Selected memory T cells infused post-haploidentical hematopoietic stem cell transplantation persist and hyperexpand.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide is a curative treatment for many hematological malignancies, yet a majority of patients still suffers from recurrent infections. Post-transplant infusion of memory T-cells could potentially enhance immunological protection without increasing the risk of eliciting acute graft-versus-host disease, which is mainly induced by naïve T-cells. Here, we performed longitudinal analysis of the lymphocyte compartment in 19 patients who underwent haplo-HSCT previously enrolled in a phase II prospective clinical trial (www.clinicaltrials.gov as #NCT04687982), in which they received post-transplant CD45RA-depleted donor lymphocyte infusions (DLI). T-cell receptor sequencing analysis showed that, surprisingly, CD45RA-depleted DLI do not increase T-cell clonal diversity, but lead to prominent expansion of a selected number of infused memory T-cell clones, suggesting recruitment of these cells in the immune response. Pathogen-specific memory T-cells, including cytomegalovirus (CMV)-specific cells, were engrafted and were able to persist for at least 1 month. Deep immunophenotyping revealed strong polyfunctional effector CMV-specific T-cell responses in the majority of patients, with their expansion correlating with the frequency of CMV-specific cells in the donor. These findings provide a rationale behind the suggested improved protection against viral infections in patients receiving CD45RA-depleted DLI.

Persistence of KIRneg NK cells after haploidentical hematopoietic stem cell transplantation protects from human cytomegalovirus infection/reactivation.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) is a therapeutic option to cure patients affected by hematologic malignancies. The kinetics and the quality of immune-reconstitution (IR) impact the clinical outcome of h-HSCT and limit the onset of life-threatening Human Cytomegalovirus (HCMV) infection/reactivation. Natural Killer (NK) cells are the first lymphocytes that recover after h-HSCT and they can provide rapid innate immune responses against opportunistic pathogens. By performing a longitudinal single-cell analysis of multiparametric flow-cytometry data, we show here that the persistence at high frequencies of CD158b1b2jneg/NKG2Apos/NKG2Cneg/NKp30pos/NKp46pos (KIRneg) NK cells is associated with HCMV infection/reactivation control. These KIRneg NK cells are "unlicensed", and are not terminal-differentiated lymphocytes appearing early during IR and mainly belonging to CD56bright/CD16neg and CD56bright/CD16pos subsets. KIRneg NK cells are enriched in oxidative and glucose metabolism pathways, produce interferon-γ, and are endowed with potent antiviral activity against HCMV ex vivo. Decreased frequencies of KIRneg NK cells early during IR are associated with clinically relevant HCMV replication. Taken together, our findings indicate that the prolonged persistence of KIRneg NK cells after h-HSCT could serve as a biomarker to better predict HCMV infection/reactivation. This phenomenon also paves the way to optimize anti-viral immune responses by enriching post-transplant donor lymphocyte infusions with KIRneg NK cells.

Implementing Pre-Therapeutic UGT1A1 Genotyping in Clinical Practice: A Real-Life Study.

Current guidelines recommend pre-therapeutic UGT1A1 genotyping to guide irinotecan dosing, but the usefulness of this approach remains to be clarified. In 247 patients with advanced gastrointestinal cancers undergoing irinotecan-based chemotherapy, we prospectively performed UGT1A1*28 genotyping and we analyzed the incidence of severe neutropenia according to genotype-guided dose reductions. Overall, 28 (11.3%) and 92 (37.2%) patients were homozygous or heterozygous UGT1A1*28 carriers, respectively. Grade ≥ 3 neutropenia was reported in 39% of homozygous patients receiving an upfront dose reduction of irinotecan (median 40%, range 22-58%), in 20% of heterozygous or wild-type patients receiving full dose (ORvs*28/*28 genotype = 0.38; 95% CI: 0.14-1.03; p = 0.058), and in 15.3% of those receiving a reduced dose for clinical reasons (OR vs*28/*28 genotype = 0.28, 95% IC: 0.12-0.67; p = 0.004). Occurrence of severe neutropenia was inversely associated with dose reduction in UGT1A1*28 homozygous carriers (ORx10 unit = 0.62, 95% CI: 0.27-1.40, p = 0.249) and UGT1A1 heterozygous or wild-type patients (ORx10 unit = 0.87, 95% CI: 0.59-1.28, p = 0.478). Incidence of severe neutropenia was related to irinotecan doses and UGT1A1 polymorphisms. Upfront irinotecan dose reductions do not reduce the burden of grade ≥ 3 neutropenia in UGT1A1*28 homozygous carriers.

Risk Factors for Early Cytomegalovirus Reactivation and Impact of Early Cytomegalovirus Reactivation on Clinical Outcomes after T Cell-Replete Haploidentical Transplantation with Post-Transplantation Cyclophosphamide.

Risk factors for cytomegalovirus (CMV) reactivation and the impact of CMV reactivation on patient outcomes have been extensively investigated after matched related or unrelated donor transplantation, but little is known in the setting of haploidentical stem cell transplantation (Haplo-SCT) with post-transplantation cyclophosphamide (PT-Cy), in which recipients are considered more severely immunocompromised. We retrospectively analyzed a cohort of 554 consecutive patients undergoing Haplo-SCT with PT-Cy at 3 different centers. Early CMV reactivation (occurring within the first 120 days post-transplantation) occurred in 242 patients, for an estimated cumulative incidence of 44%. Among those patients, 74 (30%) had recurrent CMV and 20 (8%) had CMV disease. On multivariable analysis, positive recipient CMV serostatus (hazard ratio [HR] >2.5; P < .001), disease histology (lymphoid versus myeloid: HR, 0.66; P = .003) and increasing recipient age (HR, 1.01; P = .015) were independent predictors of CMV reactivation. At a 4-month landmark analysis, CMV reactivation was associated with higher 1-year and 5-year cumulative incidence of nonrelapse mortality (NRM) relative to patients without reactivation: 13% versus 5% and 22% versus 9%, respectively (P < .001). On multivariable analysis, CMV reactivation was an independent negative predictor of NRM (HR, 2.69; P < .001) and was close to statistically significant for overall survival (HR, 1.38; P = .062). Our results suggest that CMV reactivation plays an important role at determining NRM. Because patient CMV serostatus is the main predictor of CMV reactivation, it should be considered when evaluating strategies for preventing CMV reactivation. 2022 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Single-cell profiling identifies impaired adaptive NK cells expanded after HCMV reactivation in haploidentical HSCT.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. NK cells are the first lymphocytes that recover after transplant and provide a prompt defense against human cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single-cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune reconstitution together with the expansion of CD158b1b2jpos/NKG2Aneg/NKG2Cpos/NKp30lo NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations, and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2jpos NK cells confirmed the ability of HCMV to deregulate NKG2C, NKG2A, and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits. These NK cells are characterized by the downmodulation of several gene pathways associated with cell migration, the cell cycle, and effector-functions, as well as by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-γ, a phenomenon also due to the viral-induced expression of lymphocyte-activation gene 3 (LAG-3) and programmed cell death protein 1 (PD-1) checkpoint inhibitors.

Independent expression of circulating and tissue levels of PD-L1: correlation of clusters with tumor metabolism and outcome in patients with non-small cell lung cancer.

PURPOSE: To evaluate the clinical-pathological and prognostic significance of the circulating PD-L1 level in patients with surgically treated NSCLC, by combining data for PD-L1 expression with other immune-related markers and tumor metabolism. METHODS: Overall, 40 patients with resected NSCLC (stage Ia-IIIa) who had preoperative blood storage and underwent staging PET/CT were enrolled for the study. In all cases, we determined plasma levels of PD-L1 (pg/ml), immune-reactive areas (IRA %) covered by CD3, CD68, CD20, CD8, PD-1, and PD-L1 in the tumor specimen, and metabolic parameters on PET, i.e., SUVmax, SUVpeak, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Variables were statistically analyzed to establish their association with disease-free survival (DFS). RESULTS: The circulating levels of PD-L1 in the bloodstream could be determined in 38/40 (95%) samples. The mean and median expression levels were 34.86 pg/ml and 24.83 pg/ml, respectively. We did not find any statistically significant correlation between circulating PD-L1 and tissue expression of PD-L1/PD-1. Some mild degree of positive correlation was determined between tissue PD-L1 and SUVmax (ρ = 0.390; p = 0.0148). Hierarchical clustering combining circulating, tissue, and metabolic parameters identified clusters with high metabolic tumor burden or high expression of plasma PD-L1 levels (Z score ≥ 2) as having a poor DFS (p = 0.033). The multivariate analysis detected stage and metabolism (i.e., SUVmax and SUVpeak) as independent prognostic factors for DFS. CONCLUSION: Plasma levels of PD-L1 are independent of the expression of PD-1/PD-L1 in NSCLC tumor tissue and, when combined with other clinical-pathological parameters, allow for the identification of clusters with different outcomes.

Genomic Integration of HHV-6 Mimicking Viral Reactivation after Autologous Stem Cell Transplantation.

The monitoring of Human Herpesvirus 6 (HHV-6) after allogeneic stem cell transplantation has proven to be useful in preventing life-threatening complications; however, the pathogenic role of HHV-6 after autologous transplantation is not well-characterized, although viral reactivation might be responsible for significant complications even after this type of transplant. Here we report, for the first time to our knowledge, the case of a patient with chromosomally integrated HHV-6 (ciHHV-6), presenting with high titers of HHV-6 DNA copies after autologous transplantation, mimicking HHV-6 reactivation. The presence of viral DNA in the follicle bulb confirmed the ciHHV-6 and allowed for the discontinuation of the antiviral treatment. Due to the increasing awareness of HHV-6 potential pathogenicity and the fact that ciHHV-6 is expected in 1-2% of the population, such a case might be helpful in recognizing ci HHV-6, thus avoiding unnecessary and potentially toxic antiviral therapy once the viral genomic integration is confirmed.

Human Herpesvirus 6 replication predicts Cytomegalovirus reactivation after allogeneic stem cell transplantation from haploidentical donor.

BACKGROUND AND OBJECTIVES: Since HHV-6 reactivation after transplant has been reported to increase the risk of CMV infection, we tested this hypothesis in the HLA-haploidentical hematopoietic stem cell transplantation setting. STUDY DESIGN: From February 2011 to October 2015, 75 patients received hematopoietic stem cell transplantation using a T-cell replete graft from a HLA-haploidentical donor at our Institution. RESULTS: Interestingly, 87% of HHV-6 reactivations were followed by a CMV reactivation, at a median of 15days between the two viruses. Incidence of CMV reactivation was 14.5-fold higher in those patients with prior HHV-6 reactivation vs. those without it (p-value<0.001). CONCLUSION: The present results suggest that HHV-6 can be considered as a predicting indicator of cellular immunosuppression preceding the onset of CMV infection.

Spatial distribution of B cells predicts prognosis in human pancreatic adenocarcinoma.

B-cell responses are emerging as critical regulators of cancer progression. In this study, we investigated the role of B lymphocytes in the microenvironment of human pancreatic ductal adenocarcinoma (PDAC), in a retrospective consecutive series of 104 PDAC patients and in PDAC preclinical models. Immunohistochemical analysis revealed that B cells occupy two histologically distinct compartments in human PDAC, either scatteringly infiltrating (CD20-TILs), or organized in tertiary lymphoid tissue (CD20-TLT). Only when retained within TLT, high density of B cells predicted longer survival (median survival 16.9 mo CD20-TLThi vs. 10.7 mo CD20-TLTlo; p = 0.0085). Presence of B cells within TLT associated to a germinal center (GC) immune signature, correlated with CD8-TIL infiltration, and empowered their favorable prognostic value. Immunotherapeutic vaccination of spontaneously developing PDAC (KrasG12D-Pdx1-Cre) mice with α-enolase (ENO1) induced formation of TLT with active GCs and correlated with increased recruitment of T lymphocytes, suggesting induction of TLT as a strategy to favor mobilization of immune cells in PDAC. In contrast, in an implanted tumor model devoid of TLT, depletion of B cells with an anti-CD20 antibody reinstated an antitumor immune response. Our results highlight B cells as an essential element of the microenvironment of PDAC and identify their spatial organization as a key regulator of their antitumor function. A mindfully evaluation of B cells in human PDAC could represent a powerful prognostic tool to identify patients with distinct clinical behaviors and responses to immunotherapeutic strategies.

Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or untreated with chemotherapy.

OBJECTIVE: Tumour-associated macrophages (TAMs) play key roles in tumour progression. Recent evidence suggests that TAMs critically modulate the efficacy of anticancer therapies, raising the prospect of their targeting in human cancer. DESIGN: In a large retrospective cohort study involving 110 patients with pancreatic ductal adenocarcinoma (PDAC), we assessed the density of CD68-TAM immune reactive area (%IRA) at the tumour-stroma interface and addressed their prognostic relevance in relation to postsurgical adjuvant chemotherapy (CTX). In vitro, we dissected the synergism of CTX and TAMs. RESULTS: In human PDAC, TAMs predominantly exhibited an immunoregulatory profile, characterised by expression of scavenger receptors (CD206, CD163) and production of interleukin 10 (IL-10). Surprisingly, while the density of TAMs associated to worse prognosis and distant metastasis, CTX restrained their protumour prognostic significance. High density of TAMs at the tumour-stroma interface positively dictated prognostic responsiveness to CTX independently of T-cell density. Accordingly, in vitro, gemcitabine-treated macrophages became tumoricidal, activating a cytotoxic gene expression programme, inhibiting their protumoural effect and switching to an antitumour phenotype. In patients with human PDAC, neoadjuvant CTX was associated to a decreased density of CD206(+) and IL-10(+) TAMs at the tumour-stroma interface. CONCLUSIONS: Overall, our data highlight TAMs as critical determinants of prognostic responsiveness to CTX and provide clinical and in vitro evidence that CTX overall directly re-educates TAMs to restrain tumour progression. These results suggest that the quantification of TAMs could be exploited to select patients more likely to respond to CTX and provide the basis for novel strategies aimed at re-educating macrophages in the context of CTX.

A case of esophageal squamous cell intraepithelial neoplasia with positivity for type 16 human papillomavirus successfully treated with radiofrequency ablation.

Esophageal cancer is the eight most common cancer worldwide and the sixth cause of cancer related death with squamous cell carcinoma (SCC) accounting for almost half of all esophageal cancers. Persistent human papilloma virus (HPV) infection has been suspected to play an active role in esophageal carcinogenesis but a clear association has not still been proven and no specific indications or guidelines for possible endoscopic and surgical therapeutic approaches to this clinical scenario are available. We report a case of a 62-year-old woman with histological diagnosis of high-grade intraepithelial squamous neoplasia of distal esophagus associated with cytological modifications resembling cervical HPV infection and with a positive INNO-LiPA assay for genotype 16 HPV. A single session of radiofrequency ablation (RFA) was performed on the dysplastic esophageal area with complete endoscopic eradication as confirmed by the following endoscopic, histologic and microbiologic examinations. Our report might give further strength to the hypothesis of an etiological role of HPV in selected cases of esophageal carcinogenesis and opens a discussion on the possible use of Radio Frequency Ablation as an effective and safe endoscopic treatment for both early squamous cell neoplasia and HPV esophageal colonization.

How do human cells react to the absence of mitochondrial DNA?

BACKGROUND: Mitochondrial biogenesis is under the control of two different genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA). The mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90 subunits that form the respiratory chain, the remaining ones being encoded by the nDNA. Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through alterations in nuclear gene expression, a phenomenon first defined in yeast and known as retrograde regulation. To investigate how the cellular transcriptome is modified in response to the absence of mtDNA, we used Affymetrix HG-U133A GeneChip arrays to study the gene expression profile of two human cell lines, 143BTK(-) and A549, which had been entirely depleted of mtDNA (rho(o) cells), and compared it with that of corresponding undepleted parental cells (rho(+) cells). RESULTS: Our data indicate that absence of mtDNA is associated with: i) a down-regulation of cell cycle control genes and a reduction of cell replication rate, ii) a down-regulation of nuclear-encoded subunits of complex III of the respiratory chain and iii) a down-regulation of a gene described as the human homolog of ELAC2 of E. coli, which encodes a protein that we show to also target to the mitochondrial compartment. CONCLUSIONS: Our results indicate a strong correlation between mitochondrial biogenesis and cell cycle control and suggest that some proteins could have a double role: for instance in controlling both cell cycle progression and mitochondrial functions. In addition, the finding that ELAC2 and maybe other transcripts that are located into mitochondria, are down-regulated in rho(o) cells, make them good candidates for human disorders associated with defective replication and expression of mtDNA.

Ethylmalonic encephalopathy is caused by mutations in ETHE1, a gene encoding a mitochondrial matrix protein.

Ethylmalonic encephalopathy (EE) is a devastating infantile metabolic disorder affecting the brain, gastrointestinal tract, and peripheral vessels. High levels of ethylmalonic acid are detected in the body fluids, and cytochrome c oxidase activity is decreased in skeletal muscle. By use of a combination of homozygosity mapping, integration of physical and functional genomic data sets, and mutational screening, we identified GenBank D83198 as the gene responsible for EE. We also demonstrated that the D83198 protein product is targeted to mitochondria and internalized into the matrix after energy-dependent cleavage of a short leader peptide. The gene had previously been known as "HSCO" (for hepatoma subtracted clone one). However, given its role in EE, the name of the gene has been changed to "ETHE1." The severe consequences of its malfunctioning indicate an important role of the ETHE1 gene product in mitochondrial homeostasis and energy metabolism.