CD8+HLA-DR+ T Cells Are Increased in Patients with Severe Aplastic Anemia

Abstract Abstract 4395 OBJECTIVES: To investigate the number and function of CD8+HLA-DR+ cells, which were considered to be activated CTL, in peripheral blood to further explore the pathogenesis of SAA. METHODS: The proportion of CD8+HLA-DR+T cells was analyzed by flow cytometry in peripheral blood of 38 SAA (26 untreated and 12 recovered) patients and 23 normal controls. Also the expression of perforin, granzyme B, TNF-β and FasL in CD8+HLA-DR+T cells was analyzed by flow cytometry and RT-PCR. Moreover, the apoptosis of CD3- bone marrow cells from normal persons after coculture with CD8+HLA-DR+T cells from untreated SAA patients was detected by staining with AnnexinV. The level of lactate dehydrogenase (LDH) in the supernatant were determined by automatic biochemistry analyzer. RESULTS: The ratios of CD8+HLA-DR+T cells to CD8+T cells and to CD3+T cells were 39.3 ± 8.1% and 27.8±7.1% in SAA patients, and were significantly higher than the corresponding ratios in controls (18.3 ± 6.7% and 8.5 ± 2.3%) (P < 0.05). The expression values for perforin, granzyme B, TNF-β and FasL in CD8+HLA-DR+T cells in the untreated SAA group were 8.5%, 96.1%, 94.3% and 72.1%, respectively, which were higher than the corresponding values in the control group (1.8%, 82.0%, 32.9%, 15.6%). The mRNA expression values for perforin, granzyme B, TNF-β and FasL in CD8+HLA-DR+T cells in the untreated SAA group were 0.66±0.25, 0.56±0.26, 0.61±0.16, 0.77±0.24, respectively, which were significantly higher than those in the control group (0.53±0.14, 0.40±0.13, 0.46±0.15, 0.58±0.16). (P < 0.05). The apoptosis values in SAA group 1 (CD8+HLA-DR+ T cells from untreated SAA patients and CD3- bone marrow mononuclear cells from remission patients), SAA group 2 (CD8+HLA-DR+ T cells from untreated SAA patients and CD3- bone marrow mononuclear cells from normal controls), the remission group (CD8+HLA-DR+ T cells and CD3- bone marrow mononuclear cells from remission patients) and the normal control (CD8+HLA-DR+ T cells and CD3- bone marrow mononuclear cells from normal controls) were 41.12 ± 24.84%, 45.81 ± 20.47%, 35.03 ± 22.09%, 20.95 ± 13.82%. There were no significant differences between SAA group 1, SAA group 2, and the remission group (P > 0.05). However, the apoptosis in each of these groups was higher than in the normal control (P < 0.05). The LDH levels in SAA group 1, SAA group 2, the remission group and normal control were 74.56 ± 49.13 U/L, 62.61 ± 31.76 U/L, 61.06 ± 28.41 U/L, and 28.60 ± 8.91 U/L. There were no significant differences between SAA group 1, SAA group 2, and the remission group (P > 0.05). However, the level of LDH in these groups was significantly higher than in the normal control (P < 0.05). CONCLUSION: CD8+HLA-DR+T cells might contribute to bone marrow failure in SAA. Immunosuppressive therapy dramatically reduced the quantity and function of CD8+HLA-DR+T cells, and inhibitors of CD8+HLA-DR+T cells or other factors involved in this pathway will be attractive therapeutic targets in SAA. Disclosures: No relevant conflicts of interest to declare.

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Study on the Pathways to Damage Hematopoiesis by CD8+ Effector T Cells of the Patients with Severe Aplastic Anemia

Blood ◽

10.1182/blood.v118.21.4371.4371 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4371-4371

Author(s):

Zonghong Shao ◽

Le Feng ◽

Rong Fu ◽

Jun Wang ◽

Chunyan Liu ◽

...

Keyword(s):

T Cells ◽

Aplastic Anemia ◽

Cd8 T Cells ◽

Bone Marrow Failure ◽

Granzyme B ◽

Effector T Cells ◽

Severe Aplastic Anemia ◽

Control Group ◽

Hla Dr ◽

Normal Controls

Abstract Abstract 4371 Objective To investigate the quantity and their pathways to damage hematopoietic cells of CD8+CD25+ and CD8+HLA-DR+ effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia(SAA) and explore the heterogeneous immunopathogenesis of SAA further. Methods The quantity of CD8+CD25+and CD8+HLA-DR+ cells in PB and the expressions of perforin, granzyme B, tumor necrosis factor-β(TNF -β) and FasL of 29 SAA (14 untreated and 15 recovered) patients and 12 normal controls were analyzed by flow cytometry. Results The ratio of CD8+CD25+T cells in CD8+ T cells was (3.67±2.58)% in untreated SAA patients, (5.19±4.29)% in recovered patients and (4.84±2.31)% in normal controls, and the ratios of CD8+CD25+T cells in CD3+ cells in three groups were (2.25±1.35)%, (2.98±1.35)% and (2.11±1.88)% respectively. There was no statistic difference among 3 groups(P>0.05). The ratio of CD8+HLA-DR+T cells in CD8+T cells was (39.30±8.13)% in untreated patients, which was significantly higher than that of recovered patients[(20.65±5.38%)] and controls [(18.34±6.68%)](P<0.001). There was no statistic difference between recovered patients and controls(P>0.05). CD8+HLA-DR+T cells in CD3+ cells was (27.81±7.10)% in untreated group, higher than that of recovered patients (12.02±3.03)% and controls(8.50±2.33)%(P<0.01). And the ratio in recovered group was higher than in control group(P<0.05). The expressions of perforin, granzyme B, TNF-β and FasL of CD8+HLA-DR+ T cells of untreated SAA patients were 8.51% A96.08% A72.11% and 94.25% respectively, higher than those of recovered patients(1.78% A85.20% A34.38% A51.20%)and controls(1.86% A82.09% A17.92% A32.91%). There was no statistic difference between recovered patients and controls(P>0.05). Conclusion There were elevated quantity of CD8+HLA-DR+ T cells and high expressions of perforin, granzyme B, TNF-β and FasL in SAA, which might contribute to the bone marrow failure of SAA. Disclosures: No relevant conflicts of interest to declare.

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Expansion and Characterization of Iovance Marrow Infiltrating Lymphocytes: A Potential Novel Therapeutic Strategy for the Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114825 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5683-5683

Author(s):

Lavakumar Karyampudi ◽

Ian Frank ◽

Michelle Blaskovich ◽

John C. Byrd ◽

Cecile Chartier ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Tumor Microenvironment ◽

Mononuclear Cells ◽

Immune Cell ◽

T Cell Subsets ◽

Bone Marrow Mononuclear Cells ◽

Equity Ownership ◽

Cell Fractions ◽

Infiltrating Lymphocytes

Abstract Background: Acute myeloid leukemia (AML) is a poor-prognosis malignancy arising from hematopoietic stem/progenitor cells. To date, novel immunotherapies such as checkpoint inhibitors, vaccines and adoptive cell therapy (ACT) using CAR T cells have demonstrated only modest success for the treatment of patients who are ineligible for marrow transplantation and have minimal residual disease; additional approaches are warranted (Beyar-Katz O and Gill S, Clin Cancer Res 2018). ACT with tumor infiltrating lymphocytes (TIL) has emerged as an effective treatment for patients with metastatic melanoma (Goff SL et al, J Clin Oncol 2016), likely owing to the heterogeneous population of tumor-reactive T cells that comprises the TIL products. As demonstrated for solid cancers, such tumor-reactive T cells are preferentially found in the tumor microenvironment (Gros A et al JCI 2014; Thommen DS et al Nat Med 2018). By avoiding the highly immunosuppressive tumor microenvironment, ex vivo activation of those cells rescues them from tolerance and anergic status. We hypothesized that, in the case of AML for which the bone marrow represents the tumor microenvironment, tumor antigen-specific T cells could be recovered from the patient bone marrow to produce a highly effective therapeutic product that is cytotoxic to AML tumor cells. We present findings related to the ex vivo expansion of Iovance marrow infiltrating lymphocytes (MIL) for the treatment of AML patients. Methods: Immune cell and non-immune cell fractions were sorted from bone marrow mononuclear cells. Immune cell fractions loaded with sonicated non-immune cell fractions were expanded for 14 days in the presence of αCD3/αCD28 beads and interleukin-2 (IL-2) to generate MIL products. Phenotypic and functional characteristics of the cells were determined by flow cytometry and enzyme-linked immunospot assay (ELISpot). Results: MIL were generated from isolated bone marrow mononuclear cells (n=2) with a mean expansion fold of 86 (range 78-93). Equal percentage of CD4+ and CD8+ T cell subsets constituted the MIL products. Phenotypic analysis of the cells showed that the majority of T cell subsets are effector memory and CD28 positive. Low percentages of the T cell subsets were positive for immunosuppressive markers PD-1 and LAG3. ELISpot analysis demonstrated that MIL were readily activatable and produced normal levels of IFNγ in response to CD3/CD28 stimulation. Antigen specificity of MIL is being investigated. Conclusion: We demonstrated the feasibility of MIL expansion from bone marrow mononuclear cells from AML patients. MIL are functionally active and mostly comprised of effector memory T cells. Confirmation of tumor cell antigen specificity will determine whether MIL may deploy a robust anti-tumor activity in vivo. Disclosures Karyampudi: Iovance Biotherapeutics: Employment, Equity Ownership. Frank:Iovance Biotherapeutics: Employment, Equity Ownership. Blaskovich:Iovance Biotherapeutics: Equity Ownership. Chartier:Iovance Biotherapeutics: Equity Ownership.

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Correlation of Acute GVHD with CD4+CD25+ Regulatory T Cells of the Donates and Receipients in Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v106.11.5235.5235 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5235-5235

Author(s):

Zhai Zhi Min

Keyword(s):

T Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Normal Control ◽

Acute Gvhd ◽

Hematopoietic Stem ◽

Group 2 ◽

Group 1

Abstract Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (aHSCT). CD4+CD25+ T cells are characterized in suppression of autoimmunity and induction of transplantation tolerance. Recently several studies have shown that CD4+CD25+ T cells from donor are able to effectively suppress acute GVHD (aGVHD) in murine models. However the relationship of aGVHD with the levels of CD4+CD25+ T cells in donor and receipients undergoing aHSCT in human is not clear. Here we examined the CD4+CD25+ T cells in both donors and receipients after aHSCT. Methods: donors and receipients were divided into two groups: group 1 without or with grade I aGVHD and group 2 with aGVHD above grade II. Age match with donors and receipients of normal peoples were taken as control separately. aGVHD was diagnosised and graded according to standard criteria. The peripheral blood was obtained from the donors 1 week before aHSCT, and receipients 4weeks after aHSCT for treatment of malignancy, mostly chronic myelogenous leukemia. CD4+CD25+ T cells and CD4+CD25+ bright were assessed by double color immunoflurecence labeling and FACScan. The ratio of CD4+CD25+ T cells and CD4+CD25+ bright in CD4 T cells was calculated. Results: There is no difference in CD4+CD25+ T cells between normal control and group 1 of the donors who’s stem cells were infused into the receipients underwent grade I aGVHD. CD4+CD25+ T cells were significantly decreased in group 2 of donors compared to normal control and group 1 of donors (see table 1). CD4+CD25+ T cells and the CD4+CD25bright T cells subpopulations in group 2 of receipients were significantly lower than normal control and group 1 of receipients (see table 2). Summary: the severity of aGVHD is correlated to the levels of CD4+CD25+ T cells in both donors and receipients after aHSCT. aGVHD occur less in the donor with normal levels of CD4+CD25+ T cells. Lower levels of CD4+CD25+ T cells in receipients indicate high incidents of server aGVHD. These data suggested that CD4+CD25+ T cells could have important role in prevention of aGVHD. Table 1. lymphocytes, CD4+ T cells, CD4+CD25+ and CD4+CD25bright T cells (%) in donates lymphocytes lymphocytes CD4+ CD4+CD25+ CD4+CD25bright *P<0.01 vs normal control and group 1. **P< 0.001 vs normal control and group 1 Normal control n=10 34.27±8.62 37.25±5.38 11.29±1.89 1.36±0.53 Group 1 donate n=6 33.65±10.06 36.43±9.50 11.23±1.69 1.41±0.76 Group 2 donate n=4 23.90±11.67 32.57±3.40 5.64±1.57** 0.69±0.48* Table 2. WBC count, CD4+CD25+ and the CD4+CD25bright T cells in receipients (%) WBC(×109) CD4+CD25+ CD4+CD25bright *P<0.01 vs normal control and group 1. **P< 0.001 vs normal control and group 1 Normal n= 10 6.38±1.23 11.29±1.89 1.36±0.53 Group 1 n= 6 5.16±4.49 17.10±4.19 2.23±1.52 Group 2 n=4 4.34±2.85 1.52±0.61** 0.00±0.00*

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