Transfer and loss of allergen‐specific responses via stem cell transplantation: A prospective observational study

Background Currently, no estimates can be made on the impact of hematopoietic stem cell transplantation on allergy transfer or cure of the disease. By using component‐resolved diagnosis, we prospectively investigated 50 donor‐recipient pairs undergoing allogeneic stem cell transplantation. This allowed calculating the rate of transfer or maintenance of allergen‐specific responses in the context of stem cell transplantation. Methods Allergen‐specific IgE and IgG to 156 allergens was measured pretransplantation in 50 donors and recipients and at 6, 12 and 24 months in recipients post‐transplantation by allergen microarray. Based on a mixed effects model, we determined risks of transfer of allergen‐specific IgE or IgG responses 24 months post‐transplantation. Results After undergoing stem cell transplantation, 94% of allergen‐specific IgE responses were lost. Two years post‐transplantation, recipients' allergen‐specific IgE was significantly linked to the pretransplantation donor or recipient status. The estimated risk to transfer and maintain individual IgE responses to allergens by stem cell transplantation was 1.7% and 2.3%, respectively. Allergen‐specific IgG, which served as a surrogate marker of maintaining protective IgG responses, was highly associated with the donor's (31.6%) or the recipient's (28%) pretransplantation response. Conclusion Hematopoietic stem cell transplantation profoundly reduces allergen‐specific IgE responses but also comes with a considerable risk to transfer allergen‐specific immune responses. These findings facilitate clinical decision‐making regarding allergic diseases in the context of hematopoietic stem cell transplantation. In addition, it provides prospective data to estimate the risk of transmitting allergen‐specific responses via hematopoietic stem cell transplantation

Human Adenovirus-Specific γ/δ and CD8⁺ T Cells Generated by T-Cell Receptor Transfection to Treat Adenovirus Infection after Allogeneic Stem Cell Transplantation

<div><p>Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected α/β T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into γ/δ T cells, which are not allo-reactive. TCR-transfected γ/δ T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8α-encoding mRNA. In direct comparison with TCR-transfected α/β T cells, TCR-CD8α-co-transfected γ/δ T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected α/β T cells and TCR-CD8α-co-transfected γ/δ T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only α/β T cells but also γ/δ T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.</p></div

Direct comparison of cytokine production by HAdV/A1-TCR-transfected CD8⁺ T cells and HAdV/A1-TCR-transfected γ/δ T cells.

<p>CD8⁺ T cells and γ/δ T cells of the same donors were either mock electroporated, electroporated with HAdV/A1-TCR-RNA alone, or electroporated with HAdV/A1-TCR-RNA and CD8α-RNA. These cells were stimulated with colo829 cells (<b>A</b>) or CCL cells (<b>B</b>), which were either loaded with a control peptide (contr. pept.) or the adenovirus peptide (Adeno pept.). Cytokine concentrations (IL-2, TNF, and IFNγ) in the supernatant after overnight co-incubation are depicted. Data of 3 individual experiments are shown. Bars indicate mean values. Raw data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109944#pone.0109944.s001" target="_blank">Table S5A and B in File S1</a>.</p

Cytolytic capacity of HAdV/A1-TCR-transfected CD8⁺ T cells and γ/δ T cells against adenovirus-infected target cells.

<p>CD8⁺ T cells (<b>A</b> and <b>C</b>) and γ/δ T cells (<b>B</b> and <b>D</b>) of the same donors were either electroporated with mRNA encoding the MAGE-1/A1-specific TCR (open symbols) or electroporated with mRNA encoding the HAdV/A1-specific TCR (closed symbols) either in combination with CD8α mRNA (γ/δ T cells) or not (CD8⁺ T cells). These cells were used as effector cells in standard 4–6 h cytotoxicity assays. Autologous (<b>A</b> and <b>B</b>) and allogenic (<b>C</b> and <b>D</b>) DC either untreated (non-loaded), loaded with the adenovirus peptide (Adeno pept.), or infected with adenovirus (adenovirus) were used as target cells and the percentage of lysed cells was calculated. The target to effector cell ratios were 1∶60, 1∶20, 1∶6, and 1∶2. Average values of 6 (<b>A</b> and <b>B</b>) or 4 (<b>C</b> and <b>D</b>) individual experiments (each performed in triplicates) +/− SEM are shown. P-values were calculated by the Mann-Whitney U test. For statistics, conditions were compared to the non-loaded DC condition (* and **) and to T cells transfected with the M1/A1 control TCR (^‡ and ^†). (** or ^‡ p≤0.01; *or ^† p≤0.05). Raw data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109944#pone.0109944.s001" target="_blank">Table S7A to D in File S1</a>.</p

Antigen-specific cytokine production by HAdV/A1-TCR-transfected CD8⁺ and γ/δ T cells in response to peptide-loaded target cells.

<p>CD8⁺ T cells and γ/δ T cells were either electroporated with M1/A1-TCR RNA (+CD8α RNA in case of γ/δ T cells) or with HAdV/A1-TCR-RNA (+CD8α RNA in case of γ/δ T cells) and were cryoconserved 4 h after electroporation. After thawing, these cells were stimulated with CCL cells, which were either left unloaded (w/o pept.), or were loaded with the adenovirus peptide (w/pept.). Intracellular cytokine stainings for IL-2 (white bars), TNF (grey bars), and IFNγ (black bars) were performed and analyzed by flow-cytometry. The percentages of cytokine containing cells are depicted. Average values of 4 (CD8⁺ T cells) and 3 (γ/δ T cells) individual experiments ± SEM are shown. Raw data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109944#pone.0109944.s001" target="_blank">Table S6 in File S1</a>.</p

The newly cloned HAdV/HLA-A1-specific TCR is functional in Jurkat T cells.

<p>Parental CD4⁺ and transgenic CD8⁺ Jurkat T cells were co-electroporated with RNA encoding the HAdV/A1-specific TCR or MAGE-A3/A1 (M3/A1)-specific TCR and an NFAT-inducible luciferase reporter plasmid. These Jurkat T cells were stimulated with DC either non-loaded (w/o pept.) or loaded with the adenovirus peptide (Adeno pept.) or the MAGE-A3 peptide (MAGE-A3 pept.) (as indicated). The luciferase activity was measured, and the specific activation of the Jurkat T cells was calculated as fold induction by dividing the luciferase activity induced by peptide-loaded DC by that of similarly electroporated Jurkat T cells stimulated with non-loaded DC. Data of 4 (CD4⁺ Jurkat T cells) and 5 (CD8⁺ Jurkat T cells) individual experiments are shown. Bars indicate mean values. P-values were calculated by the Mann-Whitney U test. ns  =  not significant; ** p≤0.01; * p≤0.05. Raw data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109944#pone.0109944.s001" target="_blank">Table S1 in File S1</a>.</p

Antigen-specific cytokine production by HAdV/A1-TCR-transfected CD8⁺ T cells in response to peptide-loaded and adenovirus-infected targets.

<p>CD8⁺ T cells were either mock electroporated or electroporated with HAdV/A1-TCR-RNA and were stimulated with DC, which were either left unloaded (<b>A</b>: w/o pept., <b>B</b>: w/o virus), or were loaded with the adenovirus peptide (Adeno pept.) (<b>A</b>), or were infected with adenovirus (w/virus) (<b>B</b>). Cytokine concentrations (IL-2, TNF, and IFNγ) in the supernatant after over-night co-incubation are depicted. Data of 5 (<b>A</b>) and 7 (<b>B</b>) individual experiments are shown. Bars indicate mean values. P-values were calculated by the Mann-Whitney U test. ns  =  not significant, *** p≤0.001; ** p≤0.01; * p≤0.05. Raw data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109944#pone.0109944.s001" target="_blank">Table S2A and B in File S1</a>.</p

Surface marker and TCR expression on HAdV/A1-TCR-transfected CD8⁺ and γ/δ T cells.

<p>CD8⁺ T cells (A and B; upper panels) and γ/δ T cells (A and B; lower panels) were either electroporated with M1/A1-TCR RNA (+CD8α RNA in case of γ/δ T cells) or with HAdV/A1-TCR-RNA (+CD8α RNA in case of γ/δ T cells) and were cryoconserved 4 h after electroporation. Surface marker and TCR expression on thawed T cells was determined by staining with anti-CD4, anti-CD8, anti-CD16, anti-CD19, anti-CD14, and anti-pan γ/δ TCR antibodies, and the expression of the HAdV/A1-specific TCR was determined by staining with HAdV/A1 streptamer. Expression levels were analyzed by flow-cytometry and shown as % positive cells; isotype control stainings were subtracted (<b>A</b>; average values of 4 experiments ± SEM) or as histograms (<b>B</b>; grey histogram: T cells electroporated with M1/A1-TCR RNA, black line: T cells electroporated with HAdV/A1-TCR-RNA). Raw data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109944#pone.0109944.s001" target="_blank">Table S4A and B in File S1</a>.</p

Antigen-specific cytokine production by HAdV/A1-TCR-transfected γ/δ T cells in response to peptide-loaded target cells.

<p>γ/δ T cells were either mock electroporated, electroporated with HAdV/A1-TCR-RNA alone, or electroporated with HAdV/A1-TCR-RNA and CD8α-RNA. These cells were stimulated with DC (<b>A</b>) or colo829 cells (<b>B</b>), which were either left unloaded (w/o pept.), or were loaded with the adenovirus peptide (Adeno pept.). Cytokine concentrations (IL-2, TNF, and IFNγ) in the supernatant after overnight co-incubation are depicted. Data of 3 individual experiments are shown. Bars indicate mean values. Raw data are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109944#pone.0109944.s001" target="_blank">Table S3A and B in in File S1</a>.</p

Risk assessment of relapse by lineage-specific monitoring of chimerism in children undergoing allogeneic stem cell transplantation for acute lymphoblastic leukemia

Allogeneic hematopoietic stem cell transplantation is required as rescue therapy in about 20% of pediatric patients with acute lymphoblastic leukemia. However, the relapse rates are considerable, and relapse confers a poor outcome. Early assessment of the risk of relapse is therefore of paramount importance for the development of appropriate measures. We used the EuroChimerism approach to investigate the potential impact of lineage-specific chimerism testing for relapse-risk analysis in 162 pediatric patients with acute lymphoblastic leukemia after allogeneic stem cell transplantation in a multicenter study based on standardized transplantation protocols. Within a median observation time of 4.5 years, relapses have occurred in 41/162 patients at a median of 0.6 years after transplantation (range, 0.13-5.7 years). Prospective screening at defined consecutive time points revealed that reappearance of recipient-derived cells within the CD34(+) and CD8(+) cell subsets display the most significant association with the occurrence of relapses with hazard ratios of 5.2 (P=0.003) and 2.8 (P=0.008), respectively. The appearance of recipient cells after a period of pure donor chimerism in the CD34(+) and CD8(+) leukocyte subsets revealed dynamics indicative of a significantly elevated risk of relapse or imminent disease recurrence. Assessment of chimerism within these lineages can therefore provide complementary information for further diagnostic and, potentially, therapeutic purposes aiming at the prevention of overt relapse. This study was registered at clinical. TRIALS: gov with the number NC01423747