Liver TRM Research Articles

ARTC2.2/P2RX7 Signaling during Cell Isolation Distorts Function and Quantification of Tissue-Resident CD8+ T Cell and Invariant NKT Subsets.

Peripheral invariant NKT cells (iNKT) and CD8+ tissue-resident memory T cells (TRM) express high levels of the extracellular ATP receptor P2RX7 in mice. High extracellular ATP concentrations or NAD-mediated P2RX7 ribosylation by the enzyme ARTC2.2 can induce P2RX7 pore formation and cell death. Because both ATP and NAD are released during tissue preparation for analysis, cell death through these pathways may compromise the analysis of iNKT and CD8+ TRM Indeed, ARTC2.2 blockade enhanced recovery of viable liver iNKT and TRM The expression of ARTC2.2 and P2RX7 on distinct iNKT subsets and TRM is unclear, however, as is the impact of recovery from other nonlymphoid sites. In this study, we performed a comprehensive analysis of ARTC2.2 and P2RX7 expression in iNKT and CD8+ T cells in diverse tissues, at steady-state and after viral infection. NKT1 cells and CD8+ TRM express high levels of both ARTC2.2 and P2RX7 compared with NKT2, NKT17, and CD8+ circulating memory subsets. Using nanobody-mediated ARTC2.2 antagonism, we showed that ARTC2.2 blockade enhanced NKT1 and TRM recovery from nonlymphoid tissues during cell preparation. Moreover, blockade of this pathway was essential to preserve functionality, viability, and proliferation of both populations. We also showed that short-term direct P2RX7 blockade enhanced recovery of TRM, although to a lesser degree. In summary, our data show that short-term in vivo blockade of the ARTC2.2/P2RX7 axis permits much improved flow cytometry-based phenotyping and enumeration of murine iNKT and TRM from nonlymphoid tissues, and it represents a crucial step for functional studies of these populations.

Prime-and-Trap Malaria Vaccination To Generate Protective CD8+ Liver-Resident Memory T Cells.

Tissue-resident memory CD8+ T (Trm) cells in the liver are critical for long-term protection against pre-erythrocytic Plasmodium infection. Such protection can usually be induced with three to five doses of i.v. administered radiation-attenuated sporozoites (RAS). To simplify and accelerate vaccination, we tested a DNA vaccine designed to induce potent T cell responses against the SYVPSAEQI epitope of Plasmodium yoelii circumsporozoite protein. In a heterologous "prime-and-trap" regimen, priming using gene gun-administered DNA and boosting with one dose of RAS attracted expanding Ag-specific CD8+ T cell populations to the liver, where they became Trm cells. Vaccinated in this manner, BALB/c mice were completely protected against challenge, an outcome not reliably achieved following one dose of RAS or following DNA-only vaccination. This study demonstrates that the combination of CD8+ T cell priming by DNA and boosting with liver-homing RAS enhances formation of a completely protective liver Trm cell response and suggests novel approaches for enhancing T cell-based pre-erythrocytic malaria vaccines.

CD8+ T Cell Activation Leads to Constitutive Formation of Liver Tissue-Resident Memory T Cells that Seed a Large and Flexible Niche in the Liver

Liver tissue-resident memory T (Trm) cells migrate throughout the sinusoids and are capable of protecting against malaria sporozoite challenge. To gain an understanding of liver Trm cell development, we examined various conditions for their formation. Although liver Trm cells were found in naive mice, their presence was dictated by antigen specificity and required IL-15. Liver Trm cells also formed after adoptive transfer of invitro-activated but not naive CD8+ Tcells, indicating that activation was essential but that antigen presentation within the liver was not obligatory. These Trm cells patrolled the liver sinusoids with a half-life of 36days and occupied a large niche that could be added to sequentially without effect on subsequent Trm cell cohorts. Together, our findings indicate that liver Trm cells form as a normal consequence of CD8+ Tcell activation during essentially any infection but that inflammatory and antigenic signals preferentially tailor their development.

In Vivo Blockade of Murine ARTC2.2 During Cell Preparation Preserves the Vitality and Function of Liver Tissue-Resident Memory T Cells.

On murine T cells, GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) ADP-ribosylates the P2X7 ion channel at arginine 125 in response to nicotinamide adenine dinucleotide (NAD+) released during cell preparation. We have previously shown that chronic gating of P2X7 by ADP-ribosylation reduces the vitality and function of regulatory T cells and natural killer T cells that co-express high levels of ARTC2.2 and P2X7. Here, we evaluated the expression of ARTC2.2 and P2X7 by effector and memory T cells in the liver of naïve mice and after infection with Listeria monocytogenes (Lm). We found that KLRG1−/CD69+ tissue-resident memory T cells (Trm) in the liver of naïve mice and 7 weeks after infection with Lm express high levels of ARTC2.2 and P2X7. Isolation of liver Trm and subsequent incubation at 37°C resulted in cell death of the majority of CD4+ and CD8+ Trm. Injection of the ARTC2.2-blocking nanobody s+16a 30 min prior to organ harvesting effectively prevented ADP-ribosylation of P2X7 during cell preparation and thereby prevented NAD-induced cell death of the isolated Trm upon subsequent incubation at 37°C. Consequently, preserving Trm vitality by s+16a injection enabled a highly sensitive in vitro cytokine expression profile analyses of FACS sorted liver Trm. We conclude that in vivo blockade of ARTC2.2 during cell preparation by nanobody s+16a injection represents a valuable strategy to study the role and function of liver Trm in mice.

Human intrahepatic CD69\u2009+\u2009CD8+ T cells have a tissue resident memory T cell phenotype with reduced cytolytic capacity

Tissue resident memory T cells (TRM) have been identified in various tissues, however human liver TRM to date remain unidentified. TRM can be recognized by CD69 and/or CD103 expression and may play a role in the pathology of chronic hepatitis B (CHB) and hepatitis C virus infection (CHC). Liver and paired blood mononuclear cells from 17 patients (including 4 CHB and 6 CHC patients) were isolated and CD8+ T cells were comprehensively analysed by flowcytometry, immunohistochemistry and qPCR. The majority of intrahepatic CD8+ T cells expressed CD69, a marker used to identify TRM, of which a subset co-expressed CD103. CD69 + CD8+ T cells expressed low levels of S1PR1 and KLF2 and a large proportion (>90%) was CXCR6+, resembling liver TRM in mice and liver resident NK cells in human. Cytotoxic proteins were only expressed in a small fraction of liver CD69 + CD8+ T cells in patients without viral hepatitis, however, in livers from CHB patients more CD69 + CD8+ T cells were granzyme B+. In CHC patients, less intrahepatic CD69 + CD8+ T cells were Hobit+ as compared to CHB and control patients. Intrahepatic CD69 + CD8+ T cells likely TRM which have a reduced cytolytic potential. In patients with chronic viral hepatitis TRM have a distinct phenotype.

Liver-Resident Memory CD8+ T Cells Form a Front-Line Defense against Malaria Liver-Stage Infection

In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ Tcells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.