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In classical Hodgkin lymphoma (cHL), responsiveness to immune-checkpoint blockade (ICB) is associated with specific tumor microenvironment (TME) and peripheral blood features. The role of ICB in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is not established. To gain insights into its potential in NLPHL, we compared TME and peripheral blood signatures between HLs using an integrative multiomic analysis. A discovery/validation approach in 121 NLPHL and 114 cHL patients highlighted >2-fold enrichment in programmed cell death-1 (PD-1) and T-cell Ig and ITIM domain (TIGIT) gene expression for NLPHL versus cHL. Multiplex imaging showed marked increase in intra-tumoral protein expression of PD-1+ (and/or TIGIT+) CD4+ T-cells and PD-1+CD8+ T-cells in NLPHL compared to cHL. This included T-cells that rosetted with lymphocyte predominant (LP) and Hodgkin Reed-Sternberg (HRS) cells. In NLPHL, intra-tumoral PD-1+CD4+ T-cells frequently expressed TCF-1, a marker of heightened T-cell response to ICB. The peripheral blood signatures between HLs were also distinct, with higher levels of PD-1+TIGIT+ in TH1, TH2, and regulatory CD4+ T-cells in NLPHL versus cHL. Circulating PD-1+CD4+ had high levels of TCF-1. Notably, in both lymphomas, highly expanded populations of clonal TIGIT+PD-1+CD4+ and TIGIT+PD-1+CD8+ T-cells in the blood were also present in the TME, indicating that immune-checkpoint expressing T-cells circulated between intra-tumoral and blood compartments. In in vitro assays, ICB was capable of reducing rosette formation around LP and HRS cells, suggesting that disruption of rosetting may be a mechanism of action of ICB in HL. Overall, results indicate that further evaluation of ICB is warranted in NLPHL., (© 2024 The Author(s). American Journal of Hematology published by Wiley Periodicals LLC.)

Purpose: Outcomes for Richter transformation (RT) are poor with current therapies. The efficacy and safety of anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T) for RT are not established., Methods: We performed an international multicenter retrospective study of patients with RT who received CAR-T. Patient, disease, and treatment characteristics were summarized using descriptive statistics, and modeling analyses were used to determine association with progression-free survival (PFS) and overall survival (OS). PFS and OS were estimated from the date of CAR-T infusion., Results: Sixty-nine patients were identified. The median age at CAR-T infusion was 64 years (range, 27-80). Patients had a median of four (range, 1-15) previous lines of therapy for CLL and/or RT, including previous Bruton tyrosine kinase inhibitor and/or BCL2 inhibitor therapy in 58 (84%) patients. The CAR-T product administered was axicabtagene ciloleucel in 44 patients (64%), tisagenlecleucel in 17 patients (25%), lisocabtagene maraleucel in seven patients (10%), and brexucabtagene autoleucel in one patient (1%). Eleven patients (16%) and 25 patients (37%) experienced grade ≥3 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, respectively. The overall response rate was 63%, with 46% attaining a complete response (CR). After a median follow-up of 24 months, the median PFS was 4.7 months (95% CI, 2.0 to 6.9); the 2-year PFS was 29% (95% CI, 18 to 41). The median OS was 8.5 months (95% CI, 5.1 to 25.4); the 2-year OS was 38% (95% CI, 26 to 50). The median duration of response was 27.6 months (95% CI, 14.5 to not reached) for patients achieving CR., Conclusion: CAR-T demonstrates clinical efficacy for patients with RT.

Objectives: Human cytomegalovirus (HCMV) reactivation is the leading viral complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT). Understanding of circulating cytokine/chemokine patterns which accompany HCMV reactivation and correlate with HCMV DNAemia magnitude is limited. We aimed to characterise plasma cytokine/chemokine profiles in 36 allo-HSCT patients (21 with HCMV reactivation and 15 without HCMV reactivation) at four time-points in the first 100-day post-transplant., Methods: The concentrations of 31 cytokines/chemokines in plasma samples were analysed using a multiplex bead-based immunoassay. Cytokine/chemokine concentrations were compared in patients with high-level HCMV DNAemia, low-level HCMV DNAemia or no HCMV reactivation, and correlated with immune cell frequencies measured using mass cytometry., Results: Increased plasma levels of T helper 1-type cytokines/chemokines (TNF, IL-18, IP-10, MIG) were detected in patients with HCMV reactivation at the peak of HCMV DNAemia, relative to non-reactivators. Stem cell factor (SCF) levels were significantly higher before the detection of HCMV reactivation in patients who went on to develop high-level HCMV DNAemia (810-52 740 copies/mL) vs. low-level HCMV DNAemia (< 250 copies/mL). High-level HCMV reactivators, but not low-level reactivators, developed an elevated inflammatory cytokine/chemokine profile (MIP-1α, MIP-1β, TNF, LT-α, IL-13, IL-9, SCF, HGF) at the peak of reactivation. Plasma cytokine concentrations displayed unique correlations with circulating immune cell frequencies in patients with HCMV reactivation., Conclusion: This study identifies distinct circulating cytokine/chemokine signatures associated with the magnitude of HCMV DNAemia and the progression of HCMV reactivation after allo-HSCT, providing important insight into immune recovery patterns associated with HCMV reactivation and viral control., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Chimeric antigen receptor (CAR) T cell therapy is effective in treating B cell malignancies, but factors influencing the persistence of functional CAR + T cells, such as product composition, patients' lymphodepletion, and immune reconstitution, are not well understood. To shed light on this issue, here we conduct a single-cell multi-omics analysis of transcriptional, clonal, and phenotypic profiles from pre- to 1-month post-infusion of CAR + and CAR - T cells from patients from a CARTELL study (ACTRN12617001579381) who received a donor-derived 4-1BB CAR product targeting CD19. Following infusion, CAR + T cells and CAR - T cells shows similar differentiation profiles with clonally expanded populations across heterogeneous phenotypes, demonstrating clonal lineages and phenotypic plasticity. We validate these findings in 31 patients with large B cell lymphoma treated with CD19 CAR T therapy. For these patients, we identify using longitudinal mass-cytometry data an association between NK-like subsets and clinical outcomes at 6 months with both CAR + and CAR - T cells. These results suggest that non-CAR-derived signals can provide information about patients' immune recovery and be used as correlate of clinically relevant parameters., (© 2023. The Author(s).)

Background: Corticosteroids (CSs) have previously been incorporated into graft versus host disease (GVHD) prophylaxis regimens for bone marrow (BM) and haemopoietic stem cell transplant (HSCT)., Aims: To assess the impact of prophylactic CS in HSCT using peripheral blood (PB) stem cells., Methods: Patients were identified from three HSCT centres receiving a first PB-HSCT between January 2011 and December 2015 from a fully human leukocyte antigen (HLA)-matched sibling or unrelated donor for acute myeloid leukaemia or acute lymphoblastic leukaemia. To enable meaningful comparison, patients were divided into two cohorts., Results: Cohort 1 included only myeloablative-matched sibling HSCT, where the only variation in GVHD prophylaxis was the addition of CS. In these 48 patients, there were no differences in GVHD, relapse, non-relapse mortality, overall survival or GVHD-relapse-free-survival (GRFS) at 4 years after transplant. Cohort 2 included the remaining HSCT recipients, where one group received CS-prophylaxis and the non-CS group received an antimetabolite, ciclosporin and anti-T-lymphocyte globulin. In these 147 patients, those receiving CS-prophylaxis experienced higher rates of chronic GVHD (71% vs 18.1%, P < 0.001) and lower rates of relapse (14.9% vs 33.9%, P = 0.02). Those receiving CS-prophylaxis had a lower 4-year GRFS (15.7% vs 40.3%, P = 0.002)., Conclusions: There does not appear to be a role for adding CS to standard GVHD prophylaxis regimens in PB-HSCT., (© 2023 The Authors. Internal Medicine Journal published by John Wiley & Sons Australia, Ltd on behalf of Royal Australasian College of Physicians.)

Measurable residual disease (MRD) detected by flow cytometry (FC) is well established in paediatric B- lymphoblastic leukaemia (B-ALL) and adult chronic lymphocytic leukaemia (CLL), but its utility in adult B-ALL and adult acute myeloid leukaemia (AML) is less clear. In this prospective MRD study, one of the largest in Australia to date, we examined consecutive bone marrow aspirates from adult participants with B-ALL ( n = 47) and AML ( n = 87) sent for FC-MRD testing at a quaternary referral hospital in Sydney. FC-MRD results were correlated to corresponding Mol-MRD testing where available and clinical outcomes at three-month intervals over 1 year. B-ALL showed a moderate positive correlation (r s = 0.401, p < 0.001), while there was no correlation between FC-MRD and Mol-MRD for AML (r s = 0.13, p = 0.237). Five FC-MRD patterns were identified which had significant associations with relapse (X 2 (4) = 31.17(4), p > 0.001) and survival (X 2 (4) = 13.67, p = 0.008) in AML, but not in B-ALL. The three-month MRD results were also strongly associated with survival in AML, while the association in B-ALL was less evident. There was a moderate correlation between FC-MRD and Mol-MRD in B-ALL but not AML. The association of FC-MRD with relapse and survival was stronger in AML than in B-ALL. Overall, these findings suggest divergent utilities of FC-MRD in AML and B-ALL.

Patients with post-haemopoietic stem cell transplant or chimeric antigen receptor T -cell (CAR-T) therapy face a significant risk of morbidity and mortality from coronavirus disease 2019 because of their immunosuppressed state. As case numbers in Australia and New Zealand continue to rise, guidance on management in this high-risk population is needed. Whilst we have learned much from international colleagues who faced high infection rates early in the pandemic, guidance relevant to local health system structures, medication availability and emerging therapies is essential to equip physicians to manage our patients optimally., (© 2022 The Authors. Internal Medicine Journal published by John Wiley & Sons Australia, Ltd on behalf of Royal Australasian College of Physicians.)

We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34 + stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8 + and to a lesser extent CD4 + T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3 months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability., (© 2022 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Virus-specific T-cells (VSTs) from third-party donors mediate short- and long-term antiviral effects in allogeneic hematopoietic stem cell transplant (HSCT) recipients with relapsed or refractory viral infections. We investigated early administration of third-party VSTs, together with antiviral therapy in patients requiring treatment for first cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. Thirty HSCT patients were treated with 1 to 4 VST infusions (2 × 107 cells/m2; CMV n=27, EBV n=3) at a median of 4 days after initiation of antiviral treatment. The overall viral response rate was 100%, with a complete response (CR) rate of 94%. Of the 28 patients who achieved a CR, 23 remained virus PCR negative (n=9) or below quantitation limit (n=14) for the duration of follow-up. Four patients had brief episodes of quantifiable reactivation not requiring additional therapy, and one required a second infusion after initial CR, remaining PCR negative thereafter. All 3 patients treated for EBV post-transplant lymphoproliferative disorder achieved sustained CR. Rates of aGVHD and cGVHD after infusion were 13% and 23%, respectively. There were no serious infusion-related adverse events. VST infusion was associated with rapid recovery of CD8+CD45RA-CD62L- and a slower recovery of CD4+CD45RA-CD62L- effector memory T-cells; CMV-specific T-cells comprised up to 13% of CD8+ cells. At 1 year post-transplant, non-relapse mortality was 10%, cumulative incidence of relapse was 7%, overall survival was 88% and 25 of 27 patients had ECOG status of 0 or 1. Early administration of third-party VSTs in conjunction with antiviral treatment appears safe and leads to excellent viral control and clinical outcomes. Registered on Australian New Zealand Clinical Trials Registry as #ACTRN12618000343202., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Human cytomegalovirus reactivation is a major opportunistic infection after allogeneic haematopoietic stem cell transplantation and has a complex relationship with post-transplant immune reconstitution. Here, we use mass cytometry to define patterns of innate and adaptive immune cell reconstitution at key phases of human cytomegalovirus reactivation in the first 100 days post haematopoietic stem cell transplantation. Human cytomegalovirus reactivation is associated with the development of activated, memory T-cell profiles, with faster effector-memory CD4 + T-cell recovery in patients with low-level versus high-level human cytomegalovirus DNAemia. Mucosal-associated invariant T cell levels at the initial detection of human cytomegalovirus DNAemia are significantly lower in patients who subsequently develop high-level versus low-level human cytomegalovirus reactivation. Our data describe distinct immune signatures that emerged with human cytomegalovirus reactivation after haematopoietic stem cell transplantation, and highlight Mucosal-associated invariant T cell levels at the first detection of reactivation as a marker that may be useful to anticipate the magnitude of human cytomegalovirus DNAemia., (© 2022. The Author(s).)

Purpose of Review: The clinical manifestations of the polyomaviruses BK and JC in immunocompromised patients include BK virus (BKV) induced haemorrhagic cystitis and nephropathy, and JC virus (JCV) associated progressive multifocal leukoencephalopathy (PML) and are typically a consequence of impaired adaptive immunity in the host. To date, little clinical success has been achieved with antiviral agents or other drug therapies to treat these conditions. Here we review the methods and outcomes of the most recent clinical studies utilising adoptive immunotherapy with BK and/or JC virus-specific T-cells (VST) as either prophylaxis or treatment alternatives., Recent Findings: In the last 12-18 months, several clinical trials have been published in the post-haemopoietic stem cell transplant (HSCT) setting showing good clinical success with the use of VST for treatment of BK viremia ± haemorrhagic cystitis. Between 82 and 100% clinical response has been observed in haemorrhagic cystitis using either third-party or donor-derived VST. The therapy was well tolerated with few cases of graft versus host disease in HSCT recipients, but immune mediated renal allograft loss was observed in one renal transplant recipient. Studies using BKV/JCV VST to treat PML are hindered by few patients who are sufficiently stable to receive VST. In a condition that otherwise carries such poor prognosis, VST were associated with clearance of JC virus, clinical and radiological improvement in some patients. Immune reconstitution inflammatory syndrome was a noted adverse event., Summary: Restoration of BK and JC virus immunity using VST immunotherapy has shown good clinical outcomes in BKV associated infections. Further evaluation with the administration of VST earlier in the course of disease is warranted for the treatment of BKV associated nephropathy in renal allograft and in JCV PML. In both indications, larger cohorts and standardisation of dosing and outcome measures would be of benefit., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

High-throughput single-cell technologies hold the promise of discovering novel cellular relationships with disease. However, analytical workflows constructed for these technologies to associate cell proportions with disease often employ unsupervised clustering techniques that overlook the valuable hierarchical structures that have been used to define cell types. We present treekoR, a framework that empirically recapitulates these structures, facilitating multiple quantifications and comparisons of cell type proportions. Our results from twelve case studies reinforce the importance of quantifying proportions relative to parent populations in the analyses of cytometry data - as failing to do so can lead to missing important biological insights., (© 2021. The Author(s).)

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter-driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell-derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene-modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381., (© 2021 by The American Society of Hematology.)

Cytomegalovirus (CMV) is a human herpes virus that causes significant morbidity and mortality in immunosuppressed children. CMV primary infection causes a clinically mild disease in healthy children, usually in early childhood; the virus then utilises several mechanisms to establish host latency, which allows for periodic reactivation, particularly when the host is immunocompromised. It is this reactivation that is responsible for the significant morbidity and mortality in immunocompromised children. We review CMV infection in the primary immunodeficient host, including early identification of these infants by newborn screening to allow for CMV infection prevention strategies. Furthermore, clinical CMV is discussed in the context of children treated with secondary immunodeficiency, particularly paediatric cancer patients and children undergoing haematopoietic stem cell transplant (HSCT). Treatments for CMV are highlighted and include CMV immunotherapy.