Journal of immunology最新文献

Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive immunotherapy potentially applicable to many types of solid tumors. Although gynecologic malignancies are promising targets for TIL therapy, its objective efficacy has not been established. Current TIL culture typically involves incubation of dissociated samples with high-dose IL-2 (HD-IL2) for weeks to enrich tumor-reactive T cells. While this protocol has been successfully used for melanoma TIL, it has not necessarily been optimized for other cancers. Here we investigated the method of efficiently expanding TILs derived from patients with gynecological cancers. TILs were incubated with HD-IL2 (HD-IL2-TILs) or stimulated with K562 cells expressing anti-CD3 mAb and CD80 (αCD3/CD80-TILs). We found that the αCD3/CD80-TILs showed significantly better proliferation than HD-IL2-TILs. The TIL populations that predominantly expanded upon αCD3/CD80 stimulation expressed high levels of PD-1 and CD28. CD28 co-stimulation was essential to overcome PD-1-mediated signals for growth suppression. We also identified DUSP4 as a negative regulator of TIL proliferation by downregulating ERK phosphorylation. The αCD3/CD80-TILs were reactive to tumor cells as shown by IFN-γ secretion and CD107a expression. Moreover, the αCD3/CD80-TILs were efficiently transduced with a chimeric cytokine receptor that we had previously developed to provide constitutive IL-7 signaling, resulting in superior in vivo persistence and antitumor effects without exogenous cytokine support in mouse models. Collectively, this study shows that direct stimulation of TILs with anti-CD3 mAb and CD28 co-stimulation achieves efficient expansion of tumor-reactive TILs. Genetic engineering of cytokine signaling in TILs may further enhance TIL functions and replace cytokine administration after TIL infusion.

Mucosal-associated invariant T (MAIT) cells are highly conserved innate-like T cells in mammals recognized for their high baseline frequency in human blood and cytotoxic effector functions during infectious diseases, autoimmunity, and cancer. While the majority of these cells in humans express a conserved CD8αβ+ TRAV1-2 T cell receptor (TCR) recognizing microbially derived vitamin B2 intermediates presented by the evolutionarily conserved major histocompatibility complex class I-related molecule, MR1, there is an emerging appreciation for diverse MAIT cell subsets that possess distinct functions including CD4+ MAIT cells that remain underexplored. In this study, we adopted an unbiased single-cell TCR-sequencing approach in MR1-5-OP-RU-tetramer-reactive T cells. We discovered that CD4+ MAIT cells are enriched with highly diverse TRAV1-2- TCRs. To specifically characterize this TCR repertoire, we analyzed VDJ sequences across 2 datasets and identified distinct TCR usage among CD4+ MAIT cells including TRAV21, TRAV8 (TRAV8-1, TRAV8-2, TRAV8-3), and TRAV12 families (TRAV12-2, TRAV12-3), as well as more variable J segment, CDR3α, and TRBV sequences. TRAV1-2- MAIT cell TCRs were also enriched after in vitro culture with interleukin-2 and Mycobacterium tuberculosis. These results indicate that mature human CD4+ MAIT cells adopt distinct TCR usage from the canonical TRAV1-2+ CD8+ subset and suggest that alternative MR1 ligands in addition to riboflavin intermediates may select for them.

DDX3 and other DEAD-box RNA helicases regulate nuclear export, translation, splicing, and metabolism of RNA. Perturbation of Ddx3x on the mouse X-chromosome in all hematopoietic cells resulted in a loss of natural killer (NK) cells, yet whether DDX3X is important only in progenitors or within NK cells remained unexplored. Herein, we deleted Ddx3x from committed NK cells by crossing Ddx3x-floxed mice to Ncr1-iCre mice. The resulting Cre+ offspring exhibited a profound deficiency of NK cells in the spleen and bone marrow. Ncr1-iCre-mediated deletion of Ddx3x also blocked in vitro generation of NK cells. CRISPR-mediated deletion of Ddx3x or pharmacological inhibition of DDX3 helicase activity in mature mouse NK cells resulted in rapid loss of cell viability, consistent with a role for DDX3X in NK cell survival. Indeed, perturbation of DDX3X in NK cells caused a substantial decrease in protein expression levels of the prosurvival mediator MCL1 but did not affect expression of the related prosurvival proteins BCL-2 or BCL-xL. Genetic deletion of the pro-apoptotic targets of MCL1, Bak and Bax, rescued the survival of NK cells following inhibition of DDX3. Mechanistically, expression levels of Mcl1 mRNA and proteasomal degradation of MCL1 protein were independent of DDX3. Instead, DDX3 bolstered MCL1 expression by supporting de novo translation of MCL1 protein. Collectively, these findings highlight a crucial role for the RNA helicase DDX3X in maintaining the NK cell compartment by supporting efficient translation of MCL1.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapy including immune checkpoint blockade (ICB). We previously showed that ICB selects for pancreatic tumor cells that are defective in IFN-γ-inducible MHC-I, prompting us to test the impact of IL-15 complex (IL-15C) in overcoming ICB resistance. Here, we show that IL-15C markedly expands circulating NK cells, CD8+ T cells, and CD4+Cxcr3+ T cells in an orthotopic pancreatic ductal adenocarcinoma (PDA) animal model. In tumors, IL-15C + anti-PD-L1 increased CD8+ T-cell effector cytokine production and interfered with T-cell exhaustion, including mitigating IL-10. In NK cells, IL-15C + anti-PD-L1 modulated NK cell IFN-γ production but did not alter Nkg2d, Nkg2a, Klrg1, IL-10, or granzyme B. IL-15C + anti-PD-L1 significantly prolonged animal survival, leading to tumor eradication in a subset of animals, whereas monotherapies only transiently prolonged survival. Therapeutic benefit was dependent on CD8+ T cells and independent of NK cells and Nkg2d. Together, our study supports that IL-15C improves anti-PD-L1 in PDA through sustaining antitumor T-cell function.

The γδ TCR instructively directs both lineage specification and effector programming of developing γδ T cells. However, the way in which different TCR signal strengths and other auxiliary signals coordinate downstream of the γδ TCR to regulate γδ T-cell development remains unclear. In this study we defined the role of Ras guanyl-releasing protein 1 (RasGRP1) in the development and effector programming of γδ T cells. While RasGRP1 was not necessary for bulk γδ T-cell generation, we found it was required for efficient generation of Vγ4+ thymocytes and lineage-committed CD73+ γδ T cells in the thymus and periphery. Despite a decrease in immature CD73+ γδ thymocytes, there was an expansion of the perinatally derived CD8+IFNγ+ γδ T-cell population in the absence of RasGRP1. IL-17-producing γδ T cells were significantly reduced in RasGRP1 knockout mice, with a specific loss of Vγ2+ γδ T cells that corresponded to a loss of c-Maf expression as early as the DN1d thymocyte stage. Critically, these cells undergoing γδT17 programming in adults could express c-Maf in response to CCR9 stimulation, with RasGRP1 but not MEK activity being required for CCR9-induced c-Maf expression. Thus, RasGRP1 activation serves as an important signaling hub in the effector programming of γδ T cells, which integrates signals from both non-TCR and TCR inputs to direct differentiation.

Vaccination strategies and correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have predominantly focused on the spike (S) protein and neutralizing antibodies. However, the rapid emergence of SARS-CoV-2 variants has reduced the effectiveness of spike-based vaccines and monoclonal antibodies. It remains unclear how non-neutralizing antibodies that target the nucleocapsid (N) protein contribute to protection against SARS-CoV-2 variants, especially their ability to trigger antibody effector functions. These antibodies may function by binding to infected cells and initiating antibody-dependent cellular cytotoxicity (ADCC), eliminating infected cells. In this study, we demonstrate that antibodies from individuals who recovered from coronavirus disease 2019 (COVID-19) infection and/or were vaccinated with the S protein vaccine recognize viral proteins on the surface of infected cells and mediate ADCC-mediated NK cell killing of infected cells. Notably, non-neutralizing antibodies induced in COVID-19 infection recognized non-spike proteins on the surface of SARS-CoV-2 variant-infected cells, and these non-neutralizing antibodies cleared SARS-CoV-2 infected cells following depletion of spike antibodies. We identified N and minimal membrane (M) proteins as the targets of non-neutralizing antibodies on the surface of these variant-infected cells. We show that enriched N-specific antibodies from individuals who recovered from COVID-19 infection more consistently killed SARS-CoV-2 variant-infected cells than antibodies to the spike protein. The observed cross-reactivity and robust ADCC activity mediated by N-specific antibodies across various SARS-CoV-2 variant-infected cells highlight the N protein as an important vaccine target in addition to the S protein. Targeting N may provide more comprehensive and durable immunity against SARS-CoV-2 and its evolving variants.

Hepatitis B virus (HBV) exclusively infects hepatocytes and produces large quantities of subviral particles containing its surface antigen (HBsAg). T cells play a central role in controlling HBV infection but can also mediate liver injury and contribute to disease progression. However, the mechanisms that regulate T-cell responses to eliminate the virus without causing immunopathology during acute HBV infection remain poorly defined. In this study, we established acute HBV infection models in mice by delivering the HBV genome to the liver via hydrodynamic injection or high-dose adenoviral vector administration. Single-cell RNA sequencing was performed to characterize the heterogeneity of HBsAg-specific CD4+ T cells, revealing distinct functional subsets, including follicular helper (Tfh), cytotoxic, and type 1 regulatory (Tr1) cells. These subsets were further validated by flow cytometry using representative phenotypic markers. Our findings demonstrate that Tr1 cells attenuate the cytotoxicity of both CD4+ and CD8+ T cells in response to HBsAg. Neutralization of interleukin-10 (IL-10) impaired Tr1-mediated suppression of cytotoxic T-cell responses. Notably, IL-10 deficiency in Tr1 cells led to substantial liver injury without enhancing HBsAg clearance. Together, these results highlight the critical function of Tr1 cells in safeguarding against liver immunopathology by modulating T-cell cytotoxicity during acute HBV infection in mice.

In response to an infection, the host T cell compartment develops immunological memory to ensure rapid responses upon re-infection with the same pathogen. However, these memory responses can also modulate immune reactions to unrelated pathogens through bystander activation. Herein, T cells are activated in an antigen-independent manner, often triggered by innate cytokines or Toll-like receptor ligands. To uncover new strategies for modulating immune responses, it is essential to deepen our understanding of this alternative mechanism of T cell activation, particularly in humans. Therefore, we studied the response of human CD8+ T cells to innate bystander stimuli in vitro. Thereby, we measured the induction of activation markers and proliferation using flow cytometry, and depleted or blocked several potential modulatory components to identify mediators of bystander CD8+ T-cell responses. Our study demonstrates that CD8+ T cells can be activated as bystander cells in response to innate stimuli present during a viral infection, including IL-15, IFN-α, and TLR7/8 and 9 agonists (R848 and CpG). Depletion experiments demonstrated that these bystander responses are dependent on monocytes and CD4+ T cells. In addition, we revealed that the bystander responses to the innate stimuli are highly reliant on IL-2 signaling. Altogether, our study underscores the pivotal role of IL-2 in mediating bystander responses of CD8+ T cells to innate stimuli, revealing a novel mechanism of immune response modulation during viral infections.

Human whole blood (WB) immunophenotyping may represent the in vivo immunological state with better fidelity than artificially isolated peripheral blood mononuclear cells (PBMCs). We used a deep phenomics modeling approach to elucidate the quantitative differences in major immune cell lineages in WB and PBMC compartments in a steady-state in vitro setting. We studied functional innate immune responses induced by pattern recognition receptor agonist adjuvants (PRRa). Using an optimized 49-parameter CyTOF panel and implementing machine learning (ML) algorithms, we mapped PRRa-mediated CD69, CD40, CD80, CD86 and CCR7 activation at the nodal innate immune subsets level. We also portrayed cellular origin of innate functional chemokine CCL4 and intracellular cytokine interferon γ (IFNγ) production. We mapped neutrophils as the primary source of TLR7/8 agonist (TLR7/8a) and STING agonist mediated CCL4 responses in WB. Notably, in the PBMC fraction, where neutrophils are limited, natural killer (NK) cells became the major source of innate CCL4 production. TLR7/8a-mediated IFNγ induction by early NK cells was mapped in PBMCs, which was limited in WB. Considering such distinctions, we hypothesized that deep phenomics employing a clinical sample that has not been manipulated, i.e., WB, may be additive in translating in vitro innate fingerprinting into in vivo biology.

TLR7 and myeloid-derived suppressor cells (MDSCs) play unique roles in determining host resistance to candidiasis. However, the precise mechanisms of TLR7 in MDSC differentiation and functionality during Candida albicans infection remain elusive. We found that compared with wild-type mice, kidney injuries and inflammation were significant in Tlr7 knockout mice. Tlr7 deficiency impeded the differentiation and maturation of mature myeloid cells and stimulated MDSC expansion. Furthermore, the absence of Tlr7 enhanced the immunosuppressive ability of infected MDSCs. Contrarily, the treatment of the TLR7 agonist R848 directly acted on MDSCs, leading to the differentiation and maturation of MDSCs and blocking their immunosuppressive activity. TLR7+ granulocytic MDSCs (G-MDSCs) significantly exhibited the enhanced expression of RUNX1 and KLF4. Subsequently, prevention of RUNX1 activity with Ro5-3335 or treatment with KLF4-activating agent APTO-253 affected the differentiation and maturation of G-MDSCs in vitro. Taken together, our results identified a function of TLR7 in modulating the MDSC response and suggested that RUNX1 and KLF4 were key transcription factors in regulating TLR7-mediated G-MDSC immune responses.