comparing the expression of different co-inhibitory molecules in CD8 + and CD4 + T cells in COVID-19 vs. malaria, there is a transient increase of the expression of certain inhibitory receptors like LAG-3 and TIM-3 in COVID-19 in the overall context of acute immune activation.

B cells play a central role in antiviral and antiparasitic immunity, not only as producers of antibodies, but also as APCs and mediators of inflammation. In this study, we used 16‐color flow cytometry analysis to investigate the frequency, differentiation, and activation status of peripheral B cells of patients with SARS‐CoV‐2 infection or acute Plasmodium falciparum malaria compared with the healthy individuals. As a main result, we observed an increase of the frequency of (CD27–, CD21–) atypical memory B cells and (CD19+, CD27+, CD38+) plasmablasts in malaria and COVID‐19 patients. Additionally, CD86, PD‐1, CXCR3, and CD39 expression was up‐regulated, whereas CD73 was down‐regulated on plasmablasts of COVID‐19 and malaria patients compared with the bulk B cell population. In particular, there was a more pronounced loss of CD73+ B cells in malaria. The frequency of plasmablasts positively correlated with serum levels of CRP, IL‐6, and LDH of COVID‐19 patients. In the longitudinal course of COVID‐19, a rapid normalization of the frequency of atypical memory B cells was observed. The role and function of plasmablasts and atypical memory B cells in COVID‐19 and other acute infections remain to be further investigated. The role of B cells as either “driver or passenger” of hyperinflammation during COVID‐19 needs to be clarified.

Background and Aims Autoimmune hepatitis (AIH) is a chronic liver disease that regularly relapses when immunosuppression is tapered. It is thought to be driven by T‐cells, whereas the etiologic impact of an apparently deregulated B lineage system, as evidenced by hypergammaglobulinemia and autoantibodies, remains elusive. We set out to investigate T and B cell repertoires supporting chronic inflammation in AIH. Approach and Results T and B cell receptor (TCR/BCR) and human leukocyte antigen (HLA) next‐generation immunosequencing were used to record immune signatures from a cohort of 60 patients with AIH and disease controls. Blood and liver B lineage immune metrics were not indicative of a dominant directional antigen selection apart from a slight skewing of IGHV‐J genes. More importantly, we found strong AIH‐specific TRBV‐J skewing not attributable to the HLA‐DRB1 specificities of the cohort. This TCR repertoire bias was generated as a result of peripheral T cell (de)selection and persisted in disease remission. Using a clustering algorithm according to antigenic specificity, we identified liver TCR clusters that were shared between patients with AIH but were absent or deselected in patients with other liver pathologies. Conclusions Patients with AIH show profound and persisting T‐cell architectural changes that may explain high relapse rates after tapering immunosuppression. Liver T‐cell clusters shared between patients may mediate liver damage and warrant further study.

The inhibitory receptor TIGIT, as well as theectonucleotidases CD39 and CD73 constitute potential exhaustion markers for T cells. Detailed analysis of these markers can shed light into dysregulation of the T-cell response in acute myeloid leukemia (AML) and will help to identify potential therapeutic targets. The phenotype and expression of transcription factors was assessed on different T-cell populations derived from peripheral blood (PB, n = 38) and bone marrow (BM, n = 43). PB and BM from patients with AML diagnosis, in remission and at relapse were compared with PB from healthy volunteers (HD) (n = 12) using multiparameter flow cytometry. An increased frequency of terminally differentiated (CD45R − CCR7 − )CD8 + T cells was detected in PB and BM regardless of the disease state. Moreover, we detected an increased frequency of two distinct T-cell populations characterized by the co-expression of PD-1 or CD39 on TIGIT + CD73 − CD8 + T cells in newly diagnosed and relapsed AML in comparison to HDs. In contrast to the PD-1 + TIGIT + CD73 − CD8 + T-cell population, the frequency of CD39 + TIGIT + CD73 − CD8 + T cells was normalized in remission. PD-1 + -and CD39 + TIGIT + CD73 − CD8 + T cells exhibited additional features of exhaustion by decreased expression of CD127 and TCF-1 and increased intracellular expression of the transcription factor TOX.CD8 + T cells in AML exhibit a key signature of two subpopulations, PD-1 + TOX + TIGIT + CD73 − CD8 + -and CD39 + TOX + TIGIT + CD73 − CD8 + T cells that were increased at different stages of the disease. These results provide a rationale to analyze TIGIT blockade in combination with inhibition of the purinergic signaling and depletion of TOX to improve T-cell mediated cytotoxicity in AML.

Background: T cells are thought to play a major role in conferring immunity against malaria. This study aimed to comprehensively define the breadth and specificity of the Plasmodium falciparum (P. falciparum)-specific CD4+ T cell response directed against the exported protein 1 (EXP1) in a cohort of patients diagnosed with acute malaria. Methods: Peripheral blood mononuclear cells of 44 patients acutely infected with P. falciparum, and of one patient infected with P. vivax, were stimulated and cultured in vitro with an overlapping set of 31 P. falciparum-specific 13-17-mer peptides covering the entire EXP1 sequence. EXP1-specific T cell responses were analyzed by ELISPOT and intracellular cytokine staining for interferon-γ production after re-stimulation with individual peptides. For further characterization of the epitopes, in silico and in vitro human leukocyte antigen (HLA) binding studies and fine mapping assays were performed. Results: We detected one or more EXP1-specific CD4+ T cell responses (mean: 1.09, range 0-5) in 47% (21/45) of our patients. Responses were directed against 15 of the 31 EXP1 peptides. Peptides EXP1-P13 (aa60-74) and P15 (aa70-85) were detected by 18% (n = 8) and 27% (n = 12) of the 45 patients screened. The optimal length, as well as the corresponding most likely HLA-restriction, of each of these two peptides was assessed. Interestingly, we also identified one CD4+ T cell response against peptide EXP1-P15 in a patient who was infected with P. vivax but not falciparum. Conclusions: This first detailed characterization of novel EXP1-specific T cell epitopes provides important information for future analysis with major histocompatibility complex-multimer technology as well as for immunomonitoring and vaccine design.

Thymocyte selection-associated high mobility group box (TOX) has been described to be a key regulator in the formation of CD8+ T cell exhaustion. Hepatitis C virus (HCV) infection with different lengths of antigen exposure in acute, chronic, and after resolution of HCV infection is the ideal immunological model to study the expression of TOX in HCV-specific CD8+ T cells with different exposure to antigen. HCV-specific CD8+ T cells from 35 HLA-A*01:01, HLA-A*02:01, and HLA-A*24:02 positive patients were analyzed with a 16-color FACS-panel evaluating the surface expression of lineage markers (CD3, CD8), ectoenzymes (CD39, CD73), markers of differentiation (CD45RO, CCR7, CD127), and markers of exhaustion and activation (TIGIT, PD-1, KLRG1, CD226) and transcription factors (TOX, Eomesodermin, T-bet). Here, we defined on-target T cells as T cells against epitopes without escape mutations and off-target T cells as those against a “historical” antigen mutated in the autologous sequence. TOX+HCV-specific CD8+ T cells from patients with chronic HCV and on-target T cells displayed co-expression of Eomesodermin and were associated with the formation of terminally exhausted CD127-PD1hi, CD39hi, CD73low CD8+ T cells. In contrast, TOX+HCV-specific CD8+ T cells in patients with off-target T cells represented a progenitor memory Tex phenotype characterized by CD127hi expression and a CD39low and CD73hi phenotype. TOX+HCV-specified CD8+ T cells in patients with a sustained virologic response were characterized by a memory phenotype (CD127+, CD73hi) and co-expression of immune checkpoints and Eomesodermin, indicating a key structure in priming of HCV-specific CD8+ T cells in the chronic stage, which persisted as a residual after therapy. Overall, the occurrence of TOX+HCV-specific CD8+ T cells was revealed at each disease stage, which impacted the development of progenitor Tex, intermediate Tex, and terminally exhausted T cell through an individual molecular footprint. In sum, TOX is induced early during acute infection but is modulated by changes in viral sequence and antigen recognition. In the case of antigen persistence, the interaction with Eomesodermin leads to the formation of terminally exhausted virus-specific CD8+ T cells, and there was a direct correlation of the co-expression of TOX and Eomes and terminally exhausted phenotype of virus-specific CD8+ T cells.

Immune checkpoint therapy has revolutionized the treatment of patients with cancer. Checkpoint receptors and their ligands play an important role in T cell activation and exhaustion and are currently the focus in understanding the antitumoral immune responses. In patients with acute myeloid leukemia (AML) limited data have been published that comprehensively describe the expression of checkpoint receptors on different T cell subsets. We performed multicolor flow cytometry on peripheral blood mononuclear cells (PB, PBMCs) from patients with newly diagnosed AML (n=20) and PBMCs from age matched healthy donors (HDs; n=12), focusing on differentiation, the clinically actionable exhaustion receptor T cell immunoglobulin and ITIM domain (TIGIT), the two ectoenzymes ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73). Our studies included also analysis of interleukin-7 receptor-α (CD127) and the intracellular expression of the transcription factor T cell factor 1 (TCF-1). Both markers are known to be expressed in long living memory CD8+ T cells and harboring the ability for self-renewal. The thymocyte selection-associated high mobility group box protein (TOX) was also analyzed, since this molecule has recently been described as regulator of CD8+ T cell exhaustion. Comparison of PB from patients with newly diagnosed AML vs. HDs revealed that the frequency of CD8+, CD4con (CD4+CD127+CD25-) and CD4reg (CD4+CD127-CD25+) T cells was similar among both groups. However, the frequency of CD8+ EMRA T cells (CCR7- CD45RO- CD8+ CD3+) was increased in PB from patients with AML compared to PB from HDs (39,05 ± 4,38 vs. 14,29 ± 3,83; p<0,05). TIGIT, CD39 and CD73 emerged as checkpoints of interest on CD8+ T cells. The frequency of TIGIT+ CD8+ T cells and CD39+ CD8+ T cells in PBs from patients with newly diagnosed AML was increased compared with that in HDs (42,60 ± 4,67 vs. 20,36 ± 3,68; p=0,00 and 6,54 ± 2,05 vs. 1,31 ± 0,32; p=0,05). Whereas reduced frequency of CD73+ CD8+ T cells occurred in PB from patients with AML vs. HD (41,35 ± 4,75 vs. 66,18 ± 7,28; p=0,01). Analysis between TIGIT and CD73 expression showed inverse correlation between both targets in AML (r=-0,53; p=0,01). The frequency of the TIGIT+ CD73- CD8+ T cell population was increased in AML (36,85 ± 5,17, vs. 16,23 ± 5,09 ;p=0,01). This increased frequency of TIGIT+ CD73- cells in AML was related to EM (CCR7- CD45RO+ CD8+ CD3+)T cells (42,89 ± 4,78 vs. 25,27 ± 3,39; p=0,01) and EMRA CD8+ T cells (56,01 ± 5,68 vs. 36,54 ± 4,85; p=0,03). Moreover, CD39 was aberrantly expressed on this population: we observed an increased frequency of CD39+ TIGIT+ CD73- CD8+ T cells in PB from patients with newly diagnosed AML compared to PB from HDs (13,47 ± 4,03 vs. 3,03 ± 1,21; p=0,02). Next we focused on CD127 and TCF-1 which are involved in creating long living antigen independent memory CD8+ T cells and ability to self-renewal while producing differentiated effector cells. Comparing expression of CD127 and TCF-1 on CD39+ TIGIT+ CD73- CD8+ T cells showed a significantly decreased frequency of CD127 (17,73 ± 2,16, vs. 30,72 ± 7,12; p=0,04) and TCF-1 (14,67 ± 2,90 vs. 39,74 ± 9,32; p=0,03) in PB from patients with newly diagnosed AML compared to HDs. Expression of TIGIT and TCF-1 inversely correlated in AML (r =-0,87; p<0,05). To further evaluate the exhaustion status of TIGIT+ CD73- CD8+ T cells we examined the expression of TOX, recently described as one of the key regulators governing CD8+ T cell exhaustion, the frequency of TOX+ cells was increased in AML (50,57 ± 8,21 vs. 22,14 ± 4,86; p=0,01). Analysis of co-expression showed that the TOX+ CD39+ TIGIT+ CD73- CD8+ population was significantly increased in PB from patients with newly diagnosed AML compared to their counterparts in PB from HDs (21,81 ± 3,14 vs. 3,84 ± 1,10; p=0,04). In summary, we could show that in PB from patients with newly diagnosed AML an aberrant cell population of CD39+ TIGIT+ CD73- CD8+ T cells is prevalent in contrast to the PB from HDs. In this cell population we observed elevated expression of TOX which has recently described as one of the key regulators governing CD8+ T cell exhaustion. In contrast downregulation of CD127 and TCF-1 was found in these cells. These data might contribute to CD8+ T cell exhaustion in AML and support further functional analysis to investigate the relevance of combinatorial inhibition of TIGIT and CD39. Disclosures Brauneck: Daiichi Sankyo: Consultancy, Honoraria, Other: support for meeting attendance; Novartis: Other: support for meeting attendance; Jazz Pharmaceuticals: Other: support for meeting attendance. Bokemeyer:Merck KGaA: Honoraria; Janssen-Cilag: Research Funding; Roche: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other; Karyopharm Therapeutics: Research Funding; Millenium: Research Funding; MSD: Research Funding; Nektar: Research Funding; Novartis: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Sanofi: Consultancy, Honoraria, Other: travel accomodations; Bristol-Myers Squibb: Honoraria, Other: travel accomodations, Research Funding; AstraZeneca: Honoraria, Research Funding; Merck Sharp & Dohme: Consultancy, Honoraria; Lilly/ImClone: Consultancy, Research Funding; Merck Serono: Consultancy, Other: travel accomodations; Bayer Schering Pharma: Consultancy; GSO: Consultancy; AOK Health Insurance: Consultancy; Abbvie: Research Funding; ADC Therapeutics: Research Funding; Agile Therapeutics: Research Funding; Alexion Pharmaceuticals: Research Funding; Amgen: Research Funding; Apellis Pharmaceuticals: Research Funding; Astellas: Research Funding; BerGenBio: Research Funding; Blueprint Medicines: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Gilead Sciences: Research Funding; Glycotope GmbH: Research Funding; GSK: Research Funding; Incyte: Research Funding; IO Biotech: Research Funding; Isofol Medical: Research Funding. Fiedler:Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding; ARIAD/Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Daiichi Sankyo: Other: support for meeting attendance.

Background:Myelofibrosis (MF) is a hematopoietic stem cell (HSC) neoplasm characterized by an expansion of myeloid cells followed by deposition of fibers in the bone marrow (BM) and leukemic transformation depending on the clonal evolution of MF stem cells (MF SCs). Mutations in the receptor tyrosine kinase JAK2, the endoplasmic reticulum chaperone CALR or the thrombopoietin receptor (MPL) drive the disease phenotype through cytokine‐independent activation of the JAK‐STAT signaling pathway. Patient‐derived xenograft (PDX) models have emerged as powerful tools for investigating aggressive malignancies, such as acute leukemia's. However, the development of less‐aggressive malignancies, like MF, is often limited in PDX models. We hypothesized that the constitutive expression of human cytokines and growth factors in immunocompromised mice (i.e. MISTRG mice) may provide a supportive microenvironment for MF SC development.Aims:To develop a pre‐clinical MF PDX model that faithfully recapitulates the disease phenotype and the genetic heterogeneity observed in patients.Methods:Purified peripheral blood (PB) stem and progenitor (CD34+) cells from 14 MF patients were transplanted intra‐hepatically into sub‐lethally irradiated newborn MISTRG mice and NSG mice (controls). For secondary transplantations, human CD45+ cells purified from primary animals were transplanted intra‐hepatically. Mice were sacrificed 5–26 weeks (median: 9.8 weeks) post‐transplantation and characterized by flow cytometry, immunohistochemistry, and mutational profiling.Results:MISTRG mice supported significantly higher human MF engraftment in the BM (29.20% vs 4.175%, p < 0.0001), the PB (48.70% vs. 0.73%, p < 0.0001) and the spleen (8.19% vs. 0.33%, p < 0.0001) compared to NSG mice. All 14 investigated patient samples engrafted in MISTRG mice, while 9/14 patient samples engrafted in NSG mice. MISTRG mice exhibited superior engraftment independent of risk categories (DIPSS, MIPSS70, and MYSEC), disease stage (chronic, accelerated) and diagnoses (primary, secondary MF). Both NSG and MISTRG mice supported robust monocytic and granulocytic engraftment in the BM. Furthermore, immunohistochemistry revealed human megakaryocytes in both strains. An increase in reticulin fibers was detected in MISTRG mice transplanted with 1 patient sample that was analyzed after 18 weeks. To determine whether the engrafted human cells were derived from the MF clone, NSG and MISTRG mice that showed >10% human engraftment were analyzed for driver mutations (JAK2 or CALR). JAK2 or CALR mutations were preserved in all NSG (7/7) and MISTRG (39/39) mice transplanted with the respective sample. Next, we performed targeted sequencing of 54 myeloid genes frequently mutated in myeloid neoplasms on 7 NSG mice transplanted with 4 patient samples and 22 MISTRG mice transplanted with 8 patient samples to determine whether the mutational profile of the primary sample was preserved in the corresponding engrafted xenografts. This data showed maintenance of the primary patient sample clonal composition in both NSG and MISTRG mice. Finally, purified human MF cells isolated from primary mice showed myeloid reconstitution in secondary recipients.Summary/Conclusion:These results show that MISTRG mice support robust engraftment of MF SCs across all disease stages and categories and faithfully reproduce the genetic complexity observed in patients. This novel MISTRG MF PDX model is currently being used to assess the effect of the JAK inhibitor (JAKi) Ruxolitinib on engrafted human MF cells.