Diversity and focus of CMV specific T-cell responses in patients post-HSCT and with solid tumor

Human Cytomegalovirus (CMV) is a beta-herpesvirus that commonly infects humans. Most symptoms in healthy individuals are very mild. The virus is never eliminated and remains in the individual life-long. Thus, CMV and the immune response stay in coexistence until the protective response remains. CMV disease occurs when the immune system is either immature or immunocompromised, such as in hematopoietic stem cell transplant recipients. Over the last decades, CMV has been reported in diverse cancer including glioblastoma (GBM) that could suggest a potential link between CMV infection and cancer initiation or development. The papers in this thesis investigated the CMV-specific immune response of individuals in different clinical settings: patients after hematopoietic stem cell transplantation (HSCT) and patients with brain tumor or pancreatic cancer. The thesis consists of the following studies: In Paper I, we investigated the CMV-specific interferon-gamma (IFNγ) production in a cohort of 277 patients over time post-HSCT. Impairment of the CMV-specific immune response post-HSCT is associated with CMV infection. We monitored the IFNγ production in response to CMV-pp65 over a period of 2 years post-HSCT and observed a higher IFNγ production at the first month post-HSCT. Moreover, we identified that several clinical parameters such acute graft-versus-host disease (GVHD) and CMV infection affect the IFNγ production in response to CMV-pp65. In Paper II, we aimed to characterize the CMV-specific CD8+ cytotoxic T-cells (CTL) with high affinity T-cell receptor (TCR) in patients post-HSCT using three different CMV-pp65 tetramers. High affinity CMV-CTL presented an effector memory phenotype and a stronger PD-1 expression as compared to CMV-CTL with lower affinity. Additionally, the high affinity CMV-CTLs were found at higher proportion in patients with chronic GVHD over time post-HSCT. Therefore, Paper I and II together may better characterize the CMV-specific immune response post-HSCT and potentially the bidirectional relationship between virus and GVHD. In Paper III, we investigated the plasma interleukin 7 (IL-7) and the soluble IL-7 receptor (sIL-7R) levels post-HSCT. IL-7, through its receptor, is essential for T-cell proliferation, thus, to the response to infection. Patients presenting CMV infection exhibited a lower plasma level of sIL-7R and higher level of IL-7 in the early months post-HSCT. Furthermore, the plasma level of IL-7R was reduced in patients with acute GVHD. Together these observations suggest that sIL-7R may be associated with increased risk of GVHD and potential CMV infection. In Paper IV, the clinical setting is different. We focused on the CMV-specific IFNγ production in a large cohort of patients with brain tumor and pancreatic cancer. Patients with brain tumor and more specifically those with GBM presented an impaired immune response towards viral and mitogen antigens. While survival was correlated with the CMV- specific humoral response, no correlation between survival and CMV-specific IFNγ production could be observed. Contrarily, patients with high Epstein-Barr virus (EBV)- and PHA-specific IFNγ production showed an improved survival post-operation suggesting this immune response as potential marker of general immunocompetence

Whole CMV proteome pattern recognition analysis after HSCT identifies unique epitope targets associated with the CMV status

Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/- for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the 'exclusive recognition analysis (ERA)' to identify unique CMV epitope responses for each patient group. The 'exclusive recognition analysis' of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D-/R-). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution.The work has been funded by ALF (Arbetslivfonden) to M.M. and P.L. funds from Karolinska Institutet and Vinnova, Sweden to M.M

Can we Agree? On the Rash\=omon Effect and the Reliability of Post-Hoc Explainable AI

The Rash\=omon effect poses challenges for deriving reliable knowledge from machine learning models. This study examined the influence of sample size on explanations from models in a Rash\=omon set using SHAP. Experiments on 5 public datasets showed that explanations gradually converged as the sample size increased. Explanations from <128 samples exhibited high variability, limiting reliable knowledge extraction. However, agreement between models improved with more data, allowing for consensus. Bagging ensembles often had higher agreement. The results provide guidance on sufficient data to trust explanations. Variability at low samples suggests that conclusions may be unreliable without validation. Further work is needed with more model types, data domains, and explanation methods. Testing convergence in neural networks and with model-specific explanation methods would be impactful. The approaches explored here point towards principled techniques for eliciting knowledge from ambiguous models.Comment: 13 pages, 6 figures and 6 table

Mass Spectrometric Characterization of Narcolepsy-Associated Pandemic 2009 Influenza Vaccines

The onset of narcolepsy, an irreversible sleep disorder, has been associated with 2009 influenza pandemic (pH1N1) infections in China, and with ASO3-adjuvanted pH1N1 vaccinations using Pandemrix in Europe. Intriguingly, however, the increased incidence was only observed following vaccination with Pandemrix but not Arepanrix in Canada. In this study, the mutational burden of actual vaccine lots of Pandemrix (n = 6) and Arepanrix (n = 5) sourced from Canada, and Northern Europe were characterized by mass spectrometry. The four most abundant influenza proteins across both vaccines were nucleoprotein NP, hemagglutinin HA, matrix protein M1, with the exception that Pandemrix harbored a significantly increased proportion of neuraminidase NA (7.5%) as compared to Arepanrix (2.6%). Most significantly, 17 motifs in HA, NP, and M1 harbored mutations, which significantly differed in Pandemrix versus Arepanrix. Among these, a 6-fold higher deamidation of HA146 (p.Asn146Asp) in Arepanrix was found relative to Pandemrix, while NP257 (p.Thr257Ala) and NP424 (p.Thr424Ile) were increased in Pandemrix. DQ0602 binding and tetramer analysis with mutated epitopes were conducted in Pandemrix-vaccinated cases versus controls but were unremarkable. Pandemrix harbored lower mutational burden than Arepanrix, indicating higher similarity to wild-type 2009 pH1N1, which could explain differences in narcolepsy susceptibility amongst the vaccines

Production of functional CD19 CAR T cells under hypoxic manufacturing conditions

IntroductionChronic lymphocytic leukemia (CLL) has proven difficult to treat with chimeric antigen receptor (CAR) T cell therapy. CLL cells can negatively alter T cell fitness and induce a pseudohypoxic state. We hypothesized that production of CAR T cells under restricted oxygen conditions resembling physiological oxygen levels that can be encountered in tissues (i.e. 2% O2) could promote outgrowth of hypoxia-tolerant CAR T cells.MethodsWe performed in vitro phenotypic and functional assessments of CD19-directed CAR T cells produced in either 21% (NorCAR) or 2% (HypCAR) O2 derived from healthy donors (HDs) or patients with CLL. ResultsProduction of HD-derived CAR T cells in 2% O2 promoted the enrichment of a naïve-like subset. HypCAR and NorCAR cells were functionally distinct; CD4+ HypCAR cells produced more IL-2 and tumor necrosis factor than CD4+ NorCAR cells. Production in 2% O2 was not detrimental to viability or proliferation upon cognate antigen-stimulation and led to increased activation. After chronic stimulation in hypoxia, HypCAR-product remained enriched in naïve-like cells, and demonstrated cytotoxic and cytokine production capacity. In CAR T cells derived from patients with CLL, NorCAR and HypCAR subsets were functionally and phenotypically comparable, but displayed different mitochondrial metabolism. DiscussionWe demonstrated that production in 2% O2 is not detrimental, confers subtle but lasting functional and phenotypic changes in CAR T cells warranting further research on the impact of hypoxic production on CAR T cell functionality in hypoxic tumor microenvironments

Different recovery patterns of CMV-specific and WT1-specific T cells in patients with acute myeloid leukemia undergoing allogeneic hematopoietic cell transplantation: Impact of CMV infection and leukemia relapse

In allogeneic hematopoietic cell transplantation (allo-HSCT), both virus-specific T cells and leukemia-specific T cells need to be reconstituted to protect patients from virus infections and primary disease relapse. Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality after allo-HSCT. Emerging data indicate that CMV reactivation is associated with reduced risk of leukemia relapse in patients with acute myeloid leukemia (AML) undergoing allo-HSCT. In a cohort of 24 WT1+ AML patients during the first year following HSCT, CMV specific CD8+ T cells (CMV-CTL) reconstituted much faster than WT1-specific CD8+ T cell (WT1-CTL) after allo-SCT. Moreover, CMV-CTL expressed lower levels of exhaustion markers and were more functional as identified by production of IFN-γ/TNF-α and expression of Eomes/T-bet. Interestingly, our patients with CMV reactivation presented higher frequency of CMV-CTL, lower levels of Eomes+T-bet- and higher levels of Eomes+T-bet+ expression in response to WT1 and CMV pp65 antigen during the first year after transplantation as compared to patients without CMV reactivation. Kinetics of CMV-CTL and WT1-CTL after transplantation might be associated with measurable residual disease and later leukemia relapse. Our results support that CMV reactivation, aside from the CMV-CTL reconstitution, could influence WT1-CTL reconstitution after allo-HSCT, thus potentially contributing to the remission/relapse of AML.</jats:p

Inducing expression of ICOS-L by oncolytic adenovirus to enhance tumor-specific bi-specific antibody efficacy

Abstract Background Intratumoral injection of oncolytic viruses (OVs) shows promise in immunotherapy: ONCOS-102, a genetically engineered OV that encodes Granulocyte–Macrophage Colony-Stimulating Factor (GM-CSF) demonstrated efficacy in early clinical trials, enhancing T cell infiltration in tumors. This suggests OVs may boost various forms of immunotherapy, including tumor-specific bi-specific antibodies (BsAbs). Methods Our study investigated in vitro, how ONCOS-204, a variant of ONCOS-virus expressing the ligand of inducible T-cell co-stimulator (ICOSL), modulates the process of T cell activation induced by a BsAb. ONCOS-102 was used for comparison. Phenotypic and functional changes induced by combination of different OVs, and BsAb in T cell subsets were assessed by flow cytometry, viability, and proliferation assays. Results Degranulation and IFNγ and TNF production of T cells, especially CD4 + T cells was the most increased upon target cell exposure to ONCOS-204. Unexpectedly, ONCOS-204 profoundly affected CD8 + T cell proliferation and function through ICOS-L/ICOS interaction. The effect solely depended on cell surface expression of ICOS-L as soluble ICOSL did not induce notable T cell activity. Conclusions Together, our data suggests that oncolytic adenoviruses encoding ICOSL may enhance functional activity of tumor-specific BsAbs thereby opening a novel avenue for clinical development in immunotherapeutics