Likai Tan
@cuhk.edu.hk
Research Assistant Professor, Department of Anaesthesia and Intensive Care
Faculty of Medicine
The Chinese University of Hong Kong
Scopus Publications
Scholar Citations
Scholar h-index
Scholar i10-index
Scopus Publications
Sara Terzoli, Paolo Marzano, Valentina Cazzetta, Rocco Piazza, Inga Sandrock, Sarina Ravens, Likai Tan, Immo Prinz, Simone Balin, Michela Calvi,et al.
Springer Science and Business Media LLC
Abstractγδ T cells provide rapid cellular immunity against pathogens. Here, we conducted matched single-cell RNA-sequencing and γδ-TCR-sequencing to delineate the molecular changes in γδ T cells during a longitudinal study following mRNA SARS-CoV-2 vaccination. While the first dose of vaccine primes Vδ2 T cells, it is the second administration that significantly boosts their immune response. Specifically, the second vaccination uncovers memory features of Vδ2 T cells, shaped by the induction of AP-1 family transcription factors and characterized by a convergent central memory signature, clonal expansion, and an enhanced effector potential. This temporally distinct effector response of Vδ2 T cells was also confirmed in vitro upon stimulation with SARS-CoV-2 spike-peptides. Indeed, the second challenge triggers a significantly higher production of IFNγ by Vδ2 T cells. Collectively, our findings suggest that mRNA SARS-CoV-2 vaccination might benefit from the establishment of long-lasting central memory Vδ2 T cells to confer protection against SARS-CoV-2 infection.
Zheng Song, Lara Henze, Christian Casar, Dorothee Schwinge, Christoph Schramm, Johannes Fuss, Likai Tan, and Immo Prinz
Oxford University Press (OUP)
Abstract Accurately identifying γδ T cells in large single-cell RNA sequencing (scRNA-seq) datasets without additional single-cell γδ T cell receptor sequencing (sc-γδTCR-seq) or CITE-seq (cellular indexing of transcriptomes and epitopes sequencing) data remains challenging. In this study, we developed a TCR module scoring strategy for human γδ T cell identification (i.e. based on modular gene expression of constant and variable TRA/TRB and TRD genes). We evaluated our method using 5′ scRNA-seq datasets comprising both sc-αβTCR-seq and sc-γδTCR-seq as references and demonstrated that it can identify γδ T cells in scRNA-seq datasets with high sensitivity and accuracy. We observed a stable performance of this strategy across datasets from different tissues and different subtypes of γδ T cells. Thus, we propose this analysis method, based on TCR gene module scores, as a standardized tool for identifying and reanalyzing γδ T cells from 5′-end scRNA-seq datasets.
Anne M. Hahn, Lisa Vogg, Stefanie Brey, Andrea Schneider, Simon Schäfer, Ralph Palmisano, Anna Pavlova, Inga Sandrock, Likai Tan, Alina S. Fichtner,et al.
Elsevier BV
Anne M. Hahn, Lisa Vogg, Stefanie Brey, Andrea Schneider, Simon Schäfer, Ralph Palmisano, Anna Pavlova, Inga Sandrock, Likai Tan, Alina S. Fichtner,et al.
Elsevier BV
Paolo Marzano, Simone Balin, Sara Terzoli, Silvia Della Bella, Valentina Cazzetta, Rocco Piazza, Inga Sandrock, Sarina Ravens, Likai Tan, Immo Prinz,et al.
Frontiers Media SA
IntroductionHigher frequencies of mucosal-associated invariant T (MAIT) cells were associated with an increased adaptive response to mRNA BNT162b2 SARS-CoV-2 vaccine, however, the mechanistic insights into this relationship are unknown. In the present study, we hypothesized that the TNF response of MAIT cells supports B cell activation following SARS-CoV-2 immunization.MethodsTo investigate the effects of repeated SARS-CoV-2 vaccinations on the peripheral blood mononuclear cells (PBMCs), we performed a longitudinal single cell (sc)RNA-seq and scTCR-seq analysis of SARS-CoV-2 vaccinated healthy adults with two doses of the Pfizer-BioNTech BNT162b2 mRNA vaccine. Collection of PBMCs was performed 1 day before, 3 and 17 days after prime vaccination, and 3 days and 3 months following vaccine boost. Based on scRNA/TCR-seq data related to regulatory signals induced by the vaccine, we used computational approaches for the functional pathway enrichment analysis (Reactome), dynamics of the effector cell-polarization (RNA Velocity and CellRank), and cell-cell communication (NicheNet).ResultsWe identified MAIT cells as an important source of TNF across circulating lymphocytes in response to repeated SARS-CoV-2 BNT162b2 vaccination. The TNFhigh signature of MAIT cells was induced by the second administration of the vaccine. Notably, the increased TNF expression was associated with MAIT cell proliferation and efficient anti-SARS-CoV-2 antibody production. Finally, by decoding the ligand-receptor interactions and incorporating intracellular signaling, we predicted TNFhigh MAIT cell interplay with different B cell subsets. In specific, predicted TNF-mediated activation was selectively directed to conventional switched memory B cells, which are deputed to high-affinity long-term memory.DiscussionOverall, our results indicate that SARS-CoV-2 BNT162b2 vaccination influences MAIT cell frequencies and their transcriptional effector profile with the potential to promote B cell activation. This research also provides a blueprint for the promising use of MAIT cells as cellular adjuvants in mRNA-based vaccines.
Lihua Deng, Anna Harms, Sarina Ravens, Immo Prinz, and Likai Tan
Frontiers Media SA
A Corrigendum on Systematic pattern analyses of V d 2+ TCRs reveal that shared “ public ” V d 2+ gd T cell clones are a consequence of rearrangement bias and a higher expansion status
Lihua Deng, Anna Harms, Sarina Ravens, Immo Prinz, and Likai Tan
Frontiers Media SA
BackgroundVγ9Vδ2+T cells are a major innate T cell subset in human peripheral blood. Their Vδ2+VDJ-rearrangements are short and simple in the fetal thymus and gradually increase in diversity and CDR3 length along with development. So-called “public” versions of Vδ2+TCRs are shared among individuals of all ages. However, it is unclear whether such frequently occurring “public” Vγ9Vδ2+T cell clones are derived from the fetal thymus and whether they are fitter to proliferate and persist than infrequent “private” clones.MethodsShared “public” Vδ2+TCRs were identified from Vδ2+TCR-repertoires collected from 89 individuals, including newborns (cord blood), infants, and adults (peripheral blood). Distance matrices of Vδ2+CDR3 were generated by TCRdist3 and then embedded into a UMAP for visualizing the heterogeneity of Vδ2+TCRs.ResultsVδ2+CDR3 distance matrix embedded by UMAP revealed that the heterogeneity of Vδ2+TCRs is primarily determined by the J-usage and CDR3aa length, while age or publicity-specific motifs were not found. The most prevalent public Vδ2+TCRs showed germline-like rearrangement with low N-insertions. Age-related features were also identified. Public Vδ2+TRDJ1TCRs from cord blood showed higher N-insertions and longer CDR3 lengths. Synonymous codons resulting from VDJ rearrangement also contribute to the generation of public Vδ2+TCRs. Each public TCR was always produced by multiple different transcripts, even with different D gene usage, and the publicity of Vδ2+TCRs was positively associated with expansion status.ConclusionTo conclude, the heterogeneity of Vδ2+TCRs is mainly determined byTRDJ-usage and the length of CDR3aa sequences. Public Vδ2+TCRs result from germline-like rearrangement and synonymous codons, associated with a higher expansion status.
Malte Deseke, Francesca Rampoldi, Inga Sandrock, Eva Borst, Heike Böning, George Liam Ssebyatika, Carina Jürgens, Nina Plückebaum, Maleen Beck, Ahmed Hassan,et al.
Rockefeller University Press
The innate and adaptive roles of γδ T cells and their clonal γδ T cell receptors (TCRs) in immune responses are still unclear. Recent studies of γδ TCR repertoire dynamics showed massive expansion of individual Vδ1+ γδ T cell clones during viral infection. To judge whether such expansion is random or actually represents TCR-dependent adaptive immune responses, information about their cognate TCR ligands is required. Here, we used CRISPR/Cas9-mediated screening to identify HLA-DRA, RFXAP, RFX5, and CIITA as required for target cell recognition of a CMV-induced Vγ3Vδ1+ TCR, and further characterization revealed a direct interaction of this Vδ1+ TCR with the MHC II complex HLA-DR. Since MHC II is strongly upregulated by interferon-γ, these results suggest an inflammation-induced MHC-dependent immune response of γδ T cells.
Yingshi Chen, Shu-Mei Yan, Zeyu Pu, Jinzhu Feng, Likai Tan, Yuzhuang Li, Hongrong Hu, Wenjing Huang, Yingtong Lin, Zhilin Peng,et al.
American Association for Cancer Research (AACR)
Abstract Tissue-resident memory CD8+ T (TRM) cells have been associated with robust protective antitumor immune responses and improved prognosis of patients with cancer. Therefore, therapeutic strategies that modulate either the production or activity of TRM cells could be effective for treating cancer. Using a high-throughput drug screen, we showed that the neurotransmitter dopamine drives differentiation of CD8+ T cells into CD103+ TRM cells. In murine syngeneic tumor xenograft models and clinical human colon cancer samples, DRD5 served as the major functional dopamine receptor on CD8+ T cells and positively correlated with TRM cell density. DRD5 deficiency led to a failure of CD8+ T cells to accumulate in tissues, resulting in impaired TRM cell formation, reduced effector function, and uncontrolled disease progression. Moreover, dopamine treatment promoted the antitumor activity of CD8+ T cells and suppressed colorectal cancer growth in immunocompentent mouse models, and ex vivo preconditioning with dopamine enhanced the in vivo efficacy of chimeric antigen receptor (CAR)-T cells. Finally, in a patient with colorectal cancer cohort, dopamine expression was positively associated with patient survival and CD8+ T-cell infiltration. These findings suggest that dopaminergic immunoregulation plays an important role in the differentiation of CD8+ cells into CD103+ TRM cells and thereby modulates TRM-elicited antitumor immunity in colorectal cancer. Significance: Identification of an immunostimulatory function of dopamine signaling by promoting tissue-resident memory T-cell differentiation and sustaining T-cell effector functions reveals potential therapeutic strategies and prognostic biomarkers for colorectal cancer.
Likai Tan, Daniel Inácio, Immo Prinz, and Bruno Silva-Santos
Elsevier BV
Francesca Rampoldi, Elisa Donato, Leon Ullrich, Malte Deseke, Anika Janssen, Abdi Demera, Inga Sandrock, Anja Bubke, Anna-Lena Juergens, Maxine Swallow,et al.
Elsevier BV
Christian R. Schultze-Florey, Leonie Kuhlmann, Solaiman Raha, Joana Barros-Martins, Ivan Odak, Likai Tan, Yankai Xiao, Sarina Ravens, Lothar Hambach, Letizia Venturini,et al.
American Society of Hematology
Abstract Donor lymphocyte infusion (DLI) is a standard of care for relapse of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation. Currently it is poorly understood how and when CD8+ αβ T cells exert graft-versus-leukemia (GVL) activity after DLI. Also, there is no reliable biomarker to monitor GVL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αβ T-cell clones in patients with DLI. In this prospective clinical study of 29 patients, we performed deep T-cell receptor β (TRB ) sequencing of sorted CD8+ αβ T cells to track patients’ repertoire changes in response to DLI. Upon first occurrence of GVL, longitudinal analyses revealed a preferential expansion of distinct CD8+TRB clones (n = 14). This did not occur in samples of patients without signs of GVL (n = 11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36-month study follow-up. Furthermore, absence of clonal outgrowth of the CD8+TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+TRB diversity at day 15 after DLI (n = 13) had a lower relapse incidence (P = .0040) compared with patients without clonal expansion (n = 6). In conclusion, CD8+TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.
Valentina Cazzetta, Elena Bruni, Sara Terzoli, Claudia Carenza, Sara Franzese, Rocco Piazza, Paolo Marzano, Matteo Donadon, Guido Torzilli, Matteo Cimino,et al.
Elsevier BV
Shuyong Zhu, Nancy Stanslowsky, Jorge Fernández-Trillo, Tamrat M. Mamo, Pengfei Yu, Norman Kalmbach, Birgit Ritter, Reto Eggenschwiler, Werner J.D. Ouwendijk, David Mzinza,et al.
Elsevier BV
Yiwen Zhang, Hongrong Hu, Weiwei Liu, Shu-Mei Yan, Yuzhuang Li, Likai Tan, Yingshi Chen, Jun Liu, Zhilin Peng, Yaochang Yuan,et al.
BMJ
BackgroundIn the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8+ tumor-infiltrating lymphocytes (TILs) is unclear.MethodIn this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8+ TILs.ResultsWe discovered that the Rag complex, particularly RagD, was crucial for CD8+ T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8+ TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8+ T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8+ T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo.ConclusionOur results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway.
Likai Tan, Alina Suzann Fichtner, Elena Bruni, Ivan Odak, Inga Sandrock, Anja Bubke, Alina Borchers, Christian Schultze-Florey, Christian Koenecke, Reinhold Förster,et al.
American Association for the Advancement of Science (AAAS)
Innate-like human type 3 effector γδ T cells with canonical Vγ9Vδ2+TCR develop in the earliest fetal thymus and persist into adulthood.
Alina S Fichtner, Anja Bubke, Francesca Rampoldi, Anneke Wilharm, Likai Tan, Lars Steinbrück, Christian Schultze-Florey, Constantin von Kaisenberg, Immo Prinz, Thomas Herrmann,et al.
Oxford University Press (OUP)
Abstract The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis, termed phosphoantigens (pAgs). Vγ9Vδ2 T cells are first detected at midgestation and show postnatal expansion. Interestingly, neonatal Vγ9Vδ2 T cells display a higher TCR repertoire diversity with more public clonotypes and lower pAg responsiveness than in adults. Notably, it is not known whether postnatal changes occur by TCR-dependent reactivity to pAg exposure. Here, we applied next-generation sequencing of γδ TCR repertoires to understand potential differences in the pAg-mediated response of neonatal and adult Vγ9Vδ2 T cells at the level of the expressed γδ TCR. We observed a polyclonal pAg-induced response of neonatal and adult Vγ9Vδ2 T cells, albeit neonatal γδ T cells showed less in vitro pAg responsiveness. Neonatal Vγ9Vδ2 T cells displayed a less pronounced bias for Jδ1 usage and a more frequent use of Jδ2 or Jδ3 that remained stable after pAg exposure. In addition, public and private Vδ2 TRD clones took part in the polyclonal pAg-induced response in neonates and adults. In conclusion, adult and neonatal Vγ9Vδ2 T cells both undergo polyclonal pAg-induced proliferation, whereas especially adult Vγ9Vδ2 T cells display a high stability at the level of the expressed TCR repertoire.
Nadine Eckert, Kathrin Werth, Stefanie Willenzon, Likai Tan, and Reinhold Förster
Oxford University Press (OUP)
Abstract The majority of genetically modified C57BL/6 mice contain congenic passenger DNA around the targeted gene locus as they were generated from 129-derived embryonic stem cells (ESCs) with subsequent backcrossing to the C57BL/6 genetic background. When studying the role of atypical chemokine receptor 4 (ACKR4) in the immune system, we realized that the two available Ackr4-deficient mouse strains (Ackr4−/− and Ackr4GFP/GFP) show profoundly different phenotypes: Compared to wild-type and Ackr4GFP/GFP mice, Ackr4−/− mice show a strong accumulation of plasma blasts in mesenteric lymph node and spleen as well as increased B cell proliferation after in vitro activation. This phenotype was maintained after further backcrossing to C57BL/6 mice and was even present in heterozygous Ackr4+/− animals, suggesting that a gene variant on the targeted chromosome might cause this phenotype. Exome sequencing revealed that a region of approximately 20 Mbp around the Ackr4 locus on chromosome 9 still originates from the 129 background based on high variant density observed. In activated Ackr4−/− and Ackr4GFP/GFP B cells, transcripts of genes around the Ackr4 locus were equally deregulated compared to C57BL/6 B cells, whereas increased expression of IL-6 was selectively observed in B cells of Ackr4−/− mice. Because the gene encoding for IL-6 is placed on chromosome 5 these findings suggest that passenger DNA around the Ackr4 locus has an indirect effect on B cell activation and IL-6 production. Results of the present study should not only lead to the reinterpretation of data from earlier studies using Ackr4−/− mice but should remind the scientific community about the limitations of mouse models using mice created by gene-targeting of nonsyngeneic ESCs.
Likai Tan, Inga Sandrock, Ivan Odak, Yuval Aizenbud, Anneke Wilharm, Joana Barros-Martins, Yaara Tabib, Alina Borchers, Tiago Amado, Lahiru Gangoda,et al.
Elsevier BV
Inga Sandrock, Annika Reinhardt, Sarina Ravens, Christoph Binz, Anneke Wilharm, Joana Martins, Linda Oberdörfer, Likai Tan, Stefan Lienenklaus, Baojun Zhang,et al.
Rockefeller University Press
γδ T cells are highly conserved in jawed vertebrates, suggesting an essential role in the immune system. However, γδ T cell–deficient Tcrd−/− mice display surprisingly mild phenotypes. We hypothesized that the lack of γδ T cells in constitutive Tcrd−/− mice is functionally compensated by other lymphocytes taking over genuine γδ T cell functions. To test this, we generated a knock-in model for diphtheria toxin–mediated conditional γδ T cell depletion. In contrast to IFN-γ–producing γδ T cells, IL-17–producing γδ T cells (Tγδ17 cells) recovered inefficiently after depletion, and their niches were filled by expanding Th17 cells and ILC3s. Complementary genetic fate mapping further demonstrated that Tγδ17 cells are long-lived and persisting lymphocytes. Investigating the function of γδ T cells, conditional depletion but not constitutive deficiency protected from imiquimod-induced psoriasis. Together, we clarify that fetal thymus-derived Tγδ17 cells are nonredundant local effector cells in IL-17–driven skin pathology.
Ting Pan, Zhilin Peng, Likai Tan, Fan Zou, Nan Zhou, Bingfeng Liu, Liting Liang, Cancan Chen, Jun Liu, Liyang Wu,et al.
American Society for Microbiology
Zika virus (ZIKV) infection, which causes congenital malformations, including microcephaly and other neurological disorders, has attracted global attention. We observed that several NSAIDs significantly inhibited ZIKV infection. Based on our observations, we propose a novel mechanism of action of antiviral compounds which involves the blockade of virus entry via degradation of the entry cofactor. Furthermore, NSAIDs can be practically used for preventing ZIKV infection in pregnant women, as certain NSAIDs, including ibuprofen and acetaminophen, are considered clinically safe.
Feng Huang, Jingliang Chen, Junsong Zhang, Likai Tan, Gui Lu, Yongjie Luo, Ting Pan, Juanran Liang, Qianwen Li, Baohong Luo,et al.
Wiley
Although antiviral drugs are available for the treatment of influenza infection, it is an urgent requirement to develop new antiviral drugs regarding the emergence of drug‐resistant viruses. The nucleoprotein (NP) is conserved among all influenza A viruses (IAVs) and has no cellular equivalent. Therefore, NP is an ideal target for the development of new IAV inhibitors. In this study, we identified a novel anti‐influenza compound, ZBMD‐1, from a library of 20,000 compounds using cell‐based influenza A infection assays. We found that ZBMD‐1 inhibited the replication of H1N1 and H3N2 influenza A virus strains in vitro, with an IC50 ranging from 0.41–1.14 μM. Furthermore, ZBMD‐1 inhibited the polymerase activity and specifically impaired the nuclear export of NP. Further investigation indicated that ZBMD‐1 binds to the nuclear export signal 3 (NES3) domain and the dimer interface of the NP pocket. ZBMD‐1 also protected mice that were challenged with lethal doses of A/PR/8/1934 (H1N1) virus, effectively relieving lung histopathology changes, as well as strongly inhibiting the expression of pro‐inflammatory cytokines/chemokines, without inducing toxicity effects in mice. These results suggest that ZBMD‐1 is a promising anti‐influenza compound which can be further investigated as a useful strategy against IAVs in the future.
Jun Liu, Feng Huang, Junsong Zhang, Likai Tan, Gen Lu, Xu Zhang, and Hui Zhang
Springer Science and Business Media LLC
Junsong Zhang, Feng Huang, Likai Tan, Chuan Bai, Bing Chen, Jun Liu, Juanran Liang, Chao Liu, Shaoying Zhang, Gen Lu,et al.
American Society for Microbiology
ABSTRACT The viral ribonucleoprotein (vRNP) complex of influenza A viruses (IAVs) contains an RNA-dependent RNA polymerase complex (RdRp) and nucleoprotein (NP) and is the functional unit for viral RNA transcription and replication. The vRNP complex is an important determinant of virus pathogenicity and host adaptation, implying that its function can be affected by host factors. In our study, we identified host protein Moloney leukemia virus 10 (MOV10) as an inhibitor of IAV replication, since depletion of MOV10 resulted in a significant increase in virus yield. MOV10 inhibited the polymerase activity in a minigenome system through RNA-mediated interaction with the NP subunit of vRNP complex. Importantly, we found that the interaction between MOV10 and NP prevented the binding of NP to importin-α, resulting in the retention of NP in the cytoplasm. Both the binding of MOV10 to NP and its inhibitory effect on polymerase activity were independent of its helicase activity. These results suggest that MOV10 acts as an anti-influenza virus factor through specifically inhibiting the nuclear transportation of NP and subsequently inhibiting the function of the vRNP complex. IMPORTANCE The interaction between the influenza virus vRNP complex and host factors is a major determinant of viral tropism and pathogenicity. Our study identified MOV10 as a novel host restriction factor for the influenza virus life cycle since it inhibited the viral growth rate. Conversely, importin-α has been shown as a determinant for influenza tropism and a positive regulator for viral polymerase activity in mammalian cells but not in avian cells. MOV10 disrupted the interaction between NP and importin-α, suggesting that MOV10 could also be an important host factor for influenza virus transmission and pathogenicity. Importantly, as an interferon (IFN)-inducible protein, MOV10 exerted a novel mechanism for IFNs to inhibit the replication of influenza viruses. Furthermore, our study potentially provides a new drug design strategy, the use of molecules that mimic the antiviral mechanism of MOV10.
Likai Tan, Shuo Su, David K. Smith, Shuyi He, Yun Zheng, Zhenwen Shao, Jun Ma, Huachen Zhu, and Guihong Zhang
American Society for Microbiology
ABSTRACT H6N6 viruses are commonly isolated from domestic ducks, and avian-to-swine transmissions of H6N6 viruses have been detected in China. Whether subsequent adaptation of H6N6 viruses in mammals would increase their pathogenicity toward humans is not known. To address this, we generated a mouse-adapted (MA) swine influenza H6N6 virus (A/swine/Guangdong/K6/2010 [GDK6-MA]) which exhibited greater virulence than the wild-type virus (GDK6). Amino acid substitutions in PB2 (E627K), PA (I38M), and hemagglutinin ([HA] L111F, H156N, and S263R) occurred in GDK6-MA. HA with the H156N mutation [HA(H156N)] resulted in enlarged plaque sizes on MDCK cells and enhanced early-stage viral replication in mammalian cells. PA(I38M) raised polymerase activity in vitro but did not change virus replication in either mammalian cells or mice. These single substitutions had only limited effects on virulence; however, a combination of HA(H156N S263R) with PA(I38M) in the GDK6 backbone led to a significantly more virulent variant. This suggests that these substitutions can compensate for the lack of PB2(627K) and modulate virulence, revealing a new determinant of pathogenicity for H6N6 viruses in mice, which might also pose a threat to human health. IMPORTANCE Avian H6N6 influenza viruses are enzootic in domestic ducks and have been detected in swine in China. Infections of mammals by H6N6 viruses raise the possibility of viral adaptation and increasing pathogenicity in the new hosts. To examine the molecular mechanisms of adaptation, a mouse-adapted avian-origin swine influenza H6N6 virus (GDK6-MA), which had higher virulence than its parental virus, was generated. Specific mutations were found in PB2 (E627K), PA (I38M), and HA (L111F, H156N, and S263R) and were assessed for their virulence in mice. The combination of HA(H156N S263R) and PA(I38M) compensated for the lack of PB2(627K) and showed increased pathogenicity in mice, revealing a novel mechanism that can affect the virulence of influenza viruses. H6N6 viruses should be monitored in the field for more virulent forms that could threaten human health.