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Mortality due to COVID-19 is not increased in immunosuppressed individuals after liver transplantation (OLT) compared to individuals without immunosuppression. Data on long-term protective immunity against SARS-CoV-2 in immunosuppressed convalescents, is limited. We prospectively measured immune responses against SARS-CoV-2 by quantifying antibodies against 4 different antigens (spike protein 1 and 2, receptor binding domain, nucleocapsid) and T cell responses by IFN-γ ELISPOT against 4 antigens (membrane, nucleocapsid, spike protein 1 and 2) in 24 OLT convalescents with immunosuppressive therapy longitudinally in the first year after COVID-19 including a booster vaccination in comparison to a matched cohort of non-immunosuppressed convalescents (non-IS-Con). Pre-pandemic OLT samples were retrieved from our prospective OLT biorepository (n = 16). No relevant T cell reactivity or immunoglobulin G (IgG) against SARS-CoV-2 were detectable in pre-pandemic samples of OLT recipients despite reactivity against endemic corona-viruses. OLT convalescents had a lower prevalence of IgG against nucleocapsid (54% vs. 90%) but not against spike protein domains (98-100% vs. 100%) after vaccination in the second half-year after COVID-19 compared to non-IS-Con. Also, concentrations of anti-nucleocapsid IgG were lower in OLT convalescents than in non-IS-Con. Concentration of IgG against spike protein domains was significantly increased by a booster vaccination in OLT convalescents. But concentration of IgG against two of three spike protein domains remains slightly lower compared to non-IS-Con finally. However, none of these differences was mirrored by the cellular immunity against SARS-CoV-2 that remained stable during the first year after COVID-19 and was not further stimulated by a corona vaccination in OLT convalescents. In conclusion, despite lower concentrations of anti-SARS-CoV-2 IgG in OLT convalescents anti-SARS-CoV-2 cellular immunity was as robust as in non-IS-Con.

Integrins are a large family of heterodimeric proteins that are involved in cell adhesion, migration, and proliferation. Integrin diversity and function is regulated by alternative splicing. Membrane-bound and truncated β3-integrins were shown to be key players in cancer metastasis. However, the immunomodulatory functions of the soluble (s) β3-integrin have not been investigated yet. In this study, we described a novel form of sβ3-integrin in acute myeloid leukaemia (AML) patients. Furthermore, we assessed the role of the sβ3-integrin in the modulation of natural killer (NK)-cell activity. Levels of sβ3-integrin were analysed in plasma samples of 23 AML patients and 26 healthy donors by ELISA. The capacity of sβ3-integrin to regulate NK cell activity was investigated using proliferation, cytokine secretion, and cytotoxicity assays. Circulating sβ3-integrin was detected in the plasma of 8 AML patients. NK cells showed significantly higher proliferation rates after stimulation with sβ3-integrin and IL-2, IL-15 (73%). Significant increases in the NK cells' secreted levels of TNF-α, IFN-γ were measured in presence of sβ3-integrin. In addition, sβ3-integrin caused the upregulation of Granzyme B transcripts levels as well as FasL expression levels in NK cells. Most importantly, significantly higher K562 or AML blast target cell lysis rates were observed when NK cells were exposed to sβ3-integrin. This study reports the identification of a novel sβ3-integrin in AML patients and provides novel insights into its role in the immunomodulation of NK cell activity.

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells. CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry. G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF. To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.

Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV)-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8(+) and CD4(+) HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean) expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold) compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.

Human CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and -independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4(+)CD25(-) target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4(+)CD25(-) cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.