IL-6-mediated endothelial injury impairs antiviral humoral immunity after bone marrow transplantation.

Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-γ secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-γ in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-γ-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy.

Clinical grade multiparametric cell sorting and gene-marking of regulatory T cells.

BACKGROUND AIMS: Regulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity. METHODS: We developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded in vitro and gene-marked with a clinical grade retroviral vector to enable in vivo fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs. RESULTS: We showed that Tregs could be enriched to 87- 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136-732×106 gene-marked cells, sufficient for a cell dose of at least 2 × 106 cells/kg. The expanded Tregs were highly demethylated in the FOXP3 Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive in vitro, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A. CONCLUSIONS: This study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the in vivo fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.

ANZTCT consensus position statement on ruxolitinib in steroid-refractory acute and chronic graft-versus-host disease.

This position paper provides an overview of the assessment and management of both acute and chronic graft-versus-host disease (GvHD). There is a focus on the use of ruxolitinib, a selective inhibitor of Janus kinase (JAK)1 and JAK2, for the treatment of corticosteroid-refractory and corticosteroid-dependent GvHD.

PET assessment of acute gastrointestinal graft versus host disease.

Acute gastrointestinal graft versus host disease (GI-GVHD) is a common complication following allogeneic haematopoietic cell transplantation (HCT), and is characterised by severe morbidity, frequent treatment-refractoriness, and high mortality. Early, accurate identification of GI-GVHD could allow for therapeutic interventions to ameliorate its severity, improve response rates and survival; however, standard endoscopic biopsy is inadequately informative in terms of diagnostic sensitivity or outcome prediction. In an era where rapid technological and laboratory advances have dramatically expanded our understanding of GI-GVHD biology and potential therapeutic targets, there is substantial scope for novel investigations that can precisely guide GI-GVHD management. In particular, the combination of tissue-based biomarker assessment (plasma cytokines, faecal microbiome) and molecular imaging by positron emission tomography (PET) offers the potential for non-invasive, real-time in vivo assessment of donor:recipient immune activity within the GI tract for GI-GVHD prediction or diagnosis. In this article, we review the evidence regarding GI-GVHD diagnosis, and examine the potential roles and translational opportunities posed by these novel diagnostic tools, with a focus on the evolving role of PET.

Targeting cell cycle and apoptosis to overcome chemotherapy resistance in acute myeloid leukemia.

Chemotherapy-resistant acute myeloid leukemia (AML), frequently driven by clonal evolution, has a dismal prognosis. A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockouts involving AraC metabolism and genes that regulate cell cycle arrest (cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53) as contributing to resistance. In human AML cohorts, reduced expression of CDKN2A conferred inferior overall survival and CDKN2A downregulation occurred at relapse in paired diagnosis-relapse samples, validating its clinical relevance. Therapeutically targeting the G1S cell cycle restriction point (with CDK4/6 inhibitor, palbociclib and KAT6A inhibitor, WM-1119, to upregulate CDKN2A) synergized with chemotherapy. Additionally, direct promotion of apoptosis with venetoclax, showed substantial synergy with chemotherapy, overcoming resistance mediated by impaired cell cycle arrest. Altogether, we identify defective cell cycle arrest as a clinically relevant contributor to chemoresistance and identify rationally designed therapeutic combinations that enhance response in AML, potentially circumventing chemoresistance.

A phase 3 double-blind study of the addition of tocilizumab vs placebo to cyclosporin/methotrexate GVHD prophylaxis.

We determined the efficacy of tocilizumab (TCZ) in preventing grade 2-4 acute graft-versus-host disease (aGVHD) in patients with acute leukemia or myelodysplasia undergoing matched sibling donor (MSD) or volunteer unrelated donor (VUD) allogeneic stem cell transplantation after myeloablative or reduced-intensity conditioning across 5 Australian centers. A total of 145 patients (50 MSD, 95 VUD) were randomly assigned to placebo or TCZ on day -1. All patients received T-cell-replete peripheral blood stem cell grafts and graft-versus-host disease (GVHD) prophylaxis with cyclosporin/methotrexate. A planned substudy analyzed the VUD cohort. With a median follow-up of 746 days, the incidence of grade 2-4 aGVHD at day 100 for the entire cohort was 36% for placebo vs 27% for TCZ (hazard ratio [HR], 0.69; 95% confidence interval [CI], 0.38-1.26; P = .23) and 45% vs 32% (HR, 0.61; 95% CI, 0.31-1.22; P = .16) for the VUD subgroup. The incidence of grade 2-4 aGVHD at day 180 for the entire cohort was 40% for placebo vs 29% for TCZ (HR, 0.68; 95% CI, 0.38-1.22; P = .19) and 48% vs 32% (HR, 0.59; 95% CI, 0.30-1.16; P = .13) for the VUD subgroup. Reductions in aGVHD were predominantly in grade 2 disease. For the entire cohort, transplant-related mortality occurred in 8% vs 11% of placebo-treated vs TCZ-treated patients, respectively (P = .56), and overall survival was 79% vs 71% (P = .27). Median day to neutrophil and platelet engraftment was delayed by 2 to 3 days in TCZ-treated patients, whereas liver toxicity and infectious complications were similar between groups. In this phase 3 randomized double-blind trial, TCZ showed nonsignificant trends toward reduced incidence of grade 2-4 aGVHD in recipients from HLA-matched VUDs but no improvements in long term-survival.

The NK cell granule protein NKG7 regulates cytotoxic granule exocytosis and inflammation.

Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4+ and CD8+ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8+ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4+ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy.

BACKGROUND: Analysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs. METHODS: We used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.ΔCD19. DNA from oligoclonal cell lines and polyclonal clinical samples were restriction enzyme digested with two 6-cutters, NcoI and BspHI; and the flanking genomic DNA amplified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the amplicons were sequenced on the Oxford Nanopore platform. Reads were filtered for quality, trimmed, and aligned. Custom tool was developed to cluster reads and merge overlapping clusters. RESULTS: Both inverse PCR and cassette ligation PCR could successfully amplify flanking genomic DNA, with cassette ligation PCR showing less bias. The 4.8 million raw reads were grouped into 12,186 clusters and 6410 clones. The 3'long terminal repeat (LTR)-genome junction could be resolved within a 5-nucleotide span for a majority of clusters and within one nucleotide span for clusters with ≥5 reads. The chromosomal distributions of the insertional sites and their predilection for regions proximate to transcription start sites were consistent with previous reports for gammaretroviral vector integrants as analyzed by short-read next-generation sequencing. CONCLUSION: Our study shows that it is feasible to use nanopore sequencing to map polyclonal vector integration sites. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis. Further refinement is required to reduce amplification bias and improve single nucleotide resolution.

ASC Modulates CTL Cytotoxicity and Transplant Outcome Independent of the Inflammasome.

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1ß and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.

Favourable survival in "Discordant" acute gastrointestinal graft versus host disease (GI-GVHD) is explained by mild clinical course and treatment-responsive disease.

We have previously reported that haematopoietic progenitor cell transplantation recipients with biopsy-negative acute Gastrointestinal Graft versus Host Disease (Discordant GVHD) demonstrate superior survival compared to "True Positive" cases. We aimed to elucidate this discrepancy by examining clinical and laboratory predictors of survival among patients treated for True Positive or Discordant GVHD. Data were obtained by retrospective chart review. At diagnosis, the incidence of severe symptoms, hypoalbuminaemia, hyperbilirubinaemia, and poor performance status were recorded. Following treatment, the incidence of non-response to first-line corticosteroids was assessed. Differences between cohorts were compared using Fisher's exact test. 74 patients were identified, comprising 55 (74%) True Positive and 19 (26%) Discordant GVHD cases. True Positive cases were significantly more likely to have baseline severe symptoms (84% vs. 36%; p = 0.0002) and hypoalbuminaemia (94% vs. 75%; p = 0.023). There was no significant difference between cohorts in terms of hyperbilirubinaemia or performance status. Non-response to corticosteroid therapy was observed significantly more frequently in the True Positive cohort (55% vs. 11%; p = 0.001). In summary, the superior survival observed in Discordant GVHD is explained by a less severe GI-GVHD phenotype at diagnosis and a greater likelihood of response to corticosteroids. Further research is warranted to explain biological mechanisms for these findings.

Pegylated interferon-2α invokes graft-versus-leukemia effects in patients relapsing after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation (SCT) is a curative therapy for patients with hematological malignancies related largely to an immunological graft-versus-leukemia (GVL) effect mediated by donor T cells and natural killer cells. Relapse of disease after SCT represents failure of GVL and is now the major cause of treatment failure. We sought to augment GVL effects in patients (n = 29) relapsing after SCT in a prospective phase I/II clinical trial of dose-escalated pegylated interferon-2α (peg-IFNα). The administration of peg-IFNα after reinduction chemotherapy, with or without subsequent donor lymphocyte infusion (DLI), resulted in a 2-year overall survival (OS) of 31% (95% confidence interval, 17.3%-49.2%), which rejects the null hypothesis of 7% generated by observations in an institutional historical cohort. As expected, peg-IFNα was associated with graft-versus-host disease (GVHD) and hematological toxicity, which was manageable with scheduled dose modifications. Progression-free survival (PFS) was greatest in patients who experienced GVHD, although the majority of those patients still eventually progressed. Higher PFS and OS were associated with pretreatment proportions of immune cell populations with regulatory function, including mucosal invariant T cells, regulatory T cells, and plasmacytoid dendritic cells, independent of any association with GVHD. Peg-IFNα administration after relapse thus constitutes a logical strategy to invoke GVL effects and should be studied in a larger, multicenter cohort. This trial was registered at www.anzctr.org.au as #ACTRN12612000728831.

Adoptive T Cell Therapy Following Haploidentical Hematopoietic Stem Cell Transplantation.

Delayed immune reconstitution and the consequently high rates of leukemia relapse and infectious complications are the main limitations of haploidentical hematopoietic stem cell transplantation. Donor T cell addback can accelerate immune reconstitution but the therapeutic window between graft-vs.-host disease and protective immunity is very narrow in the haploidentical transplant setting. Hence, strategies to improve the safety and efficacy of adoptive T cell transfer are particularly relevant in this setting. Adoptive T cell transfer strategies in haploidentical transplantation include the use of antigen-specific T cells, allodepletion and alloanergy induction, immune modulation by the co-infusion of regulatory cell populations, and the use of safety switch gene-modified T cells. Whilst common principles apply, there are features that are unique to haploidentical transplantation, where HLA-mismatching directly impacts on immune reconstitution, and shared vs. non-shared HLA-allele can be an important consideration in antigen-specific T cell therapy. This review will also present an update on safety switch gene-modified T cells, which can be conditionally deleted in the event of severe graft- vs.-host disease or other adverse events. Herpes Virus Simplex Thymidine Kinase (HSVtk) and inducible caspase-9 (iCasp9) are safety switches that have undergone multicenter studies in haploidentical transplantation with encouraging results. These gene-modified cells, which are trackable long-term, have also provided important insights on the fate of adoptively transferred T cells. In this review, we will discuss the biology of post-transplant T cell immune reconstitution and the impact of HLA-mismatching, and the different cellular therapy strategies that can help accelerate T cell immune reconstitution after haploidentical transplantation.

Effect of plasmapheresis on ATG (Thymoglobulin) clearance prior to adoptive T cell transfer.

The effect of anti-thymocyte globulin (ATG) on the outcome of hematopoietic stem cell transplantation (SCT) is dependent on formulation, dose and exposure. However, ATG levels are not routinely measured and therapeutic levels are not well defined. In ex vivo T cell-deplete SCT, the potential effect of residual ATG has important implications on the timing of adoptive T cell transfer. Here we measured active rabbit ATG concentration using a flow cytometry-based method that can be implemented in any laboratory. Three adult patients received 6 mg/kg Thymoglobulin over 4 days, leading to peak plasma active ATG concentration of 20.8 ± 1.4 µg/mL, suggesting volume of distribution of 16-19 L. The half-life of active ATG was 6.1 ± 0.7 days and plasmapheresis at Day 25 ± 1 post-transplant reduced mean plasma concentration from 1.25 to 0.61 µg/mL. Total ATG and active ATG do not have a constant relationship because of differences in volumes of distribution and half-lives. Thymoglobulin can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro at concentrations as low as 0.03 µg/mL, with a log-linear relationship between ATG concentration and ADCC. Plasmapheresis can remove ATG but likely has modest biological impact when performed 4 weeks after 6 mg/kg ATG.