Low Percent of Granzyme B Positive T-Regulatory Cells (CD4+CD25high) As a Predictor of Acute Graft-Versus-Host Disease (GVHD) after Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5871-5871
Author(s):  
Mikhail Drokov ◽  
Elena N. Parovichnikova ◽  
Larisa A Kuzmina ◽  
Irina Galtseva ◽  
Vera Vasilieva ◽  
...  
Keyword(s):  
T Regulatory Cells ◽  
Conflicts Of Interest ◽  
Granzyme B ◽  
Geometric Mean ◽  
Target Cells ◽  
Unrelated Donor ◽  
Regulatory Cells ◽  
Cd4 Cells ◽  
Becton Dickinson ◽  
Hematopoietic Stem

Abstract Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B – mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (Tregs) influence on T-effectors cells. It is a well-known fact that Tregs participate in a pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence. No clinical data exist regarding the Granzyme B positive Tregs in this setting. Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30, +60, +90 after allo-HSCT and at day of acute GVHD onset from 27 patients with hematological malignancies (AML=12, ALL n=12, LPD=2, MDS =1; 6 with active disease, 21 - in CR) after allo-HSCT (from matched unrelated donor n=23, from matched related donor n=4; MAC = 10, RIC=17). The anti-CD4-APC-Cy7, anti-CD25-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population as CD4+CD25high. We did not include anti-FoxP3 antibodies as FoxP3 is not so specific in humans and particularly after allo-HSCT due to technical difficulties, several isoforms, FoxP3-positivity on activated nonregulatory CD4+ cells. CD4- lymphocytes (CD8+ and NK-cells certainly containing granzyme B) were used as internal positive control to define the percent of CD4+CD25highGranzymeB+ cells and gMFI (geometric mean fluorescence intensity) among the mononuclear cell fraction. 50000 of CD4+ cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA). Results. 11 patients (42%) have developed aGVHD (II-IV) at a median time of +26 day (8 - 88) after HSCT. The percent of CD4+CD25highGranzymeB+ cells among CD4+cells at day +30 after allo-HSCT in patients who never developed aGVHD was statistically higher (9,49±2,79; p=0.003) than in patients at day of aGVHD onset (3,8±1,78). It’s worth to note that in 4 patients who did not have signs of a GVHD on day +30 but developed it later (on day +40, +45, +74, +88), the percent of CD4+CD25highGranzymeB+ cells at day +30 was as low (3,38±1,47) as at day of aGVHD onset and significantly lower (p=0.003) than in patients who never developed aGVHD, thus predicting the occurrence of aGVHD in the future. Conclusion Despite the fact that the analyzed group is small, we suggest that low relative amount of CD4+CD25highGranzymeB+ cells after allo-HSCT may contribute to the development aGVHD and even predict it onset. This fact, of course, needs further investigation. Disclosures No relevant conflicts of interest to declare.

Granzyme B Expression in T-Regulatory Cells Is a Strong Predictor of Acute Graft-Versus-Host Disease after Day +30 in Patients with "Classic" Immunosuppression after Allo-HSCT

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2238-2238
Author(s):  
Mikhail Yu. Drokov ◽  
Elena N. Parovichnikova ◽  
Julia Davydova ◽  
Larisa A. Kuzmina ◽  
Irina V Galtseva ◽  
...  
Keyword(s):  
Roc Curve ◽  
T Regulatory Cells ◽  
Conflicts Of Interest ◽  
Granzyme B ◽  
Conditioning Regimen ◽  
Curve Analysis ◽  
Unrelated Donor ◽  
Regulatory Cells ◽  
Roc Curve Analysis ◽  
Becton Dickinson

Abstract Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (T-regs) influence on T-effectors cells. Despite the well-known fact that T-regs participate in pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence, data about functional status of T-regs and aGVHD is still limited. Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30 after allo-HSCT. We use PBMC from 29 patients with hematological malignancies obtained by density gradient media. This method was used due to lymphopenia in this group of patients. Group with no aGVHD consist of 22 patients (AML=12, ALL n=5, LPD=1, CML=2, AA=1, MDS=1) after allo-HSCT n=17 from MUD, n=5 from mismatch unrelated donor. MAC conditioning regimen was used in 17 cases, 5 patients receive RIC. The group of aGVHD after day +30 include 7 patients. (AML=4, ALL n=3) after allo-HSCT from MUD (n=5), mismatch unrelated donor (n=1) and n=1 from sibling HLA-identical donor. Two patients receive MAC and 5 patients receive RIC conditioning regimen. All patients in both groups receive standard immunosuppression (MMF+CSA+ATG). All patients developed II-IV grade aGVHD (II (n=1); III (n=4); IV (n=2)) with a median time onset on day +50 (34-150). The anti-CD4-APC-Cy7, anti-CD25-APC, anti-CD127-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population. A Bland-Altman plot (difference plot) was used in analyzing the agreement between the two different methods of T-regs assay (CD4+CD25high and CD4+CD25highCD127low) - differences were not found (p=0,942). Due to this fact in our experiments CD4+CD25high cells were identified as T-reg cells (Gregg et al., 2005). 30000 of CD4+ cells were analyzed on a BD FACSCanto II to achieve sufficient statistical power (Becton Dickinson, USA). Results. As we can see on chart 1 level of Granzyme B was higher in patients who never developed aGVHD. In accordance with chart 2 percentage of Granzyme B positive T-regs in group of patients who never developed aGVHD was 7,26±1,89% in comparison with 2,04±0,93% in group who developed aGVHD after day +30 (p=0,02*). We should note that according to our previous experiments bacterial or viral infection (e.g CMV) and HLA-disparity does not affect the level of granzyme B expression in T-regs. Using ROC- curve analysis (see Chart 3) we obtained area under curve (AUC) 0,74. Conclusion. Our data shows that Granzyme B in T-reg cells after allo-HSCT in patients with standard IST may predict aGVHD onset after day +30 with satisfactory test results (AUC=0,74, «cut-off» - 4,15%; sensitivity - 85,71%; specificity - 45,45%).). According to data of our transplant center (for 2006-2016), "granzyme B-based strategy" can help us to predict up to 47,3% of all aGVHD. Disclosures: No relevant conflicts of interest to declare. Chart 1: Example of Granzyme B expression (right; shown on horizontal axis) in T-regs (left; upper right quadrant) on day +30 after allo-HSCT in group with standard immunosuppression Chart 2: Percentage of Granzyme B positive T-reg cells in patient without aGVHD and patients who develop aGVHD after day +30. Chart 3: ROC curve analysis of Granzyme B in T-regs as predictor of aGVHD. Figure Figure. Figure Figure. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells

Blood ◽  
2004 ◽  
Vol 104 (9) ◽  
pp. 2840-2848 ◽  
Author(s):  
William J. Grossman ◽  
James W. Verbsky ◽  
Benjamin L. Tollefsen ◽  
Claudia Kemper ◽  
John P. Atkinson ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Nk Cells ◽  
T Regulatory Cells ◽  
Lymphocyte Subsets ◽  
Granzyme B ◽  
Human Lymphocytes ◽  
Target Cells ◽  
Regulatory Cells ◽  
Cd8 T Lymphocytes ◽  
Tr1 Cells

Abstract Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the perforin/granzyme pathway as a major mechanism to kill pathogen-containing cells and tumor cells.1,2 Dysregulation of this pathway results in several human diseases, such as hemophagocytic lymphohistiocytosis. Here we characterize the single-cell expression pattern of granzymes A and B in human lymphocytes using a flow cytometry-based assay. We demonstrate that most circulating CD56+8- NK cells, and approximately half of circulating CD8+ T lymphocytes, coexpressed both granzymes A and B. In contrast, few circulating CD4+ T lymphocytes expressed granzymes A or B. Activation of CD8+ T lymphocytes with concanavalin A (ConA)/interleukin-2 (IL-2), and activation of CD4+ T lymphocytes with antibodies to CD3/CD28 or CD3/CD46 (to generate T regulatory [Tr1] cells), induced substantial expression of granzyme B, but not granzyme A. Naive CD4+CD45RA+ cells stimulated with antibodies to CD3/CD46 strongly expressed granzyme B, while CD3/CD28 stimulation was ineffective. Finally, we show that granzyme B-expressing CD4+ Tr1 cells are capable of killing target cells in a perforin-dependent, but major histocompatibility complex (MHC)/T-cell receptor (TCR)-independent, manner. Our results demonstrate discordant expression of granzymes A and B in human lymphocyte subsets and T regulatory cells, which suggests that different granzymes may play unique roles in immune system responses and regulation.

scholarly journals Immune Disorders in Hashimoto’s Thyroiditis: What Do We Know So Far?

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Aleksandra Pyzik ◽  
Ewelina Grywalska ◽  
Beata Matyjaszek-Matuszek ◽  
Jacek Roliński
Keyword(s):  
Hashimoto’S Thyroiditis ◽  
T Regulatory Cells ◽  
Hashimoto Thyroiditis ◽  
Hashimoto's Thyroiditis ◽  
Regulatory Cells ◽  
Review Of Literature ◽  
Immune Disorders ◽  
Cd4 Cells ◽  
Immune Defect

This review of literature attempts to identify the factors that are involved in the pathogenesis of Hashimoto thyroiditis, an immune defect in an individual with genetic susceptibility accompanied with environmental factors. The frequency of Hashimoto’s disease is a growing trend and among Caucasians it is estimated at approximately 5%. The dysfunction of the gland may be clinically evident (0.1–2% of the population) or subclinical (10–15%). The pathology is diagnosed five to ten times more often in women than men and its incidence increases with the age (the peak of the number of cases is between 45 and 65); however, it can also be diagnosed in children. The pathogenesis of Hashimoto’s thyroiditis is still not fully comprehended. In the etiology of Hashimoto thyroiditis excessively stimulated T CD4+ cells are known to play the most important role. Recent research has demonstrated an increasing role of newly discovered cells such as Th17 (CD4+IL-17+) or T regulatory cells (CD4+CD25+highFoxP3+) in the induction of autoimmune disorders. The process of programmed cell death also plays an equally important role in the pathogenesis and the development of hypothyroidism.

Proportion of IL-17A Producing CD4+ Cells Increases in Response to Alloreactivity as Part of a Network Involving Memory and Regulatory Cells Post HSCT.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1160-1160
Author(s):  
Andrzej Lange ◽  
Dorota Dlubek ◽  
Krzysztof Suchnicki ◽  
Sylwia Madej ◽  
Eliza Turlej
Keyword(s):  
Cell Population ◽  
Conflicts Of Interest ◽  
Disease Onset ◽  
Wilcoxon Test ◽  
Cd4 Cells ◽  
Similar Time ◽  
Ifn Gamma ◽  
Hematopoietic Stem ◽  
Cd4 Cell ◽  
Cd4 Lymphocytes

Abstract Abstract 1160 Poster Board I-182 Acute GvHD is a major factor affecting the outcome of hematopoietic stem cell transplantation (HSCT). In this study the focus was on the presence of IL-17-producing lymphocytes in patients post-HSCT with respect to FoxP3-positive lymphocytes and those with IFNgamma generation potential. The cytoplasmic expressions of IL-17, FoxP3, and IFN-gamma were studied in stimulated PBMCs (brefeldin A, Ionomycin, and PMA) of alloHSCT patients (43 patients, median age: 45 yrs, range; 1.0-64 yrs, with 41 hematological malignancies and 2 SCID).The cells were labeled with CD4, IL-17A, IFN-gamma, and FoxP3 MoAbs and analyzed in the CD4+ lymphocyte subpopulation. Twenty patients received MUD and 23 SIB transplants. Twenty developed aGvHD, among them 7 patients with aGvHD seen at a later stage after hematological reconstitution and 4 patients with recurrent clinical manifestation of aGvHD. It was found that: (1) the percentage of IL-17-producing CD4+ lymphocytes was higher in patients without aGvHD at the beginning of hematological reconstitution than in healthy controls (0.65±0.11% vs. 0.19±0.06%, p=0.02), (2) aGvHD cases with disease onset at the time of hematological reconstitution had lower proportions of IL-17A-producing CD4+ lymphocytes than those without aGvHD at a similar time after HSCT (0.31±0.1% vs. 0.65±0.11%, p=0.04), (3) when individual patients were followed it became apparent that the proportion of IL-17A-producing CD4+ lymphocytes, increased one week prior to aGvHD compared with the preceding observation (1.16±0.43% vs. 0.77±0.33%) and then a significant decrease was seen when symptoms became apparent (1.16±0.43% vs. 0.36±0.16%, p=0.017, Wilcoxon Test), (4) a high degree of correlation was found between the percentages of CD4+IL-17A+ and IFNgamma+CD4+ lymphocytes in blood at the time of hematological recovery (r=0.55, p=0.007), (5) patients with a higher percentage of IL-17A-producing cells in the CD4+ cell population more frequently had a higher proportion of FoxP3 in CD4+ cells (chi2=5.67, p=0.017), (6) the percentage of IFNgamma-producing CD4+ cells correlated with the proportion of FoxP3+ cells in the CD4+ cell population (r=0.48, p=0.02), and (7) the percentage of IL-17A-generating CD4+ cells rather inversely correlated with the proportion of CCR7+ lymphocytes in the blood of patients at the beginning of hematological recovery (r=-0.35, p=0.10). In conclusion Allogeneic HSCT induces the generation of IL-17A by CD4+ lymphocytes, especially in populations with lower proportions of naïve (CCR7+) lymphocytes, in response to alloantigens (the highest values were seen prior to the overt manifestation of aGvHD), which correlates with an increase in FoxP3+CD4+ cells. IL-17A-producing cells are likely to contribute to target organ lesions as they disappear from the blood at the time of overt manifestation of aGvHD. Supported by Grant no. 2P05E 037 30 from the Polish Ministry of Science & Higher Education. Disclosures No relevant conflicts of interest to declare.

Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3584-3584
Author(s):  
Takahiro Shima ◽  
Yoshikane Kikushige ◽  
Toshihiro Miyamoto ◽  
Koichi Akashi
Keyword(s):  
Stem Cells ◽  
Acute Myelogenous Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Myelogenous Leukemia ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Kit Mutation ◽  
Acute Myelogenous

Abstract Abstract 3584 The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo. Disclosures: No relevant conflicts of interest to declare.

Selective Accumulation of CD45RO+CCR7- in the Marrow Aspirates in Patients with Essential Thrombocythemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5155-5155
Author(s):  
Andrzej Lange ◽  
Mariola Sedzimirska ◽  
Aleksandra Klimczak ◽  
Daria Drabczak Skrzypek
Keyword(s):  
Essential Thrombocythemia ◽  
T Regulatory Cells ◽  
Conflicts Of Interest ◽  
Wilcoxon Test ◽  
Effector Memory ◽  
Present Observation ◽  
Becton Dickinson ◽  
Normal Representation ◽  
Selective Accumulation ◽  
Cd4 Lymphocytes

Abstract Abstract 5155 Patients with newly diagnosed essential thrombocythemia (ET) were investigated for selective accumulation/expansion of CD4+ lymphocytes subpopulations in the marrow, to verifie the hypothesis that the neoplastic process associated factor(s) are recognized by the immune system. Sixteen patients (F/M: 12/4, age 28–72 - median 60 yrs) with ET diagnosed according to the WHO criteria were studied. They had platelets from 496–809 x 10̂3/ul (median 644 × 10̂3 ), (12 heterozygotes, 2 homozygotes). Cytological evaluation revealed normal differentiation of erythroid and myeloid lineages, in contrast an increased number of megakaryocytes often enlarged, hyperlobulated and in clusters were seen in all patients. Trephine biopsy revealed activation of megakaryopoiesis with enlarged numerous megakaryocytes with normal representation of other lineages and a lack of an increase in the reticulin fibers. Marrow cellularity was from 9 – 126 x10̂3/ul (median 42 × 10̂3/ul). The whole populations of marrow and blood cells were labeled for a purpose of this study with the use CD4, CD25, CD45RO and CCR7 MoAb (Becton Dickinson, San Jose, CA, UmarSA) followed by detection and analysis with the use of flow cytometry (FACS Calibur, Becton Dickinson) equipped with PCLysis programe. Immunostaining of trephine biopsies was used for detection of FOXP3 cells. It was found: (i) Lymphocytes in the marrow were in proportions from 4,5%-15,7% (median 7,25%), in numbers from1377/ul to 8245/ul (median 3870/ul) what was significantly higher as compared to blood from 1300/ul to 3900/ul (median 2100/ul) (Wilcoxon Matched Pairs Test, p=0.010). (ii) CD45RO+CCR7- lymphocytes were higher in numbers in the marrow aspirates as compared to blood (median 1281/ul vs 631,2/ul, p=0,04 Wilcoxon Test for pairs). (iii) In contrast numbers in the marrow aspirates of CD45 RO-CCR7+ (median 297,4/ul vs median 238,2/ul), as well as CD45 RO+CCR7+ (median 149,6/ul vs 146,4/ul) did not differ from those in blood. (iv) It was a significant increase of CD4+CD25high+ cells in the marrow aspirates as compared to blood (median 36,2/ul vs 9,1/ul; p=0,02 Wilcoxon Test for pairs). Indeed in 11 out of 14 trephine biopsies investigated FoxP3+ cells were identified. In conclusion an increase, in the marrow of ET patients, of effector/memory CD4 lymphocytes (CD45RO+CCR7-) but not those of naïve and central memory lymphocytes phenotypes points out on the selective accumulation of these cells in the marrows. Concomitant increase of T regulatory cells (CD4+CD25+high) suggests that an active immunological is taking place at the site. It is possible that effector memory cells are attracted to come to the marrows by ET process associated factors and if so the present observation constitute the primary observation building the rationale behind immunotherapy in ET patients Disclosures: No relevant conflicts of interest to declare.

Correlation Between Severity of cGvHD and Transplant Outcome: A Retrospective Monocenter Study Based on NIH Classification,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4088-4088
Author(s):  
Colombe Saillard ◽  
Roberto Crocchiolo ◽  
Sabine Furst ◽  
Jean El-Cheikh ◽  
Luca Castagna ◽  
...  
Keyword(s):  
Stem Cells ◽  
Disease Risk ◽  
Conflicts Of Interest ◽  
Immunosuppressive Treatment ◽  
Conditioning Regimen ◽  
Unrelated Donor ◽  
Extensive Form ◽  
Hematopoietic Stem ◽  
Transplant Outcome ◽  
Significant Difference

Abstract Abstract 4088 Background: Chronic graft-versus-host disease (cGvHD) after allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies is associated with lower relapse rate, due to graft-versus-tumor effect. We know that the extensive form is associated with higher transplant-related mortality after myeloablative conditioning regimen, mainly due to infectious complications as a consequence of immunosuppressive treatment. Beside the “classical” Seattle classification (limited or extensive form), a recent classification (from National Institute of Health, NIH) distinguishes three levels of severity: limited, moderate and severe. We compare here both classifications for patients receiving reduced-intensity conditioning (RIC) transplant and looked for any association of cGvHD severity with transplant outcome. Patients and Methods: We evaluated data on all adult patients with hematological lymphoid or myeloid malignancies who received HSCT from related or unrelated donor, using peripheral blood stem cells, after RIC regimens (with fludarabine-busulfan-ATG) between 1998 and 2010 at the Institut Paoli-Calmettes (Marseille, France). Data on main pre- and post-transplant variables were collected; cGvHD was classified according to its presentation and severity (with both Seattle and NIH classifications) and was correlated with overall survival (OS), non relapse mortality (NRM), and relapse. cGvHD was considered as time-dependent variable, and was included in uni- and multivariate models, after adjusting for age, disease risk, HLA compatibility, graft source and comorbidity score. Relapse or death before cGvHD was considered as a competing event. Results: 283 patients were evaluated, 121 have developed cGvHD (27 limited forms and 94 extensive forms), 162 have not, for an incidence rate of 10% and 33% of limited and extensive forms respectively. Median follow up was 607 days, patients had a median age of 50 years, transplanted for acute leukemia (55), lymphoma (78), multiple myeloma (49), myelodysplastic syndrome (24), CLL (12), CML (16) or others malignancies (19). Peripheral stem cells were mostly used (294 versus 20 bone marrow graft). We had 241 related donors and 77 unrelated donors. The median day of cGvHD occurrence was 132, we found 52 de novo forms, 40 quiescent and 26 progressive forms. After reclassification with NIH criteria, we obtained 28 mild, 52 moderate and 41 severe forms. 22 of 27 limited forms were classified as mild, the extensive forms were divided into 49 moderate and 39 severe forms. In multivariate analysis, mild and moderate forms were associated with better OS compared with other groups. Severe cGvHD was associated with significant increase in NRM. Among the other variables, only age was statistically significative in OS and NRM models. Although the incidence of relapse was lower in patients with cGvHD compared with those without, no significant difference was seen between the 3 groups of patients presenting it. Conclusion: Following a fludarabine-busulfan-ATG RIC, it seems that mild to moderate cGVHD forms are associated with better OS than patients without or with severe cGVHD. This is related to lower NRM than patients with severe cGVHD and at least a comparable antitumoral effect with respect to patients without cGVHD. This invites developing strategies limiting severity but not abrogating the effect of cGVHD. Disclosures: No relevant conflicts of interest to declare.

Favorable Results of Allo-HSCT from MRD in Patients with Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5934-5934
Author(s):  
Miroslaw Markiewicz ◽  
Monika Dzierzak-Mietla ◽  
Patrycja Zielinska ◽  
Agata Wieczorkiewicz-Kabut ◽  
Sylwia Mizia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Collagen Type ◽  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Graft Loss ◽  
Chronic Phase ◽  
Curative Treatment ◽  
Unrelated Donor ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract Introduction: Myelofibrosis (MF), chronic myeloid malignancy associated with shortened survival, in majority of patients develops de novo as Primary MF, but also polycythemia vera (PV) or essential thrombocythemia (ET) may progress into post-PV or post-ET MF. Although management of MF includes several treatment options, the only potentially curative treatment approach in MF is allogeneic hematopoietic stem cell transplantation (allo-HSCT). Aim of this study was to evaluate the results of allo-HSCT in patients with MF treated in Katowice, Poland. Material and Methods: 27 pts (14 male and 13 female) with median age 51 years (range 21–63) were treated with allo-HCT due to PMF (20), post-PV (4) or post-ET (3) MF. 11,7,11,26 and 41% of pts had DIPSS 0,1,2,3 and 4, respectively. Median bone marrow cellularity was 70% (10-100%), fibrosis was collagen-type (14 pts including 2 with osteosclerosis), reticulin (10) or it was not specified (3). Splenomegaly was present in all pts: 13-20 cm (14 pts), > 20 cm (13 pts). JAK2V617F point mutation was present in 18 pts. Karyotype was available in 14 pts: in 9 normal, in 5 with variable abnormalities. Median time from diagnosis to allo-HCT was 1.5 (0.4–9.5) years. 16 pts (59.3%) received cells from HLA-matched related donor (MRD), 11 pts (40.7%) from unrelated donor: 10/10 (9) or 9/10 (2) HLA-A,B,C,DR,DQ alleles matched. Reduced intensity conditioning (RIC) was used in 26 pts, 1 patient received myeloablative conditioning (MC). Sources of stem cells were: peripheral blood (21), bone marrow (4) and both (2). All pts but one had chronic phase of MF at time of transplantation. Results: 14/27 (52%) pts are alive at median 3.4 (0.4-5.4) years after allo-HSCT: 11/16(69%) from MRD and 3/11(27%) from MUD, p=0.032. Graft failure, graft loss or PRCA were observed in 3, 5 and 1 pt, respectively. Absolute neutrophil count >0.5×109/L and platelet count >50×109/L were achieved at median 16 and 28 days, respectively. 12/27 (44%) pts reached complete blood count of Hb>10 g/dl, Plt>100 G/l and WBC>3.5 G/l; 11 of them (92%) are alive. 6/27 (22%) pts remained either RBC or PLT transfusions dependent post-transplant; 3 of them (50%) died. 9/27 (33%) pts remained both RBC and PLT transfusion dependent and all of them died. JAK2V617F mutation was completely eradicated in 11/16 evaluated previously positive patients (69%), decreased in 4 (25%) and stable in 1(6%) pt. Acute graft-versus-host disease (aGVHD) III-IV developed in 5/27 (19%) and extensive chronic GVHD in 5/19 (26%) pts. Relapse occurred in 4 pts and was treated with subsequent second transplant (in 1 pt thereafter by 3-rd allo-HSCT). Spleen length decreased at median by 5 (0.3-9.2) cm. Out of 7 pts with initial collagen fibrosis who were evaluated post-transplant, 1 had no fibrosis, 5 reticulin type and only in 1 pt collagen fibrosis was stable. Out of 3 pts with initial reticulin fibrosis it disappeared in 2 and progressed to collagen type in 1. Causes of death were GVHD (5 pts: 3 aGVHD, 2 cGVHD) and pancytopenia with either infection (7 pts) or CNS hemorrhage (1 pt). Conclusions: Allo-HSCT, the only curative treatment of myelofibrosis, provides chance of long survival, regression of the disease (lower stage of fibrosis, JAK2V617F eradication) and improved quality of life (transfusion independency, decreased splenomegaly). Transfusion independency may indicate good outcome. Favorable results are observed after allo-HSCT from MRD. Disclosures No relevant conflicts of interest to declare.

HLA and Kir Mismatch Transplant Affect Cytopenia Beyond Day 28 after Thalassemia Transplant: A Primary Manifestation of Chronic Graft Versus Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5929-5929
Author(s):  
Xiaohui Zhou ◽  
Chunfu Li ◽  
Jianyun Liao ◽  
Xiangjun Liu
Keyword(s):  
Conflicts Of Interest ◽  
Hla Class I ◽  
Thalassemia Major ◽  
Class I ◽  
Unrelated Donor ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Hla Antibody ◽  
Cmv Status

Abstract Background Cytopenia beyond day 28 post-transplant (CB-28PT) following hematopoietic stem cell transplantation (HSCT) with β-thalassemia major (TM) rarely was reported. The exact mechanism for the development of CB-28PT is not well known. Aim To find out causes of CB-28PT cytopenia. Method We retrospectively analyzed data (HLA mismatch status, HLA antibody status of patients, KIR gene mismatch status, KIR-ligand matching status, donor/patient CMV status, donor/patient age and sex) of 93 TM patients underwent HLA 8/8 fully matched or 7/8 matched unrelated donor HSCT. All the patients used sole NF-08-TM protocol with median follow-up time of 19 (r: 2-44) months. Results Results show a significant association between DRB1 mismatch and CB-28PT (P = 0.012). In addition, presence of Class I HLA antibody in the patient’s sera seems increase the chance of CB-28PT. Finally, the matching between inhibitory KIR2DL1 and their corresponding ligand HLA-C2 has a protective effect for CB-28PT. Conclusion We propose that CB-28PT may be a primary manifestation of cGVHD in pediatric TM patients undergoing HSCT positive influenced by HLA DRB1 mismatch, HLA class I antibody and negatively affected by KIR-ligand match. Disclosures No relevant conflicts of interest to declare.

Low Density Granulocytes in Patients after Allogeneic Hematopoietic Stem Cells Transplantation (allo-HSCT): A Distinct Class of Neutrophils in Systemic Alloimmunity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4614-4614
Author(s):  
Ekaterina Mikhaltsova ◽  
Valeri G. Savchenko ◽  
Larisa A. Kuzmina ◽  
Mikhail Drokov ◽  
Vera Vasilyeva ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Peripheral Blood ◽  
Hematological Malignancies ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Unrelated Donor ◽  
Low Density ◽  
Hematopoietic Stem

Abstract Introduction It's generally considered that all alloimmune process such as acute graft-versus host disease (aGVHD) after allo-HSCT are mostly controlled by lymphocytes. The role of neutrophils in systemic alloimmunity after allo-HSCT is still illusive. In 1987 a distinct subset of proinflammatory, low-density granulocytes (LDGs) isolated from the peripheral blood mononuclear cell fractions of patients with system lupus erythematosus has been described. There is no LDG's in healthy donors. While the origin and role of LDGs still needs to be fully characterized, we try to describe this population in patients with hematological malignancies after allo-HSCT Patients and methods. Peripheral blood samples were collected in EDTA-tubes before allo-HSCT, on day +30,+60,+90 after allo-HSCT and at day of aGVHD from 47 patients with hematological malignancies (AML=22, ALL n=17, LPD=3, MDS =2; CML=2; 17 with active disease, 30 - in CR) after allo-HSCT (from matched unrelated donor n=34, from matched related donor n=13; MAC = 13, RIC=34). Isolation of mononuclear cells from human peripheral blood was made by standard protocol using Lympholyte®-M Cell Separation Media (Cedarlane Labs). The anti-CD66b-PE (Biolegend, USA) antibodies and FSC/SSC were used to determine LDGs cells as FSChigh \SSChigh \CD66b+. 100000 of cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA). Results. Results of blood evaluation of 47 patients with hematological malignancies, whose blood was examined after allo-HSCT presented in table 1. Conclusion Despite the fact that we don't get significant differences. LDG's detection in allo-HSCT patients need further investigation. Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD Disclosures No relevant conflicts of interest to declare.

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