marrow (BM) samples from adult patients displaying monocytosis were assessed with the "monocyte assay" by ten ELN iMDS Flow working group centers (6 equipped with BD FACSCanto™ II (BD Biosciences), 3 with Navios™ (Beckman Coulter) and one with BD™ LSRII (BD Biosciences)) with harmonized protocols. The corresponding files were reanalyzed in a blind fashion by a skilled operator and the cMo (CD14 ++CD16 -) percentages obtained by both analyses were compared. Information regarding age, gender, complete blood count, marrow cytomorphology, cytogenetics and molecular analysis was collected. Confirmed diagnoses were collected when available as well as follow-up for CMML patients. Results The comparison between cMo percentages from 267 PB files provided by the 10 centers and the centralized cMo percentages showed a good global significant correlation (r=0.88; p&lt;0.0001; FigA) with no bias (FigB). Confirmed diagnoses were available for 212 files, namely 101 CMML according to the WHO criteria, 99 reactive monocytosis, and 12 MPN with monocytosis. A phenotype in favor of CMML, either classical with accumulation of cMo ≥94% or a bulbous aspect (FigC), was observed respectively in 81 and 14 patients. Hence, a total of 95 out of the 101 CMML patients translated into a sensitivity of 94% (FigD). Assessment of C reactive protein counts were available in seven of the 14 patients with the characteristic bulbous profile and correlated with an inflammatory state, showing a median of 93.0 [7.0-157.4] mg/L. Conversely, a phenotype not in favor of CMML (FigC) was observed in 83 of the 99 patients with reactive monocytosis and in 10 of 12 patients with MPN with monocytosis, leading to a 84% specificity (FigD). We established a Receiver Operator Curve (ROC) and again obtained a 94% cut-off value of cMo with an area under the ROC curve (AUC) of 0.865 (FigE). The second aim of this multicenter study was to assess the feasibility of the monocyte assay on 117 BM samples provided by 7 out of the 10 ELN centers, 43 of which being paired to PB samples. The comparison between cMo percentages provided by the 7 centers and the centralized cMo percentages showed a lower global significant correlation compared to PB samples (r=0.74; p&lt;0.0001; FigF) with a slight underestimation of cMo percentage by the participating centers (FigG). The comparison between PB and BM samples cMo% obtained by centralized reanalysis showed an excellent global correlation (r=0.93; p&lt;0.0001; FigH) with a higher percentage in the marrow (FigI). Seventy-nine files were associated to a confirmed diagnosis, as expected mostly CMML (n=69), only seven reactive monocytosis and three MPN with monocytosis. Thus, we determined a sensitivity of the "monocyte assay" on BM samples of 87% (a phenotype in favor of CMML being observed in 60 out of the 69 CMML with 6 bulbous aspect profiles) and a specificity of 80% (a phenotype not in favor of CMML being observed in 5 of the 7 patients with reactive monocytosis and in 3 of the 3 patients with MPN with monocytosis). Conclusions This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership.

Isolated immune complexes from sera of 49 out of 67 human immunodeficiency virus‐1‐positive (HIV‐1+) patients (CIC–HIV+), composed of anti‐HIV–HIV‐Ag, could induce apoptosis on normal phytohaemagglutinin (PHA)‐activated lymphocytes. DNA degradation was detected by propidium iodide staining. This activity is directed against CD4+ lymphocytes as demonstrated by double binding of CIC–HIV+ and anti‐CD4 on apoptotic cells. Expression of Fas antigen is prior to apoptotic phenomena. CIC–HIV+ apoptosis inducers belong mainly to asymptomatic HIV‐infected patients, indicating that immune complexes from these patients can destroy CD4+ lymphocytes.

Patients with chronic renal failure undergoing hemodialysis who are infected with hepatitis C virus (HCV) may test consistently anti-HCV negative. Because CD4(+) T-cells provide help for antibody production virus-specific effector CD4(+) T-cell responses were investigated in relation to anti-HCV positivity in 15 hemodialysis patients grouped according to HCV antibody and viremia. CD4(+) T-cell reactivity was studied in peripheral blood mononuclear cells by standard lymphocyte proliferation assay and phenotypic/functional characterization (cell-surface staining/cytokine secretion) by flow cytometry. HCV-specific CD4(+) T-cell proliferation in viremic hemodialysis patients was weak or absent independently of their anti-HCV status. Virus-specific CD4(+) T-cells displayed a memory phenotype and showed low to undetectable capacity to secrete effector interferon (IFN)-gamma. Impaired activation-induced cytokine secretion appeared to be Th1 (IFN-gamma) but not Th2 (interleukin-4)-directed and was virus-specific as cytomegalovirus responses were preserved. The frequency ex vivo of CD3(+)CD4(+)IFN-gamma(+) T-cells was independent of the HCV antibody status and comparable between viremic (range: 0.08-1.54%) or non-viremic (0.11-3.2%) hemodialysis patients and healthy donors (0.13-1.10%; P = 0.58). The numbers of CD3(+)CD4(+)IFN-gamma(+) T-cells augmented slightly (P = 0.047) in HCV-infected hemodialysis patients but markedly in only one (greater than ninefold) after HCV stimulation. In conclusion, hemodialysis patients show limited HCV-specific effector CD4(+) Th1-cell responses which nonetheless seem unrelated to the anti-HCV status and are not more impaired due to the ongoing hemodialysis.

SUMMARYWe assessed whether antiretroviral regimes are able to diminish apoptosis and markers of lymphocyte activation and restore lymphocyte proliferation. T-cell subset, spontaneous and induced apoptosis, CD95 and soluble Fas antigen and cell proliferation were analysed in 41 human immunodeficiency virus type 1-positive patients. Twenty-five were in asymptomatic stage A and 16 were in stage B/C. Thirty-five received antiretroviral treatment: 18 received two inhibitors of reverse transcriptase and one protease inhibitor and 17 received three inhibitors of reverse transcriptase. Six patients did not receive treatment, for different reasons, but continued to participate in the study. Studies were performed at baseline, 3, 6 and 12 months. Levels of CD4 increased slightly until 6 months of antiretroviral treatment, as a whole, in all the patients treated. Naïve CD4 lymphocytes, as well as memory CD4 lymphocytes, remained constant. Spontaneous apoptosis of lymphocytes, after 72 hr of culture, decreased in all patients treated, but to a much smaller extent than phytohaemagglutinin-induced apoptosis. In both groups treated, levels of soluble Fas decreased until 6 months of treatment and then increased again. Lymphocyte proliferation reached normal levels after 1 year of treatment. In patients without treatment CD4 cells decreased slowly and no modification in activation markers was found. Antiretroviral regimes decrease immune activation as well as viral load and this deactivation restores lymphocyte proliferation.