Tracking the kinetics of intrahepatic immune responses by repeated fine needle aspiration of the liver

Liver disease is an increasing global health burden. The final sequalae of cirrhosis, liver failure and hepatocellular carcinoma are often the result of inflammation driven by intrahepatic lymphocytes. Accurate assessment of organ-specific diseases ideally employs tissue sampling though this is rarely performed. Here we report our experiences of utilising repeated fine needle aspirations (FNAs) to assess liver-derived leukocytes. In 88 patient samples, we obtained a mean of 36,959 lymphocytes from each FNA-derived biopsy (SD 22,319 cells, range 5034–91,242 cells) measured by flow cytometry. This quick technique required minimal analgesia compared to liver biopsy (p = 0.03); was well tolerated and safe, and hence repeated sampling up to 3 times within a week was feasible. We detail the technique to rapidly derive a single cell suspension suitable for multiparameter flow cytometry analysis. Finally we illustrate the importance of organ-derived sampling by showing that natural killer (NK) cells from FNA samples have a markedly altered phenotype compared to those assessed in peripheral blood. In combination these data validate FNA as a powerful and well-tolerated method of sampling intrahepatic lymphocytes to study the immunology of acute and chronic liver diseases

Re-directing CD4+ T cell responses with the flanking residues of MHC class II-bound peptides: The core is not enough

Recombinant αβ T cell receptors, expressed on T cell membranes, recognize short peptides presented at the cell surface in complex with MHC molecules. There are two main subsets of αβ T cells: CD8(+) T cells that recognize mainly cytosol-derived peptides in the context of MHC class I (pMHC-I), and CD4(+) T cells that recognize peptides usually derived from exogenous proteins presented by MHC class II (pMHC-II). Unlike the more uniform peptide lengths (usually 8–13mers) bound in the MHC-I closed groove, MHC-II presented peptides are of a highly variable length. The bound peptides consist of a core bound 9mer (reflecting the binding motif for the particular MHC-II type) but with variable peptide flanking residues (PFRs) that can extend from both the N- and C-terminus of the MHC-II binding groove. Although pMHC-I and pMHC-II play a virtually identical role during T cell responses (T cell antigen presentation) and are very similar in overall conformation, there exist a number of subtle but important differences that may govern the functional dichotomy observed between CD8(+) and CD4(+) T cells. Here, we provide an overview of the impact of structural differences between pMHC-I and pMHC-II and the molecular interactions with the T cell receptor including the functional importance of MHC-II PFRs. We consider how factors such as anatomical location, inflammatory milieu, and particular types of antigen presenting cell might, in theory, contribute to the quantitative (i.e., pMHC ligand frequency) as well as qualitative (i.e., variable PFR) nature of peptide epitopes, and hence offer a means of control and influence of a CD4(+) T cell response. Lastly, we review our recent findings showing how modifications to MHC-II PFRs can modify CD4(+) T cell antigen recognition. These findings may have novel applications for the development of CD4(+) T cell peptide vaccines and diagnostics

Home sweet home: The tumor microenvironment as a haven for regulatory T cells

CD4+Foxp3+ regulatory T cells (Tregs) have a fundamental role in maintaining immune balance by preventing autoreactivity and immune-mediated pathology. However this role of Tregs extends to suppression of anti-tumor immune responses and remains a major obstacle in the development of anti-cancer vaccines and immunotherapies. This feature of Treg activity is exacerbated by the discovery that Treg frequencies are not only elevated in the blood of cancer patients, but are also significantly enriched within tumors in comparison to other sites. These observations have sparked off the quest to understand the processes through which Tregs become elevated in cancer-bearing hosts and to identify the specific mechanisms leading to their accumulation within the tumor microenvironment. This manuscript reviews the evidence for specific mechanisms of intra-tumoral Treg enrichment and will discuss how this information may be utilized for the purpose of manipulating the balance of tumor-infiltrating T cells in favor of anti-tumor effector cells

In silico and structural analyses demonstrate that intrinsic protein motions guide T cell receptor complementarity determining region loop flexibility

T-cell immunity is controlled by T cell receptor (TCR) binding to peptide major histocompatibility complexes (pMHCs). The nature of the interaction between these two proteins has been the subject of many investigations because of its central role in immunity against pathogens, cancer, in autoimmunity, and during organ transplant rejection. Crystal structures comparing unbound and pMHC-bound TCRs have revealed flexibility at the interaction interface, particularly from the perspective of the TCR. However, crystal structures represent only a snapshot of protein conformation that could be influenced through biologically irrelevant crystal lattice contacts and other factors. Here, we solved the structures of three unbound TCRs from multiple crystals. Superposition of identical TCR structures from different crystals revealed some conformation differences of up to 5 Å in individual complementarity determining region (CDR) loops that are similar to those that have previously been attributed to antigen engagement. We then used a combination of rigidity analysis and simulations of protein motion to reveal the theoretical potential of TCR CDR loop flexibility in unbound state. These simulations of protein motion support the notion that crystal structures may only offer an artifactual indication of TCR flexibility, influenced by crystallization conditions and crystal packing that is inconsistent with the theoretical potential of intrinsic TCR motions

Suppression of tumour-specific CD4+ T cells by regulatory T cells is associated with progression of human colorectal cancer

Background. There is indirect evidence that T cell responses can control the metastatic spread of colorectal cancer (CRC). However, an enrichment of CD4(+)Foxp3(+) regulatory T cells (Tregs) has also been documented. Objective. To evaluate whether CRC promotes Treg activity and how this influences anti-tumour immune responses and disease progression. Methods. A longitudinal study of Treg activity on a cohort of patients was performed before and after tumour resection. Specific CD4(+) T cell responses were also measured to the tumour associated antigens carcinoembryonic antigen (CEA) and 5T4. Results. Tregs from 62 preoperative CRC patients expressed a highly significant increase in levels of Foxp3 compared to healthy age-matched controls (p=0.007), which returned to normal after surgery (p=0.0075). CD4(+) T cell responses to one or both of the tumour associated antigens, CEA and 5T4, were observed in approximately two-thirds of patients and one third of these responses were suppressed by Tregs. Strikingly, in all patients with tumour recurrence at 12 months, significant preoperative suppression was observed of tumour-specific (p=0.003) but not control CD4(+) T cell responses. Conclusion. These findings demonstrate that the presence of CRC drives the activity of Tregs and accompanying suppression of CD4(+) T cell responses to tumour-associated antigens. Suppression is associated with recurrence of tumour at 12 months, implying that Tregs contribute to disease progression. These findings offer a rationale for the manipulation of Tregs for therapeutic intervention

Resolution of persistent SARS-CoV-2 infection with prolonged intravenous remdesivir and vaccination in a patient post CAR-T

SARS-CoV-2 virus is a single-stranded enveloped RNA virus, which causes coronavirus disease. Most of the immunocompetent patients with SARS-CoV-2 infection do have mild to moderate respiratory illness; however, in immunocompromised patients, the course of infection is unpredictable with high rates of infectivity and mortality. So, it is important to identify the immunocompromised patients early and establish the course of treatment accordingly. Here, we describe a 25-year-old male with background of B cell ALL, post-BMT and CAR-T therapy who received treatment with remdesivir and vaccination and was followed up for six months from the onset of symptoms to post vaccination, which showed resolution of symptoms and improvement of immunological markers. Here, we review the literature concerning the course and treatment of SARS-CoV-2 infection aimed at achieving cure in this patient

Human leukocyte antigen (HLA) class II peptide flanking residues tune the immunogenicity of a human tumor-derived epitope

CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111–130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR–pHLA-II interactions

Rising incidence, progression and changing patterns of liver disease in Wales 1999-2019

BACKGROUND Liver disease incidence and hence demand on hepatology services is increasing. AIM To describe trends in incidence and natural history of liver diseases in Wales to inform effective provision of hepatology services. METHODS The registry is populated by International Classification of Diseases-10 (ICD-10) code diagnoses for residents derived from mortality data and inpatient/day case activity between 1999-2019. Pseudo-anonymised linkage of: (1) Causative diagnoses; (2) Cirrhosis; (3) Portal hypertension; (4) Decompensation; and (5) Liver cancer diagnoses enabled tracking liver disease progression. RESULTS The population of Wales in 2019 was 3.1 million. Between 1999 and 2019 73054 individuals were diagnosed with a hepatic disorder, including 18633 diagnosed with cirrhosis, 10965 with liver decompensation and 2316 with hepatocellular carcinoma (HCC). Over 21 years the incidence of liver diseases increased 3.6 fold, predominantly driven by a 10 fold increase in non-alcoholic fatty liver disease (NAFLD); the leading cause of liver disease from 2014. The incidence of cirrhosis, decompensation, HCC, and allcause mortality tripled. Liver-related mortality doubled. Alcohol-related liver disease (ArLD), autoimmune liver disease and congestive hepatopathy were associated with the highest rates of decompensation and all-cause mortality. CONCLUSION A 10 fold increase in NAFLD incidence is driving a 3.6 fold increase in liver disease in Wales over 21 years. Liver-related morbidity and mortality rose more slowly reflecting the lower progression rate in NAFLD. Incidence of ArLD remained stable but was associated with the highest rates of liver-related and all-cause mortality

Effect of modified vaccinia Ankara–5T4 and low-dose cyclophosphamide on antitumor immunity in metastatic colorectal cancer: A randomized clinical trial

Importance The success of immunotherapy with checkpoint inhibitors is not replicated in most cases of colorectal cancer; therefore, different strategies are urgently required. The oncofetal antigen 5T4 is expressed in more than 90% of cases of metastatic colorectal cancer (mCRC). Preliminary data using modified vaccinia Ankara–5T4 (MVA-5T4) in mCRC demonstrated that it safely induced serologic and T-cell responses. Objective To determine whether antitumor immunity in mCRC could be increased using MVA-5T4, metronomic low-dose cyclophosphamide, or a combination of both treatments. Design, Setting, and Participants In this randomized clinical trial, 55 patients with inoperable mCRC and prior stable disease after standard chemotherapy were enrolled at a single center and randomized to watch and wait (n = 9), cyclophosphamide treatment only (n = 9), MVA-5T4 only (n = 19), and a combination of MVA-5T4 and cyclophosphamide (n = 18). Patients were enrolled and treated from July 9, 2012, through February 8, 2016, and follow-up was completed on December 13, 2016. Data were analyzed based on intention to treat. Interventions Patients randomized to a cyclophosphamide group received 50 mg twice daily on treatment days 1 to 7 and 15 to 21. Patients randomized to a MVA-5T4 group received an intramuscular injection at a dose of 1 × 109 50% tissue culture infectious dose on treatment days 22, 36, 50, 64, 78, and 106. Main Outcomes and Measures The predefined primary end point was the magnitude of anti-5T4 immune responses (5T4-specific T-cell and antibody levels) generated at treatment week 7. Secondary end points included analysis of the kinetics of anti-5T4 responses, progression-free survival (PFS), and overall survival (OS). Results Fifty-two patients (38 men and 14 women; mean [SD] age, 64.2 [10.1] years) were included in the study analysis. The 5T4-specific antibody immune responses were significantly increased in the MVA-5T4 (83.41 [36.09] relative units [RU]; P = .02) and combination treatment (65.81 [16.68] RU; P = .002) groups compared with no treatment (20.09 [7.20] RU). Cyclophosphamide depleted regulatory T cells in 24 of 27 patients receiving MVA-5T4, independently prolonging PFS (5.0 vs 2.5 months; hazard ratio [HR], 0.48; 95% CI, 0.21-1.11; P = .09). MVA-5T4 doubled baseline anti-5T4 responses in 16 of 35 patients, resulting in significantly prolonged PFS (5.6 vs 2.4 months; HR, 0.21; 95% CI, 0.09-0.47; P < .001) and OS (20.0 vs 10.3 months; HR, 0.32; 95% CI, 0.14-0.74; P = .008). No grade 3 or 4 adverse events were observed. Conclusions and Relevance This initial randomized clinical immunotherapy study demonstrates a significant survival benefit in mCRC. Prior depletion of regulatory T cells by cyclophosphamide did not increase immune responses generated by MVA-5T4 vaccination; however, cyclophosphamide and MVA-5T4 each independently induced beneficial antitumor immune responses, resulting in prolonged survival without toxic effects. Larger clinical trials are planned to further validate these data