The transcription regulator ID3 maintains tumor-specific memory CD8+ T cells in draining lymph nodes during tumorigenesis.

During tumorigenesis, the recently identified tumor-specific memory T cells in draining lymph nodes (TdLN-TTSM cells) play a pivotal role in tumor repression that gives rise to progenitor exhausted T (TPEX) cells and further replenishes tumor-specific CD8+ T cells residing in the tumor microenvironment (TME). However, how TTSM cells are maintained in TdLN is largely unknown. Here, we show that the transcription regulator ID3 (inhibitor of DNA binding 3) is highly expressed by TTSM cells compared with other CD8+ T cell subsets. The deficiency of ID3 significantly interrupts the maintenance of TTSM and TPEX cells, resulting in decreased tumor-infiltrating CD8+ T cells and impaired tumor control. Consistent with this, overexpression of ID3 in CD8+ T cells increases the TTSM cell population and enhances the anti-tumor immune response.

Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor.

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ T cells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ T cells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ T cell subset by coupled T cell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ T cells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ T cell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ T cell exhaustion and could be targeted to potentiate cancer immunotherapy.

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice.

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

Mycobacterium tuberculosis produces D-serine under hypoxia to limit CD8+ T cell-dependent immunity in mice.

Adaptation to hypoxia is a major challenge for the survival of Mycobacterium tuberculosis (Mtb) in vivo. Interferon (IFN)-γ-producing CD8+ T cells contribute to control of Mtb infection, in part by promoting antimicrobial activities of macrophages. Whether Mtb counters these responses, particularly during hypoxic conditions, remains unknown. Using metabolomic, proteomic and genetic approaches, here we show that Mtb induced Rv0884c (SerC), an Mtb phosphoserine aminotransferase, to produce D-serine. This activity increased Mtb pathogenesis in mice but did not directly affect intramacrophage Mtb survival. Instead, D-serine inhibited IFN-γ production by CD8+ T cells, which indirectly reduced the ability of macrophages to restrict Mtb upon co-culture. Mechanistically, D-serine interacted with WDR24 and inhibited mTORC1 activation in CD8+ T cells. This decreased T-bet expression and reduced IFN-γ production by CD8+ T cells. Our findings suggest an Mtb evasion mechanism where pathogen metabolic adaptation to hypoxia leads to amino acid-dependent suppression of adaptive anti-TB immunity.

An oncolytic virus delivering tumor-irrelevant bystander T cell epitopes induces anti-tumor immunity and potentiates cancer immunotherapy.

Tumor-specific T cells are crucial in anti-tumor immunity and act as targets for cancer immunotherapies. However, these cells are numerically scarce and functionally exhausted in the tumor microenvironment (TME), leading to inefficacious immunotherapies in most patients with cancer. By contrast, emerging evidence suggested that tumor-irrelevant bystander T (TBYS) cells are abundant and preserve functional memory properties in the TME. To leverage TBYS cells in the TME to eliminate tumor cells, we engineered oncolytic virus (OV) encoding TBYS epitopes (OV-BYTE) to redirect the antigen specificity of tumor cells to pre-existing TBYS cells, leading to effective tumor inhibition in multiple preclinical models. Mechanistically, OV-BYTE induced epitope spreading of tumor antigens to elicit more diverse tumor-specific T cell responses. Remarkably, the OV-BYTE strategy targeting human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory efficiently inhibited tumor progression in a human tumor cell-derived xenograft model, providing important insights into the improvement of cancer immunotherapies in a large population with a history of SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination.

Bile acids modified by the intestinal microbiota promote colorectal cancer growth by suppressing CD8+ T cell effector functions.

Concentrations of the secondary bile acid, deoxycholic acid (DCA), are aberrantly elevated in colorectal cancer (CRC) patients, but the consequences remain poorly understood. Here, we screened a library of gut microbiota-derived metabolites and identified DCA as a negative regulator for CD8+ T cell effector function. Mechanistically, DCA suppressed CD8+ T cell responses by targeting plasma membrane Ca2+ ATPase (PMCA) to inhibit Ca2+-nuclear factor of activated T cells (NFAT)2 signaling. In CRC patients, CD8+ T cell effector function negatively correlated with both DCA concentration and expression of a bacterial DCA biosynthetic gene. Bacteria harboring DCA biosynthetic genes suppressed CD8+ T cells effector function and promoted tumor growth in mice. This effect was abolished by disrupting bile acid metabolism via bile acid chelation, genetic ablation of bacterial DCA biosynthetic pathway, or specific bacteriophage. Our study demonstrated causation between microbial DCA metabolism and anti-tumor CD8+ T cell response in CRC, suggesting potential directions for anti-tumor therapy.

Role of tumor cell pyroptosis in anti-tumor immunotherapy.

Peripheral tumor-specific CD8+ T cells often fail to infiltrate into tumor parenchyma due to the immunosuppression of tumor microenvironment (TME). Meanwhile, a significant portion of tumor-specific CD8+ T cells infiltrated into TME are functionally exhausted. Despite the enormous success of anti-PD-1/PD-L1 immune-checkpoint blockade (ICB) treatment in a wide variety of cancer types, the majority of patients do not respond to this treatment largely due to the failure to efficiently drive tumor-specific CD8+ T cell infiltration and reverse their exhaustion states. Nowadays, tumor cell pyroptosis, a unique cell death executed by pore-forming gasdermin (GSDM) family proteins dependent or independent on inflammatory caspase activation, has been shown to robustly promote immune-killing of tumor cells by enhancing tumor immunogenicity and altering the inflammatory state in the TME, which would be beneficial in overcoming the shortages of anti-PD-1/PD-L1 ICB therapy. Therefore, in this review we summarize the current progresses of tumor cell pyroptosis in enhancing immune function and modulating TME, which synergizes anti-PD-1/PD-L1 ICB treatment to achieve better anti-tumor effect. We also enumerate several strategies to better amply the efficiency of anti-PD-1/PD-L1 ICB therapy by inducing tumor cell pyroptosis.

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis.

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

The role of CD4+ T cells in tumor and chronic viral immune responses.

Immunotherapies are mainly aimed to promote a CD8+ T cell response rather than a CD4+ T cell response as cytotoxic T lymphocytes (CTLs) can directly kill target cells. Recently, CD4+ T cells have received more attention due to their diverse roles in tumors and chronic viral infections. In antitumor and antichronic viral responses, CD4+ T cells relay help signals through dendritic cells to indirectly regulate CD8+ T cell response, interact with B cells or macrophages to indirectly modulate humoral immunity or macrophage polarization, and inhibit tumor blood vessel formation. Additionally, CD4+ T cells can also exhibit direct cytotoxicity toward target cells. However, regulatory T cells exhibit immunosuppression and CD4+ T cells become exhausted, which promote tumor progression and chronic viral persistence. Finally, we also outline immunotherapies based on CD4+ T cells, including adoptive cell transfer, vaccines, and immune checkpoint blockade. Overall, this review summarizes diverse roles of CD4+ T cells in the antitumor or protumor and chronic viral responses, and also highlights the immunotherapies based on CD4+ T cells, giving a better understanding of their roles in tumors and chronic viral infections.

Functional diversification and dynamics of CAR-T cells in patients with B-ALL.

Understanding of cellular evolution and molecular programs of chimeric antigen receptor-engineered (CAR)-T cells post-infusion is pivotal for developing better treatment strategies. Here, we construct a longitudinal high-precision single-cell transcriptomic landscape of 7,578 CAR-T cells from 26 patients with B cell acute lymphoblastic leukemia (B-ALL) post-infusion. We molecularly identify eight CAR-T cell subtypes, including three cytotoxic subtypes with distinct kinetics and three dual-identity subtypes with non-T cell characteristics. Remarkably, long-term remission is coincident with the dominance of cytotoxic subtypes, while leukemia progression is correlated with the emergence of subtypes with B cell transcriptional profiles, which have dysfunctional features and might predict relapse. We further validate in vitro that the generation of B-featured CAR-T cells is induced by excessive tumor antigen stimulation or suppressed TCR signaling, while it is relieved by exogenous IL-12. Moreover, we define transcriptional hallmarks of CAR-T cell subtypes and reveal their molecular changes along computationally inferred cellular evolution in vivo. Collectively, these results decipher functional diversification and dynamics of peripheral CAR-T cells post-infusion.

Dopamine Receptor 1 Impedes ILC2-Mediated Antitumor Immunity.

Ever-growing evidence has revealed that group 2 innate lymphoid cells (ILC2s) exhibit pleiotropic effects in antihelminth immunity, allergy, tissue protection, and cancer. Currently, the role of ILC2s in cancer is highly controversial regarding the intricate tumor microenvironment (TME), and the tumor-promoting or antitumor immunological mechanisms of ILC2s remain largely unknown. In this study, we report that dopamine receptor 1 (DRD1) restrains ILC2 activity in the TME. DRD1 deficiency promotes ILC2 activation, which irritates eosinophil recruitment and cytotoxic CD8+ T cell expansion during ongoing malignancy. Consequently, DRD1-deficient mice exhibit delayed tumor growth and reduced tumor progression. Furthermore, fenoldopam, a selective DRD1 agonist, restrains the ILC2 response in the TME and aggravates tumor burden in mice. Taken together, our data elaborate that the DRD1 signal acts as an excitatory rheostat in regulating ILC2-dependent antitumor immunity.

The TRIM37 variants in Mulibrey nanism patients paralyze follicular helper T cell differentiation.

The Mulibrey (Muscle-liver-brain-eye) nanism caused by loss-of-function variants in TRIM37 gene is an autosomal recessive disorder characterized by severe growth failure and constrictive pericarditis. These patients also suffer from severe respiratory infections, co-incident with an increased mortality rate. Here, we revealed that TRIM37 variants were associated with recurrent infection. Trim37 FINmajor (a representative variant of Mulibrey nanism patients) and Trim37 knockout mice were susceptible to influenza virus infection. These mice showed defects in follicular helper T (TFH) cell development and antibody production. The effects of Trim37 on TFH cell differentiation relied on its E3 ligase activity catalyzing the K27/29-linked polyubiquitination of Bcl6 and its MATH domain-mediated interactions with Bcl6, thereby protecting Bcl6 from proteasome-mediated degradation. Collectively, these findings highlight the importance of the Trim37-Bcl6 axis in controlling the development of TFH cells and the production of high-affinity antibodies, and further unveil the immunologic mechanism underlying recurrent respiratory infection in Mulibrey nanism.

Aged hematopoietic stem cells entrap regulatory T cells to create a prosurvival microenvironment.

Although DNA mutation drives stem cell aging, how mutation-accumulated stem cells obtain clonal advantage during aging remains poorly understood. Here, using a mouse model of irradiation-induced premature aging and middle-aged mice, we show that DNA mutation accumulation in hematopoietic stem cells (HSCs) during aging upregulates their surface expression of major histocompatibility complex class II (MHCII). MHCII upregulation increases the chance for recognition by bone marrow (BM)-resident regulatory T cells (Tregs), resulting in their clonal expansion and accumulation in the HSC niche. On the basis of the establishment of connexin 43 (Cx43)-mediated gap junctions, BM Tregs transfer cyclic adenosine monophosphate (cAMP) to aged HSCs to diminish apoptotic priming and promote their survival via activation of protein kinase A (PKA) signaling. Importantly, targeting the HSC-Treg interaction or depleting Tregs effectively prevents the premature/physiological aging of HSCs. These findings show that aged HSCs use an active self-protective mechanism by entrapping local Tregs to construct a prosurvival niche and obtain a clonal advantage.

PD-1/PD-L1 blockade restores tumor-induced COVID-19 vaccine bluntness.

The COVID-19 vaccinations are crucial in protecting against the global pandemic. However, accumulating studies revealed the severely blunted COVID-19 vaccine effectiveness in cancer patients. The PD-1/PD-L1 immune checkpoint blockade (ICB) therapy leads to durable therapeutic responses in a subset of cancer patients and has been approved to treat a wide spectrum of cancers in the clinic. In this regard, it is pivotal to explore the potential impact of PD-1/PD-L1 ICB therapy on COVID-19 vaccine effectiveness during ongoing malignancy. In this study, using preclinical models, we found that the tumor-suppressed COVID-19 vaccine responses are largely reverted in the setting of PD-1/PD-L1 ICB therapy. We also identified that the PD-1/PD-L1 blockade-directed restoration of COVID-19 vaccine effectiveness is irrelevant to anti-tumor therapeutic outcomes. Mechanistically, the restored COVID-19 vaccine effectiveness is entwined with the PD-1/PD-L1 blockade-driven preponderance of follicular helper T cell and germinal center responses during ongoing malignancy. Thus, our findings indicate that PD-1/PD-L1 blockade will greatly normalize the responses of cancer patients to COVID-19 vaccination, while regardless of its anti-tumor efficacies on these patients.

An RBD bispecific antibody effectively neutralizes a SARS-CoV-2 Omicron variant.

Potent neutralizing antibodies (nAbs) against SARS-CoV-2 are a promising therapeutic against the ongoing COVID-19 pandemic. However, the continuous emergence of neutralizing antibody escape variants makes it challenging for antibody therapeutics based on monospecific nAbs. Here, we generated an IgG-like bispecific antibody (bsAb), Bi-Nab, based on a pair of human neutralizing antibodies targeting multiple and invariant sites of the spike receptor binding domain (RBD): 35B5 and 32C7. We demonstrated that Bi-Nab exhibited higher binding affinity to the Delta spike protein than its parental antibodies and presented an extended inhibition breadth of preventing RBD binding to angiotensin-converting enzyme 2 (ACE2), the cellular receptor of SARS-CoV-2. In addition, pseudovirus neutralization results showed that Bi-Nab improved the neutralization potency and breadth with a lower half maximum inhibitory concentration (IC50) against wild-type SARS-CoV-2, variants being monitored (VBMs) and variants of concern (VOCs). Notably, the IgG-like Bi-Nab enhanced the neutralizing activity against Omicron variants with potent capabilities for transmission and immune evasion in comparison with its parental monoclonal antibody (mAb) 32C7 and a cocktail (with the lowest IC50 values of 31.6 ng/mL against the Omicron BA.1 and 399.2 ng/mL against the Omicron BA.2), showing evidence of synergistic neutralization potency of Bi-Nab against the Omicron variants. Thus, Bi-Nab represents a feasible and effective strategy against SARS-CoV-2 variants of concern.

The G allele of SNP rs3922 reduces the binding affinity between IGF2BP3 and CXCR5 correlating with a lower antibody production.

Effective vaccines that function through humoral immunity seek to produce high-affinity antibodies. Our previous research identified the single-nucleotide polymorphism rs3922G in the 3'UTR of CXCR5 as being associated with nonresponsiveness to the hepatitis B vaccine. The differential expression of CXCR5 between the dark zone (DZ) and light zone (LZ) is critical for organizing the functional structure of the germinal center (GC). In this study, we report that the RNA-binding protein IGF2BP3 can bind to CXCR5 mRNA containing the rs3922 variant to promote its degradation via the nonsense-mediated mRNA decay pathway. Deficiency of IGF2BP3 leads to increased CXCR5 expression, which results in the disappearance of CXCR5 differential expression between DZ and LZ, disorganized GCs, aberrant somatic hypermutations, and reduced production of high-affinity antibodies. Furthermore, the affinity of IGF2BP3 for the rs3922G-containing sequence is lower than that for the rs3922A counterpart, which may explain the nonresponsiveness to the hepatitis B vaccination. Together, our findings suggest that IGF2BP3 plays a crucial role in the production of high-affinity antibodies in the GC by binding to the rs3922-containing sequence to regulate CXCR5 expression.

Identification and Comparison of the Sialic Acid-Binding Domain Characteristics of Avian Coronavirus Infectious Bronchitis Virus Spike Protein.

Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSDM41-S1, rSDM41-206, rGDM41-S1, and rGDM41-206 strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design. IMPORTANCE To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.

A novel mAb broadly neutralizes SARS-CoV-2 VOCs in vitro and in vivo, including the Omicron variants.

Novel immune escape variants have emerged as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. Many of the variants cause breakthrough infections in vaccinated populations, posing great challenges to current antiviral strategies targeting the immunodominance of the receptor-binding domain within the spike protein. Here, we found that a novel broadly neutralizing monoclonal antibody (mAb), G5, provided efficient protection against SARS-CoV-2 variants of concern (VOCs) in vitro and in vivo. A single dose of mAb G5 could significantly inhibit the viral burden in mice challenged with the mouse-adapted SARS-CoV-2 or SARS-CoV-2 Omicron BA.1 variant, as well as the body weight loss and cytokine release induced by mouse-adapted SARS-CoV-2. The refined epitope recognized by mAb G5 was identified as 1148 FKEELDKYF1156 in the stem helix of subunit S2. In addition, a human-mouse chimeric mAb was generated based on the variable region of heavy chain and VL genes of mAb G5. Our study provides a broad antibody drug candidate against SARS-CoV-2 VOCs and reveals a novel target for developing pan-SARS-CoV-2 vaccines.