scholarly journals In vitro tests for distinguishing possible immune-mediated aplastic anemia from transfusion-induced sensitization

Blood ◽  
1980 ◽  
Vol 55 (2) ◽  
pp. 211-215
Author(s):  
BJ Torok-Storb ◽  
C Sieff ◽  
R Storb ◽  
J Adamson ◽  
ED Thomas
Keyword(s):  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Colony Growth ◽  
Cell Depletion ◽  
In Vitro Tests ◽  
T Cell Depletion ◽  
Immune Mediated ◽  
Before And After

Forty-two patients with aplastic anemia (AA) were studied to determine whether or not transfusion-induced sensitization is responsible for the in vitro inhibition by patient lymphocytes of HLA-identical erythroid burst-forming units (BFU-E). The results indicate that lymphocytes from 12 of 34 transfused patients inhibited normal colony growth. In contrast, lymphocytes from none of the 8 untransfused patients demonstrated inhibition. These data were interpreted to mean that coculture studies would not be useful for identifying immune-mediated AA in transfused patients. Therefore, in order to identify possible immune-related AA, we assayed BFU-E from patient blood before and after T-cell depletion. In all 32 patients studied, BFU-E failed to grow from peripheral blood cells before T-cell depletion, but in 8 cases, normal- appearing BFU-E grew after T cells had been removed. Growth of patient BFU-E colonies was inhibited in 6 cases when patient T cells were added back to the culture, indicating that in these 6 patients, an “autoimmune” mechanism may have been present.

scholarly journals In vitro tests for distinguishing possible immune-mediated aplastic anemia from transfusion-induced sensitization

Blood ◽  
1980 ◽  
Vol 55 (2) ◽  
pp. 211-215 ◽  
Author(s):  
BJ Torok-Storb ◽  
C Sieff ◽  
R Storb ◽  
J Adamson ◽  
ED Thomas
Keyword(s):  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Colony Growth ◽  
Cell Depletion ◽  
In Vitro Tests ◽  
T Cell Depletion ◽  
Immune Mediated ◽  
Before And After

Abstract Forty-two patients with aplastic anemia (AA) were studied to determine whether or not transfusion-induced sensitization is responsible for the in vitro inhibition by patient lymphocytes of HLA-identical erythroid burst-forming units (BFU-E). The results indicate that lymphocytes from 12 of 34 transfused patients inhibited normal colony growth. In contrast, lymphocytes from none of the 8 untransfused patients demonstrated inhibition. These data were interpreted to mean that coculture studies would not be useful for identifying immune-mediated AA in transfused patients. Therefore, in order to identify possible immune-related AA, we assayed BFU-E from patient blood before and after T-cell depletion. In all 32 patients studied, BFU-E failed to grow from peripheral blood cells before T-cell depletion, but in 8 cases, normal- appearing BFU-E grew after T cells had been removed. Growth of patient BFU-E colonies was inhibited in 6 cases when patient T cells were added back to the culture, indicating that in these 6 patients, an “autoimmune” mechanism may have been present.

scholarly journals Human bone marrow and peripheral blood T lymphocyte depletion: efficacy and effects of both T cells and monocytes on growth of hematopoietic progenitors

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 663-679
Author(s):  
L Levitt ◽  
TJ Kipps ◽  
EG Engleman ◽  
PL Greenberg
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocyte ◽  
Peripheral Blood ◽  
Cell Depletion ◽  
Target Cells ◽  
Facs Analysis ◽  
Hematopoietic Progenitors ◽  
T Cell Depletion

The efficacy of four separate methods of human bone marrow T lymphocyte depletion was assessed, and the effect of T cells and monocytes on in vitro growth of marrow (CFU-GEMM, BFU-E, and CFU-GM) and peripheral blood (BFU-E) hematopoietic progenitors was determined. Extent of T cell depletion was assessed by multiparameter fluorescent cell sorter (FACS) analysis and by functional studies. Cells staining positively by FACS analysis for one or more of three separate fluorescent pan-T cell monoclonal antibodies (MCAbs) comprised 8.4% to 9.5% of control marrow mononuclear cells (MNCs). T cells constituted 3.2% to 5.1% of marrow following single, sequential, or combination treatment with two different pan-T cell MCAbs (Leu 1 and TM1) plus complement, 1.5% to 2.2% of marrow following solid-phase immunoabsorption (“panning”), 0.2% of marrow after sheep cell rosetting, and only 0.05% of marrow after FACS selective cell sorting and gated separation. T cells made up 59% to 73% of control peripheral blood MNCs and 0.8% to 2.8% of peripheral MNCs following sheep cell rosetting plus treatment with Leu 1 MCAb and complement. Mitogen (PHA, Con A) and allogeneic MLC-induced blastogenic responses (stimulation indices, experimental/control or E/C) revealed a concordant decrement in marrow T cell function after MCAb plus complement (E/C of 3.9 to 9.0), after panning (E/C of 1.6 to 3.5) and after sheep cell rosetting (E/C of 0.7 to 1.3), compared with control marrow (E/C of 5.3 to 15.7). After T cell depletion, marrow BFU-E growth was 95% to 120% of control, CFU-GM growth was 90% to 108% of control, and CFU-GEMM growth was 89% to 111% of control. Marrow T cell and/or monocyte depletion did not alter erythropoietin-dependent BFU-E growth in the absence of Mo-conditioned medium (81% to 95% of control), and the addition of as many as 50 to 100 X 10(3) purified marrow monocytes or T cells to 10(5) autologous nonadherent T cell-depleted marrow target cells had a negligible (P greater than .1) effect on marrow BFU-E growth in vitro. Peripheral blood (PB) BFU-E/10(5) T- depleted target cells were 106% +/- 19% of expected; PB BFU-E growth was significantly diminished after monocyte depletion alone (7% +/- 6% of expected) or after monocyte plus T cell depletion (8% +/- 4% of expected).(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Impact of Different T Cell Depletion Protocols on Immune Reconstitution Following Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-44
Author(s):  
Amandine Pradier ◽  
Adrien Petitpas ◽  
Anne-Claire Mamez ◽  
Federica Giannotti ◽  
Sarah Morin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Cell Depletion ◽  
Unrelated Donor ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapeutic modality for a variety of hematological malignancies and congenital disorders. One of the major complications of the procedure is graft-versus-host-disease (GVHD) initiated by T cells co-administered with the graft. Removal of donor T cells from the graft is a widely employed and effective strategy to prevent GVHD, although its impact on post-transplant immune reconstitution might significantly affect anti-tumor and anti-infectious responses. Several approaches of T cell depletion (TCD) exist, including in vivo depletion using anti-thymocyte globulin (ATG) and/or post-transplant cyclophosphamide (PTCy) as well as in vitro manipulation of the graft. In this work, we analyzed the impact of different T cell depletion strategies on immune reconstitution after allogeneic HSCT. Methods We retrospectively analysed data from 168 patients transplanted between 2015 and 2019 at Geneva University Hospitals. In our center, several methods for TCD are being used, alone or in combination: 1) In vivo T cell depletion using ATG (ATG-Thymoglobulin 7.5 mg/kg or ATG-Fresenius 25 mg/kg); 2) in vitro partial T cell depletion (pTCD) of the graft obtained through in vitro incubation with alemtuzumab (Campath [Genzyme Corporation, Cambridge, MA]), washed before infusion and administered at day 0, followed on day +1 by an add-back of unmanipulated grafts containing about 100 × 106/kg donor T cells. The procedure is followed by donor lymphocyte infusions at incremental doses starting with 1 × 106 CD3/kg at 3 months to all patients who had received pTCD grafts with RIC in the absence of GVHD; 3) post-transplant cyclophosphamide (PTCy; 50 mg/kg) on days 3 and 4 post-HSCT. Absolute counts of CD3, CD4, CD8, CD19 and NK cells measured by flow cytometry during the first year after allogeneic HSCT were analyzed. Measures obtained from patients with mixed donor chimerism or after therapeutic DLI were excluded from the analysis. Cell numbers during time were compared using mixed-effects linear models depending on the TCD. Multivariable analysis was performed taking into account the impact of clinical factors differing between patients groups (patient's age, donor type and conditioning). Results ATG was administered to 77 (46%) patients, 15 (9%) patients received a pTCD graft and 26 (15%) patients received a combination of both ATG and pTCD graft. 24 (14%) patients were treated with PTCy and 26 (15%) patients received a T replete graft. 60% of patients had a reduced intensity conditioning (RIC). 48 (29%) patients received grafts from a sibling identical donor, 94 (56%) from a matched unrelated donor, 13 (8%) from mismatched unrelated donor and 13 (8%) received haploidentical grafts. TCD protocols had no significant impact on CD3 or CD8 T cell reconstitution during the first year post-HSCT (Figure 1). Conversely, CD4 T cells recovery was affected by the ATG/pTCD combination (coefficient ± SE: -67±28, p=0.019) when compared to the T cell replete group (Figure 1). Analysis of data censored for acute or chronic GVHD requiring treatment or relapse revealed a delay of CD4 T cell reconstitution in the ATG and/or pTCD treated groups on (ATG:-79±27, p=0.004; pTCD:-100±43, p=0.022; ATG/pTCD:-110±33, p<0.001). Interestingly, pTCD alone or in combination with ATG resulted in a better reconstitution of NK cells compared to T replete group (pTCD: 152±45, p<0.001; ATG/pTCD: 94±36, p=0.009; Figure 1). A similar effect of pTCD was also observed for B cells (pTCD: 170±48, p<.001; ATG/pTCD: 127±38, p<.001). The effect of pTCD on NK was confirmed when data were censored for GVHD and relapse (pTCD: 132±60, p=0.028; ATG/pTCD: 106±47, p=0.023) while only ATG/pTCD retained a significant impact on B cells (102±49, p=0.037). The use of PTCy did not affect T, NK or B cell reconstitution when compared to the T cell replete group. Conclusion Our results indicate that all TCD protocols with the only exception of PTCy are associated with a delayed recovery of CD4 T cells whereas pTCD of the graft, alone or in combination with ATG, significantly improves NK and B cell reconstitution. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals A novel protocol for haploidentical hematopoietic SCT without in vitro T-cell depletion in the treatment of severe acquired aplastic anemia

2012 ◽  
Vol 47 (12) ◽  
pp. 1507-1512 ◽  
Author(s):  
L P Xu ◽  
K Y Liu ◽  
D H Liu ◽  
W Han ◽  
H Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Aplastic Anemia ◽  
Cell Depletion ◽  
T Cell Depletion

scholarly journals Human bone marrow and peripheral blood T lymphocyte depletion: efficacy and effects of both T cells and monocytes on growth of hematopoietic progenitors

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 663-679 ◽  
Author(s):  
L Levitt ◽  
TJ Kipps ◽  
EG Engleman ◽  
PL Greenberg
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocyte ◽  
Peripheral Blood ◽  
Cell Depletion ◽  
Target Cells ◽  
Facs Analysis ◽  
Hematopoietic Progenitors ◽  
T Cell Depletion

Abstract The efficacy of four separate methods of human bone marrow T lymphocyte depletion was assessed, and the effect of T cells and monocytes on in vitro growth of marrow (CFU-GEMM, BFU-E, and CFU-GM) and peripheral blood (BFU-E) hematopoietic progenitors was determined. Extent of T cell depletion was assessed by multiparameter fluorescent cell sorter (FACS) analysis and by functional studies. Cells staining positively by FACS analysis for one or more of three separate fluorescent pan-T cell monoclonal antibodies (MCAbs) comprised 8.4% to 9.5% of control marrow mononuclear cells (MNCs). T cells constituted 3.2% to 5.1% of marrow following single, sequential, or combination treatment with two different pan-T cell MCAbs (Leu 1 and TM1) plus complement, 1.5% to 2.2% of marrow following solid-phase immunoabsorption (“panning”), 0.2% of marrow after sheep cell rosetting, and only 0.05% of marrow after FACS selective cell sorting and gated separation. T cells made up 59% to 73% of control peripheral blood MNCs and 0.8% to 2.8% of peripheral MNCs following sheep cell rosetting plus treatment with Leu 1 MCAb and complement. Mitogen (PHA, Con A) and allogeneic MLC-induced blastogenic responses (stimulation indices, experimental/control or E/C) revealed a concordant decrement in marrow T cell function after MCAb plus complement (E/C of 3.9 to 9.0), after panning (E/C of 1.6 to 3.5) and after sheep cell rosetting (E/C of 0.7 to 1.3), compared with control marrow (E/C of 5.3 to 15.7). After T cell depletion, marrow BFU-E growth was 95% to 120% of control, CFU-GM growth was 90% to 108% of control, and CFU-GEMM growth was 89% to 111% of control. Marrow T cell and/or monocyte depletion did not alter erythropoietin-dependent BFU-E growth in the absence of Mo-conditioned medium (81% to 95% of control), and the addition of as many as 50 to 100 X 10(3) purified marrow monocytes or T cells to 10(5) autologous nonadherent T cell-depleted marrow target cells had a negligible (P greater than .1) effect on marrow BFU-E growth in vitro. Peripheral blood (PB) BFU-E/10(5) T- depleted target cells were 106% +/- 19% of expected; PB BFU-E growth was significantly diminished after monocyte depletion alone (7% +/- 6% of expected) or after monocyte plus T cell depletion (8% +/- 4% of expected).(ABSTRACT TRUNCATED AT 400 WORDS)

Low Incidence of Post-Transplant EBV-Related Disease After Alemtuzumab-Mediated T Cell Depletion Is Explained by the Differential Susceptibility to Alemtuzumab of B Cells and Protective CD8 and CD4 T Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2330-2330
Author(s):  
Constantijn J.M. Halkes ◽  
Inge Jedema ◽  
Judith Olde Wolbers ◽  
Esther M van Egmond ◽  
Peter A. Von Dem Borne ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Depletion ◽  
T Cell Depletion

Abstract Abstract 2330 In vivo T cell depletion with anti-thymocyte globulin (ATG) or alemtuzumab (anti-CD52) before reduced intensity allogeneic stem cell transplantation (alloSCT) in combination with in vitro T cell depletion with alemtuzumab reduces the risk of GVHD. Detectable levels of circulating antibodies are present up to several months after the alloSCT, leading to a delayed immune reconstitution which is associated with an increased incidence of opportunistic infections and early relapses. Prior to 2007, combined in vitro (Alemtuzumab 20 mg added “to the bag”) and in vivo T cell depletion with horse-derived ATG (h-ATG) resulted in good engraftment without GVHD in the absence of GVHD prophylaxis after reduced intensity alloSCT using conditioning with fludarabine and busulphan. Due to the unavailability of h-ATG, rabbit-derived ATG (r-ATG) 10–14 mg/kg was introduced in the conditioning regimen in 2007. Strikingly, in this cohort of patients, early EBV reactivation and EBV-associated post-transplantation lymphoproliferative disease (PTLD) was observed in 10 out of 18 patients at a median time of 6 weeks after alloSCT (range 5 to 11 weeks) in the absence of GVHD or immunosuppressive treatment. Analysis of T and B cell recovery early after transplantation revealed preferential depletion of T cells as compared to B cells, thereby allowing unrestricted proliferation of EBV infected B cells. Due to this unacceptable high incidence of EBV-related complications, in the conditioning regimen r-ATG was replaced by low dose alemtuzumab (15 mg i.v. day -4 and -3) in 2008. In this cohort of 60 patients, only 2 patients experienced transient EBV reactivation during the first 3 months after alloSCT and one patient developed an EBV-associated lymphoma 4 weeks after alloSCT. To investigate the mechanisms underlying the low incidence of EBV reactivation using alemtuzumab for T cell depletion, we studied the in vivo and in vitro effects of alemtuzumab on different lymphocyte subsets. First, lineage-specific reconstitution was studied in 20 patients from the alemtuzumab cohort with known CD52 negative diseases (11 AML and 9 multiple myeloma) to exclude the confounding effect of antibody absorption by malignant cells. Whereas at 3 weeks after alloSCT detectable numbers of circulating NK cells and T cells were observed (medians 71 (range 6–378), and 12 (range 1–1164)E6/L, respectively), no circulating B cells could be detected (median 0, range 0–1 E6/L). At 6 weeks after alloSCT, NK and T cell numbers further increased (medians 212 (52-813), and 130 (range 25–1509)E6/L, respectively), whereas B cell numbers still remained low in the majority of patients (median 15, range 0–813E6/L). In all patients, T cells were detectable before the appearance of circulating B cells. Furthermore, the expression of CD52 and the sensitivity to alemtuzumab-mediated complement-dependent cell lysis (CDC) of B cells, T cells and NK cells was measured in vitro. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with 10ug/mL alemtuzumab and rabbit complement. NK cells showed a significantly lower CD52 expression (MFI 41), which was also reflected by a lower susceptibility to alemtuzumab-mediated CDC (62% lysis). Interestingly, differential expression of CD52 was observed on CD4 and CD8 T cells (MFI 120 and 101, respectively). Cytotoxicity analysis revealed relative protection of CD8 compared to CD4 T cells against alemtuzumab-mediated CDC, resulting in 52% and 90% lysis, respectively. Based on these results, we investigated in detail the presence and phenotype of the CD4 and CD8 subsets and EBV-specific CD8 T cells using tetramer staining at 6 weeks after alloSCT. In accordance with the in-vitro expression and susceptibility data, circulating CD52+ CD8 T cells including EBV-specific T cells were detectable. Interestingly, the majority of circulating CD4 T cells (64-93%, n=4) lacked CD52 expression, explaining their capacity to persist in the presence of alemtuzumab. We conclude that in vivo and in vitro T cell depletion with alemtuzumab is associated with a relatively low risk of EBV-associated PTLD because of efficient B cell depletion and persistent EBV immunity allowed by the relative insusceptibility for alemtuzumab of CD8 T cells and the development of CD52 negative escape variants of CD4 T cells. Disclosures: No relevant conflicts of interest to declare.

In Vivo T Cell Depletion Using Rabbit Derived ATG Leads to An Increased EBV-PTLD Risk Due to An Induced Imbalance Between B and T Cell Recovery Which Is Not Seen After Horse Derived ATG or Alemtuzumab

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1979-1979 ◽  
Author(s):  
C.J.M. Halkes ◽  
J.H.F. Falkenburg ◽  
H.M. van Egmond ◽  
J. Olde Wolbers ◽  
C.W.J. Starrenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd8 T Cells ◽  
Cell Depletion ◽  
Cell Recovery ◽  
Post Transplantation ◽  
T Cell Depletion

Abstract Abstract 1979 Control of replication of endogenous viruses like CMV and EBV is fully dependent on CMV or EBV specific T cells after allogeneic stem cell transplantation (alloSCT). In the absence of specific CD8 T cell control, proliferation of EBV infected B cells can lead to post transplantation lymphoproliferative disease (PTLD). In an initial cohort of patients treated with horse derived anti thymocyte globulin (h-ATG), no early PTLD was observed. However, due to unavailability in Europe, h-ATG had to be replaced by rabbit derived ATG (r-ATG), leading to an unacceptable high incidence of EBV-PTLD (26% during first 3 months after alloSCT). Replacement of r-ATG by alemtuzumab (ALT) significantly reduced the incidence of EBV-PTLD (3 months incidence of EBV-PTLD 2%). To determine the immunological basis of these findings we performed a detailed analysis of immune reconstitution in these three cohorts of transplanted patients. The first cohort (41 patients) received h-ATG (Lymphoglobulin) 10 mg/kg/day for 4 days. The second cohort (19 patients) received r-ATG (Thymoglobulin) 2.0 or 3.5 mg/kg/day for 4 days and the third cohort (60 patients) received ALT, 15 mg/day for 2 days. All grafts consisted of PBSC to which 20 mg of ALT was added for in vitro T cell depletion. All patients received a fludarabin and busulphan based conditioning regimen. No standard post transplantation immunosuppressive treatment was given. In the r-ATG cohort, early EBV-PTLD occurred after a median of 7 weeks (range 4–12 weeks) post alloSCT. Three r-ATG treated patients died while high levels of circulating EBV-DNA were present (> log 4.0 copies/mL). Incidence of CMV disease was not significantly different in the three cohorts (5%, 6% and 0%, respectively). In contrast to the other 2 cohorts, immune reconstitution in the r-ATG cohort was characterized by an imbalance between recovery of B cells and CD8 T cells. Already 3 weeks after alloSCT, the majority (67%) of r-ATG patients showed a more rapid reconstitution of B cells than CD8 T cells, leading to B cells outnumbering CD8 T cells. This was seen in only a small minority of patients after h-ATG and ALT (17% and 6%, respectively, p<0.01 versus r-ATG). Because rapid recovery of T cells in the alemtuzumab patients was frequently found in the presence of circulating ALT (mean concentration 0.43 μg/mL and 0.12 μg/mL after 3 and 6 weeks, respectively), the phenotype of circulating CD4 and CD8 T cells at 6 weeks after ALT was analyzed. The majority of circulating CD8 and CD4 T cells lacked CD52 expression (56% (range 0–99%) and 81% (range 0–93%), respectively). Using tetramer staining, cytotoxicity assays and analysis of cytokine production, we demonstrated the presence of functional CD52 negative as well as CD52 positive CMV and EBV specific CD8 T cells. Based on FLAER negativity, it was demonstrated that the CD52 negative T cells are GPI anchor deficient, representing a PNH-like clone escaping ALT induced cell lysis. Because almost half of the circulating CD8 T cells were CD52 positive, we examined expression of CD52 and the in-vitro sensitivity to ALT-mediated complement-dependent cell lysis (CDC) of B cells, CD4 and CD8 T cells of healthy donors. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with ALT and complement. Differential expression of CD52 was observed on CD4 and CD8 T cells, MFI 120 and 101 respectively, resulting in relative protection of CD52 positive CD8 compared to CD4 T cells against ALT-mediated CDC (52% and 90% lysis). We conclude that the high incidence of EBV-PTLD after in-vivo T cell depletion with r-ATG is caused by an induced imbalance between B and T cell recovery, which is not seen after h-ATG or ALT. In-vivo T cell depletion with ALT is associated with a relatively low risk of EBV disease because of efficient B cell depletion and persistent EBV immunity due to the relative insusceptibility for ALT of CD8 T cells and the development of functional CD52 negative escape variants of CD4 and CD8 T cells. Disclosures: Off Label Use: Alemtuzumab and Anti Thymocyte Globulin used for in vivo T cell depletion prior to allogeneic stem cell transplantation.

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