Severe Immunodeficiency Research Articles

A clinical case of disseminated BCG infection with a fatal outcome

Complications after the introduction of the tuberculosis vaccine develop extremely rarely. They become accidentally vaccinated infants with undiagnosed severe immunodeficiency. The article considers a clinical case of the course of disseminated BCG infection with multiple organ damage in congenital immunodeficiency in a 9-month-old boy. The disease manifested itself acutely, the child’s condition worsened within a week, and its outcome was the death of the baby. Primary immunodeficiency was confirmed by the results of a pathoanatomic examination. The discussion provides a classification of complications after the introduction of BCG vaccine, the algorithm of the doctor’s actions in case of a suspected complication after vaccination, and also describes the main signs of immunodeficiency.

Multiplex Real-Time PCR-Based Newborn Screening for Severe Primary Immunodeficiency and Spinal Muscular Atrophy in Osaka, Japan: Our Results after 3 Years.

In newborn screening (NBS), it is important to consider the availability of multiplex assays or other tests that can be integrated into existing systems when attempting to implement NBS for new target diseases. Recent developments in innovative testing technology have made it possible to simultaneously screen for severe primary immunodeficiency (PID) and spinal muscular atrophy (SMA) using quantitative real-time polymerase chain reaction (qPCR) assays. We describe our experience of optional NBS for severe PID and SMA in Osaka, Japan. A multiplex TaqMan qPCR assay was used for the optional NBS program. The assay was able to quantify the levels of T-cell receptor excision circles and kappa-deleting recombination excision circles, which is useful for severe combined immunodeficiency and B-cell deficiency screening, and can simultaneously detect the homozygous deletion of SMN1 exon 7, which is useful for NBS for SMA. In total, 105,419 newborns were eligible for the optional NBS program between 1 August 2020 and 31 August 2023. A case each of X-linked agammaglobulinemia and SMA were diagnosed through the optional NBS and treated at early stages (before symptoms appeared). Our results show how multiplex PCR-based NBS can benefit large-scale NBS implementation projects for new target diseases.

Клинический случай прогрессирующего микобактериоза легких, потенциально связанный с генетической предрасположенностью, у пациентки с легким течением бронхиальной астмы

Mycobacteriosis is an infectious disease caused by opportunistic non-tuberculous microbacteria in patients with chronic respiratory pathology and decreased immunological reactivity. The number of patients with this pathology is increasing not only in Russia, but throughout the world. Scientists' attention was drawn to the fact that the isolation of non-tuberculous mycobacteria from patient’s sputum samples does not correlate with the incidence of non-tuberculous mycobacteriosis and the need for its treatment. This may be due to the presence of gene polymorphisms that affect the structure and functional characteristics of innate immune factors. The article presents a rare clinical case of non-tuberculous mycobacteriosis caused by Mycobacterium avium in a 50-year-old woman without severe concomitant diseases and secondary immunodeficiency. The genetic study revealed polymorphic gene alleles, which may predispose this patient for the infection and severe course of this disease.

Chimeric antigen receptor T-cell therapy after COVID-19 in refractory high-grade B-cell lymphoma

Although chimeric antigen receptor T-cell (CAR-T) therapies have dramatically improved the outcomes of relapsed/refractory B-cell malignancies, recipients suffer from severe humoral immunodeficiencies. Furthermore, patients with coronavirus disease 2019 (COVID-19) have a poor prognosis, as noted in several case reports of recipients who had COVID-19 before the infusion. We report the case of a 70-year-old woman who developed COVID-19 immediately before CAR-T therapy for high-grade B-cell lymphoma. She received Tixagevimab−Cilgavimab chemotherapy and radiation therapy but never achieved remission. She was transferred to our hospital for CAR-T therapy, but developed COVID-19. Her symptoms were mild and she was treated with long-term molnupiravir. On day 28 post-infection, lymphodepleting chemotherapy was restarted after a negative polymerase chain reaction (PCR) test was confirmed. The patient did not experience recurrence of COVID-19 symptoms or severe cytokine release syndrome. Based on the analysis and comparison of the previous reports with this case, we believe that CAR-T therapy should be postponed until a negative PCR test is confirmed. In addition, Tixagevimab−Cilgavimab and long term direct-acting antiviral agent treatment can be effective prophylaxis for severe COVID-19 and shortening the duration of infection.

PANUVEITIS AS A MANIFESTATION OF OCULAR SYPHILIS IN PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUS : A CASE SERIES

Introduction : Syphilis is a sexually transmitted disease caused by the spirochete bacterium Treponema pallidum. Syphilis can result in multiple organ involvement, including the eyes, and demonstrates various clinical findings. Ocular syphilis is an important cause of uveitis in the new era of co-infection with the human immunodeficiency virus (HIV).
 Case Illustration : Case 1: A 29-year-old male HIV-positive with 5 months of blurred vision, pain, and redness in left eye (LE) with skin rashes on the palms and foots (Figure 1). The visual acuity was 1/300 in LE, anterior segment showed flare cell (Figure 2). Fundus examination (Figure 3) revealed multiple exudative lesion of right eye (RE) and vitreus opacity of LE due to inflammation. VDRL and TPHA tests were positive. Case 2: 38-year-old male, HIV-positive and tuberculosis-positive, with 2 months of blurred vision,pain and redness in both eyes with skin rashes on the palms and foots (Figure 1). The visual acuity of the RE was 1/60 and 1,5/60 in the LE on the initial visit. Anterior segment eye examination revealed flare and cell (Figure 2). Fundus examination revealed (Figure 3) preretinal hemorrhage and exudate of both eyes. VDRL and TPHA testing were positive, CD4 count showed severe immunodeficiency.
 Discussion : This study depicts clinical features and outcomes of two cases of ocular syphilis with HIV co- infection. Poor visual outcome was associated with posterior segment-involving uveitis at presentation.
 Conclusion : An accurate diagnosis is crucial, treatment is adjusted according to the clinical stage. Comprehensive education is needed for patients and their families.

The City Clinic Cohort Study: Hepatitis B, HTLV-III/LAV, and CDC AIDS Project 24.

Anticipating the availability of a safe vaccine, scientists at the Center for Disease Control (CDC) planned for a multicenter study of the prevalence, incidence, and efficacy of an experimental vaccine for hepatitis B in 1977, conducted the study among homosexual male volunteers in five collaborating sexually transmitted infection (STI) clinics in the United States from April 1978 through 1980, and concluded that the candidate vaccine was highly efficacious in preventing infections with the hepatitis B virus. Then something completely unexpected and portentous happened. Some successfully vaccinated as well as other homosexual and bisexual men began to show signs and symptoms of a rare cancer, Kaposi's sarcoma, and opportunistic infections typically associated with severe immunodeficiency. As early as October 1983, members of the Hepatitis B study cohort in San Francisco were invited to return to the city STI clinic for further examinations, testing, and confidential interviews about their sexual and other practices. CDC AIDS Project 24 was designed to help describe the natural history of AIDS, define risk factors, and predict future trends. It produced some of the earliest and most convincing scientific evidence about the seriousness and extent of the AIDS epidemic among homosexual and bisexual men in the United States. How the City Clinic Cohort Study came about and evolved is the focus of this commentary.

Stem Cell Transplant in Immune-deficiency-associated Vaccine-derived Poliovirus.

Patients with severe primary immunodeficiency are at risk for complications from live-attenuated vaccines. Here, we report a case of a vaccine-associated paralytic polio and Bacille Calmette-Guérin disease in a 6-month-old girl with severe combined immunodeficiency resulting from homozygous recombinant activating gene 1 deficiency. The patient was successfully treated with intravenous immunoglobulins and oral pocapavir for poliovirus, and antimycobacterial therapy for regional Bacille Calmette-Guérin disease, allowing stem cell transplant. Following transplantation, poliovirus type 3 with 13 mutations was detected from cerebrospinal fluid but not from stool, indicating ongoing viral evolution in the central nervous system despite pocapavir treatment. Clinical improvement and immune reconstitution allowed the patient to be successfully discharged with no further detection of poliovirus.

Humanization with CD34-positive hematopoietic stem cells in NOG-EXL mice results in improved long-term survival and less severe myeloid cell hyperactivation phenotype relative to NSG-SGM3 mice.

NSG-SGM3 and NOG-EXL mice combine severe immunodeficiency with transgenic expression of human myeloid stimulatory cytokines, resulting in marked expansion of myeloid populations upon humanization with CD34+ hematopoietic stem cells (HSCs). Humanized NSG-SGM3 mice typically develop a lethal macrophage activation syndrome and mast cell hyperplasia that limit their use in long-term studies (e.g., humanization followed by tumor xenotransplantation). It is currently unclear to what extent humanized NOG-EXL mice suffer from the same condition observed in humanized NSG-SGM3 mice. We compared the effects of human CD34+ HSC engraftment in these two strains in an orthotopic patient-derived glioblastoma model. NSG-SGM3 mice humanized in-house were compared to NOG-EXL mice humanized in-house and commercially available humanized NOG-EXL mice. Mice were euthanized at humane or study endpoints, and complete pathological assessments were performed. A semiquantitative multiparametric clinicopathological scoring system was developed to characterize chimeric myeloid cell hyperactivation (MCH) syndrome. NSG-SGM3 mice were euthanized at 16 weeks after humanization because of severe deterioration of clinical conditions. Humanized NOG-EXL mice survived to the study endpoint at 22 weeks after humanization and showed less-severe MCH phenotypes than NSG-SGM3 mice. Major differences included the lack of mast cell expansion and limited tissue/organ involvement in NOG-EXL mice compared to NSG-SGM3 mice. Engraftment of human lymphocytes, assessed by immunohistochemistry, was similar in the two strains. The longer survival and decreased MCH phenotype severity in NOG-EXL mice enabled their use in a tumor xenotransplantation study. The NOG-EXL model is better suited than the NSG-SGM3 model for immuno-oncology studies requiring long-term survival after humanization.

Combined treatment with allogeneic Epstein–Barr- and human polyomavirus 1 specific T-cells in progressive multifocal leukoencephalopathy and EBV infection: a case report

Opportunistic viral infections in individuals with severe immunodeficiency can lead to fatal conditions such as progressive multifocal leukoencephalopathy (PML), for which treatment options are limited. These infections pose significant risks, especially when co-infections with other viruses occur. We describe a combined therapy approach using directly isolated allogeneic Human Polyomavirus 1 (also known as BKV) and Epstein–Barr virus (EBV) specific cytotoxic T-cells for the treatment of PML in conjunction with identified EBV in the cerebrospinal fluid (CSF) of a male patient infected with human immunodeficiency virus (HIV). A 53-year-old HIV-positive male, recently diagnosed with PML, presented with rapidly worsening symptoms, including ataxia, tetraparesis, dysarthria, and dysphagia, leading to respiratory failure. The patient developed PML even after commencing highly active antiretroviral therapy (HAART) 3 months prior. Brain magnetic resonance imaging (MRI) revealed multifocal demyelination lesions involving the posterior fossa and right thalamus suggestive of PML. In addition to the detection of human polyomavirus 2 (also known as JCV), analysis of CSF showed positive results for EBV deoxyribonucleic acid (DNA). His neurological condition markedly deteriorated over the following 2 months. Based on MRI, there was no evidence of Immune Reconstitution Inflammatory Syndrome contributing to this decline. The patient did not have endogenous virus-specific T-cells. We initiated an allogeneic, partially human leukocyte antigen-matched transfer of EBV and utilizing the cross-reactivity between BKV and JCV–BKV specific T-cells. This intervention led to notable neurological improvement and partial resolution of the MRI lesions within 6 weeks. Our case of a patient with acquired immune deficiency syndrome demonstrates that PML and concurrent EBV co-infection can still occur despite undergoing HAART treatment. This innovative experimental therapy, involving a combination of virus-specific T-cells, was demonstrated to be an effective treatment option in this patient.

Особенности поражения легких при коинфекции ВИЧ/SARS-CoV-2 у пациентов с разной степенью иммунодефицита

Introduction.The development of the COVID-19 pandemic along with the ongoing pandemic of HIV infection has raised the question of how these two diseases interact. This study examines morphological changes that occur in lung tissue during HIV/SARS-CoV-2 coinfection in patients with varying levels of immune status. Materials and methods. The study included 6 deceased HIV-infected patients who had a clinical diagnosis of COVID-19, which was confirmed postmortem by the detection of SARS-CoV-2 RNA in lung samples obtained during biopsy using polymerase chain reaction. Three patients were with newly diagnosed HIV infection; three patients had a relatively intact immune status (CD4+ more than 200 cells/μl) and no opportunistic infection and received effective antiretroviral therapy. To perform histologic examination, we used histochemical stains by Van Gieson, Grocott, Ziehl-Neelsen, PAS reaction as well as immunohistochemistry (IHC) with antibodies against SARS-CoV-2 nucleocapsid and cytomegalovirus. Results. The macroscopic picture in all cases was characterized by a decreased airiness and compaction of the lung tissue. Microscopically, patients with newly diagnosed HIV demonstrated diffuse alveolar damage (DAD) with pneumocystis pneumonia and cytomegalovirus infection. Nucleocapsid of SARS-CoV-2 was revealed in hyaline membranes and alveolar cells. In patients with controlled HIV, we detected manifestations of various phases of DAD and/or disseminated intravascular coagulation (DIC) syndrome with a predominant distribution of SARS-CoV-2 in macrophages and blood vessels. Conclusion. Lung damage in severe HIV-associated immunodeficiency was determined by the combination of direct cytopathic effect of coronavirus and opportunistic pathogens. In controlled HIV and relatively intact immune status, immune mediated action of SARS-CoV-2 could take place manifesting in diffuse alveolar damage and making the morphological picture comparable to that in HIV-negative patients.

Hem1 inborn errors of immunity: waving goodbye to coordinated immunity in mice and humans.

Inborn errors of immunity (IEI) are a group of diseases in humans that typically present as increased susceptibility to infections, autoimmunity, hyperinflammation, allergy, and in some cases malignancy. Among newly identified genes linked to IEIs include 3 independent reports of 9 individuals from 7 independent kindreds with severe primary immunodeficiency disease (PID) and autoimmunity due to loss-of-function mutations in the NCKAP1L gene encoding Hematopoietic protein 1 (HEM1). HEM1 is a hematopoietic cell specific component of the WASp family verprolin homologous (WAVE) regulatory complex (WRC), which acts downstream of multiple immune receptors to stimulate actin nucleation and polymerization of filamentous actin (F-actin). The polymerization and branching of F-actin is critical for creating force-generating cytoskeletal structures which drive most active cellular processes including migration, adhesion, immune synapse formation, and phagocytosis. Branched actin networks at the cell cortex have also been implicated in acting as a barrier to regulate inappropriate vesicle (e.g. cytokine) secretion and spontaneous antigen receptor crosslinking. Given the importance of the actin cytoskeleton in most or all hematopoietic cells, it is not surprising that HEM1 deficient children present with a complex clinical picture that involves overlapping features of immunodeficiency and autoimmunity. In this review, we will provide an overview of what is known about the molecular and cellular functions of HEM1 and the WRC in immune and other cells. We will describe the common clinicopathological features and immunophenotypes of HEM1 deficiency in humans and provide detailed comparative descriptions of what has been learned about Hem1 disruption using constitutive and immune cell-specific mouse knockout models. Finally, we discuss future perspectives and important areas for investigation regarding HEM1 and the WRC.

Comparative Study of Immunodeficient Rat Strains in Engraftment of Human-Induced Pluripotent Stem Cell-Derived Airway Epithelia.

The airway epithelia (AE) play a role in the clearance of foreign substances through ciliary motility and mucus secreted. We developed an artificial trachea that is made of collagen sponges and polypropylene mesh for the regeneration of the tracheal defect, and it was used for a clinical study. Then, a model in which the luminal surface of an artificial trachea was covered with a human-induced pluripotent stem cell-derived AE (hiPSC-AE) was transplanted into the tracheal defect of nude rats to promote epithelialization. In the future, this model was expected to be applied to research on infectious diseases and drug discovery as a trachea-humanized rat model. However, at present, sufficient engraftment has not been achieved to evaluate functional recovery in transplanted cells. Therefore, this study focused on immunosuppression in recipient rats. Nude rats lack T cell function and are widely used for transplantation experiments; however, more severe immunosuppressed recipients are preferred for xenotransplantation. Several strains of immunodeficient rats were created as rats that exhibit more severe immunodeficiency until now. In this study, to establish a trachea-humanized rat model in which human AE function can be analyzed to improve engraftment efficiency, engraftment efficiency in nude rats and X-linked severe combined immunodeficiency (X-SCID) rats following hiPSC-AE transplantation was compared. In the analysis of the proportion of engrafted cells in total cells at the graft site, the engraftment efficiency of epithelial cells tended to be high in X-SCID rats, although no statistical difference was found between the two groups, whereas the engraftment efficiency of mesenchymal cells was higher in X-SCID rats. Furthermore, the number of immune cells that accumulated in the grafts showed that a pan T cell marker, that is, CD3-positive cells, did not differ between the two strains; however, CD45-positive cells and major histocompatibility complex (MHC) class II-positive cells significantly decreased in X-SCID rats. These results indicate that X-SCID rats are more useful for the transplantation of hiPSC-AE into the tracheae to generate trachea-humanized rat models.

Genetic Characteristics of Patients with Young-Onset Myelodysplastic Neoplasms.

Myelodysplastic neoplasm (MDS) is a heterogeneous group of myeloid neoplasms affected by germline and somatic genetic alterations. The incidence of MDS increases with age but rarely occurs at a young age. We investigated the germline and somatic genetic alterations of Korean patients with young-onset MDS (<40 years). Among the thirty-one patients, five (16.1%) had causative germline variants predisposing them to myeloid neoplasms (three with GATA2 variants and one each with PGM3 and ETV variants). We found that PGM3 deficiency, a subtype of severe immunodeficiency, predisposes patients to MDS. Somatic mutations were identified in 14 patients (45.2%), with lower rates in patients aged < 20 years (11.1%). Nine (29%) patients had U2AF1 S34F/Y mutations, and patients with U2AF1 mutations showed significantly worse progression-free survival (p < 0.001) and overall survival (p = 0.006) than those without U2AF1 mutations. A UBA1 M41T mutation that causes VEXAS syndrome was identified in a male patient. In conclusion, a germline predisposition to myeloid neoplasms occurred in ~16% of young-onset MDS patients and was largely associated with primary immunodeficiencies, including GATA2 deficiency. Furthermore, the high frequency of somatic U2AF1 mutations in patients with young-onset MDS suggests the presence of a distinct MDS subtype.

Systematic analysis of variants escaping nonsense-mediated decay uncovers candidate Mendelian diseases

Protein-truncating variants (PTVs) near the 3' end of genes may escape nonsense-mediated decay (NMD). PTVs in the NMD-escape region (PTVescs) can cause Mendelian disease but are difficult to interpret given their varying impact on protein function. Previously, PTVesc burden was assessed in an epilepsy cohort, but no large-scale analysis has systematically evaluated these variants in rare disease. We performed a retrospective analysis of 29,031 neurodevelopmental disorder (NDD) parent-offspring trios referred for clinical exome sequencing to identify PTVesc de novo mutations (DNMs). We identified 1,376 PTVesc DNMs and 133 genes that were significantly enriched (binomial p<0.001). The PTVesc-enriched genes included those with PTVescs previously described to cause dominant Mendelian disease (e.g., SEMA6B, PPM1D, and DAGLA). We annotated ClinVar variants for PTVescs and identified 948 genes with at least one high-confidence pathogenic variant. Twenty-two known Mendelian PTVesc-enriched genes had no prior evidence of PTVesc-associated disease. We found 22 additional PTVesc-enriched genes that are not well established to be associated with Mendelian disease, several of which showed phenotypic similarity between individuals harboring PTVesc variants in the same gene. Four individuals with PTVesc mutations in RAB1A had similar phenotypes including NDD and spasticity. PTVesc mutations in IRF2BP1 were found in two individuals who each had severe immunodeficiency manifesting in NDD. Three individuals with PTVesc mutations in LDB1 all had NDD and multiple congenital anomalies. Using a large-scale, systematic analysis of DNMs, we extend the mutation spectrum for known Mendelian disease-associated genes and identify potentially novel disease-associated genes.

Impact of COVID-19 on immunocompromised populations during the Omicron era: insights from the observational population-based INFORM study.

Immunocompromised individuals are not optimally protected by COVID-19 vaccines and potentially require additional preventive interventions to mitigate the risk of severe COVID-19. We aimed to characterise and describe the risk of severe COVID-19 across immunocompromised groups as the pandemic began to transition to an endemic phase. COVID-19-related hospitalisations, intensive care unit (ICU) admissions, and deaths (01/01/2022-31/12/2022) were compared among different groups of immunocompromised individuals vs the general population, using a retrospective cohort design and electronic health data from a random 25% sample of the English population aged ≥12 years (Registration number: ISRCTN53375662). Overall, immunocompromised individuals accounted for 3.9% of the study population, but 22% (4585/20,910) of COVID-19 hospitalisations, 28% (125/440) of COVID-19 ICU admissions, and 24% (1145/4810) of COVID-19 deaths in 2022. Restricting to those vaccinated with ≥3 doses of COVID-19 vaccine (∼84% of immunocompromised and 51% of the general population), all immunocompromised groups remained at increased risk of severe COVID-19 outcomes, with adjusted incidence rate ratios (aIRR) for hospitalisation ranging from 1.3 to 13.1. At highest risk for COVID-19 hospitalisation were individuals with: solid organ transplant (aIRR 13.1, 95% confidence interval [95% CI] 11.2-15.3), moderate to severe primary immunodeficiency (aIRR 9.7, 95% CI 6.3-14.9), stem cell transplant (aIRR 11.0, 95% CI 6.8-17.6), and recent treatment for haematological malignancy (aIRR 10.6, 95% CI 9.5-11.9). Results were similar for COVID-19 ICU admissions and deaths. Immunocompromised individuals continue to be impacted disproportionately by COVID-19 and have an urgent need for additional preventive measures beyond current vaccination programmes. These data can help determine the immunocompromised groups for which targeted prevention strategies may have the highest impact. This study was funded by AstraZeneca UK.

Targeting Aberrantly Activated Glycolysis in Natural Killer T Cell Lymphomas

Background and aims: With advances in the diagnosis and treatment of malignant lymphoma, improvements in long-term survival have been achieved for most common lymphoma types. However, natural killer/T-cell lymphoma (NKTL), which is rare in Europe and North America (0.3%) but relatively common in Asia and Latin America, has been left behind as a subtype of lymphoma with a high relapse rate and a poor prognosis. In particular, the outcome of patients with advanced or recurrent NKTL is dismal, with an overall survival of only a few months. This is mainly attributed to the intrinsic resistance of NKTL cells to anthracycline due to the high expression of P-glycoprotein. Molecular-targeted drugs used for other hematological malignancies, such as anti-CD30 and anti-CD52 monoclonal antibodies, have only limited effects on NKTL, and conversely, these drugs can cause severe immunodeficiency and severe autoimmune diseases, often with a fatal course. The development of NKTL-specific and effective molecular-target therapies is expected to improve the prognosis of NKTL. Methods: We performed a gene expression array targeting genes involved in glucose cell metabolism to compare the gene signatures of NK cell lines (NKL, NK92, YT, and KYHG1) and freshly isolated NK cells from three healthy donors (FNK). Results: The expression of various glucose metabolism genes was higher in NK cell lines than in FNK cells, and in particular, the PKM gene was highly enriched in NK cell lines. The PKM gene encodes the pyruvate kinase muscle isozyme 2 (PKM2), which is responsible for ATP production within the glycolytic sequence and plays an essential role in aerobic glycolysis (the Warburg effect), a dominant metabolic pathway used by cancer cells. Immunohistochemistry studies revealed high PKM2 protein expression in 17 out of 20 tumor tissues derived from patients with extranodal NK/T cell lymphoma. Treatment of NKL, NK92, YT, and KYHG1 cells with Benserazide or Shikonin, two known PKM2 inhibitors, robustly induced cell apoptosis in a time- and dose-dependent manner and inhibited colony formation in in vitro clonogenic assays. Western blotting studies showed a dose-dependent inhibition of STAT1 and STAT3 in NK cell lines exposed to PKM2 inhibitors. A colorimetric assay showed that PKM2 glycolytic activity correlated with the PKM2 gene expression in NK cells, with significantly higher glycolytic activity in malignant NK cell lines than in normal FNK cells. Further mechanistic studies showed that in normal FNK cells, PKM2 is mainly in the tetrameric form, where glucose is converted to pyruvate to generate ATP. In contrast, in malignant NK cell lines, PKM2 is mainly expressed in its dimeric form, with a low affinity for pyruvate and a higher affinity for glycolytic intermediates that promote the synthetic processes and thereby lead to persistent cell proliferation. Conclusions: These results substantiate the importance of aerobic glycolysis in malignat NK cells and indicate that targeting this pathway by blocking PKM2 activity has therapeutic potential for NKTL.

Platelet Factor 4 Binds to Low-Density Lipoprotein Receptor to Block Leukemic Stem Cell Expansion and Recurrence

Healthy hematopoietic stem cells (HSCs) reside in highly specialized microenvironments within the bone marrow, commonly referred to as niches. HSC activity is tightly regulated by various cellular and molecular niche constituents that ensure healthy hematopoiesis. However, through the accumulation of mutations, hematopoietic stem and progenitor cells can give rise to leukemia-initiating cells or leukemic stem cells (LSCs), which play a vital role in the onset, progression, and treatment outcome of acute myelogenous leukemia (AML). In AML, the expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity. Current therapeutic options mainly eliminate the bulk of leukemic cells but do not efficiently target LSCs, leading to the high rates of relapse seen in AML patients. Recent studies suggest that LSCs hijack and alter the healthy bone marrow niche to better support their maintenance and proliferation. Understanding how LSCs interact with the bone marrow microenvironment is crucial for eradicating LSCs and preventing AML relapse. Our previous work showed that platelet factor 4 (PF4), a chemokine stored in platelet alpha-granules and secreted by bone marrow megakaryocytes, can induce quiescence in healthy myeloid-biased HSCs ( Nature Medicine 2014; Developmental Cell 2018). Here, we investigate the regulation of malignant LSCs by PF4 using mouse and human AML models. In an aggressive mouse AML model, MLL-AF9, our results show that recombinant PF4 blocks the proliferation of phenotypic LSCs (Lineage − IL7Rα − Sca1 − MLL-AF9-GFP + c-Kit high CD34 + FcγRII/III high) in vitro (70% reduction; P=0.0279). Strikingly, cell cycle analyses revealed that in vivo, both LSCs and bulk AML proliferation are significantly inhibited 24 hours post-PF4 (80 mg/kg) injection, resulting in a significantly lower leukemic burden. By contrast, Pf4 -/- mice exhibited rapid AML disease progression via increased LSC proliferation. Furthermore, using a genetic mouse model ( Pf4-Cre; iDTR) where PF4-secreting megakaryocyte cells can be depleted in vivo in leukemic mice, our results revealed that megakaryocyte depletion led to poor AML survival due to accelerated disease progression (Log-rank P&amp;lt;0.0017). These results demonstrate that megakaryocyte-derived PF4 can directly regulate the proliferation of leukemia cells. Interestingly, our results indicate that AML drives striking alterations in bone marrow megakaryocytes, including a lower frequency, reduced cell maturation, and reduced secretion of PF4 (~80% reduction, P&amp;lt;0.0001) compared to healthy controls. Accordingly, PF4 levels are reduced in the serum of myelodysplastic syndrome patients where hyperproliferative malignant cells are found alongside dysplastic megakaryocytes. To assess whether recombinant PF4 can alter the course of AML relapse in a preclinical experimental setting, we treated moribund leukemic mice with the chemotherapeutic drug Cytarabine followed by 3 daily I.V. injections of PF4 (80 mg/kg) or saline. Strikingly, this short treatment with PF4 preferentially reduced the frequency of LSCs in vivo during the relapse phase of AML (75% reduction; P=0.05). Furthermore, mice transplanted with MLL-AF9 AML cells that were pre-treated with Cytarabine and PF4 in vitro had prolonged overall survival (Log-rank P&amp;lt;0.0132) compared to Cytarabine alone. Importantly, two of six mice had no detectable AML up to 150 days post-transplantation. Finally, single-cell RNA sequencing experiments revealed that PF4 signals through Low-Density Lipoprotein Receptor (LDLR) on LSCs, whose expression is significantly upregulated upon Cytarabine treatment, during the relapse phase. Functional studies with fluorescently labeled low-density lipoprotein (LDL) confirmed that PF4 limits LSC uptake of LDL via LDLR to block LSC proliferation ( P=0.0044), suggesting an important role for PF4 in AML metabolism. Altogether, our studies reveal an unknown function for PF4 in AML and highlight PF4's potential as an effective adjuvant therapy in preventing AML relapse by inhibiting the recurrence of chemotherapeutic-resistant LSCs.

Heterogeneous Genetic Landscape of Chronic Idiopathic Neutropenia Revealed By Whole Exome Sequencing

Background: Adultpatients with persistent, unexplained neutropenia who do not fulfill the diagnostic criteria of any underlying disease are characterized as chronic idiopathic neutropenia (CIN) cases.It is an exclusion diagnosis that can be established after a thorough clinical/laboratory investigation for any underlying causes, including negative anti-neutrophil antibody testing, inconclusive bone marrow (BM) aspiration/biopsy and normal cytogenetics. CIN patients regardless of their absolute neutrophil counts (ANC) usually display a benign and uncomplicated clinical course. Although the pathogenesis of CIN remains largely unknown, we hypothesize that a constitutional/congenital background might exist, at least in some cases, that have potentially escaped diagnosis during childhood. Aim of the study: To investigate the genetic landscape of neutropenia in CIN by conducting whole exome sequencing (WES) analysis in a number of adult CIN patients. Methods: We have performed WES in 16 adult (median age 59 years, range 30-72 years) patients (11 females and 5 males) with CIN according to previously published criteria (Fioredda F, et al Hemasphere. 2023). The patients had median ANC 1.2 x 10 9/L (range 0.4-1.6 x 10 9/L) for prolonged period and unknown neutropenia onset in most cases. Genomic DNA was extracted from peripheral blood samples, coding fragment libraries were prepared, sequencing performed on Illumina NovaSeq 6000 System, and data subjected to an in-house pipeline for bioinformatic analysis for variant calling and annotations. Singleton analyses were performed focusing on genes associated with congenital defects of phagocytes, BM failure (BMF) and immune deficiency and dysregulation. Mean depth of coverage was 70x and the mean number of variants/sample was 22553. Results: WES analysis identified 10 pathogenic/likely pathogenic (P/LP) variants in 10 patients. The variants consisted of 4 frameshift mutations, 3 nonsense mutations, 1 missense mutation, 1 splice-site mutation and 1 splice-region mutation. Pathogenic variants in congenital neutropenia (CN)-related genes included only a heterozygous mutation in G6PC3:c.511C&amp;gt;T,p.Gln171Ter (previously detected in one of our CN patients; Nikolouzakis TK et al, Ann Hematol. 2022). Pathogenic variants in genes associated with BMF were detected in two genes, namely FANCM:c.1492C&amp;gt;T, p.Gln498Ter and CTC1:c.3058C&amp;gt;T,p.Gln1020Ter, respectively. The majority of the pathogenic variants were found in genes associated with immune deficiency and dysregulation. These involved: (i) a heterozygous mutation in DCLRE1C:c.1784dup,p.Ile596fs (associated with immunodeficiencies affecting cellular and humoral immunity); (ii) a LP mutation in ORAI1:c.132_133del,p. Arg45fs and a pathogenic variant in SPINK5:c.1302+4A&amp;gt;T (related to combined immunodeficiencies with associated or syndromic features); (iii) a pathogenic variant in MEFV:c.2080A&amp;gt;G, p.Met694Val and a LP variant in PSMG2:c.703-2A&amp;gt;T (both genes associated with autoinflammatory disorders); (iv) a LP variant in IRF7:c.1089dup,p.Pro364Alafs (associated with defects in intrinsic and innate immunity); and (v) a LP variant in PEPD:c.738_739insGT,p.Ser247fs (associated with immune dysregulation). Regarding variants of undetermined significance (VUS) an average of 4.5 VUS variants per patient were detected in genes related to CN ( HAX1, VPS13B, LYST), BMF ( GATA2, SAMD9, CXCR4) or immunodeficiency ( TCF3). However, none of these patients display clinical symptoms typical of these conditions. Specifically, the majority of the patients presented with mild/moderate neutropenia and rarely with severe neutropenia, they have relatively normal BM biopsies and do not display major symptoms or signs related to severe immunodeficiency or immune dysregulation syndromes. Conclusions: This study is the first attempt of WES analysis in adult CIN patients and shows that there is a heterogeneous genetic landscape underlying the disease. More studies are required so that the detected P/LP variants and VUS can be associated with the clinical phenotypes to more accurately interpret their potential contribution to the neutropenia phenotype.

Prevention of CMV/EBV Reactivation By Double-Specific T Cells in Patients after Allogeneic Stem Cell Transplantation: Results from the Randomized Phase I/IIa Multivir-01 Study

Allogeneic stem cell transplantation (aSCT) is used to cure hematologic malignancies or deficiencies of the hematopoietic system. It is associated with severe immunodeficiency of the host early after transplant and therefore early reactivation of latent herpesviruses such as CMV and EBV within the first 100 days are frequent. Small studies and case series indicated that application of herpes virus specific T cells can control and prevent disease in this patient population. Study Design: A randomized, controlled, phase I/IIa multi-center study was conducted in 5 centers in Germany between 2013 and 2018 (ClinicalTrials.gov Identifier: NCT02227641). The study aimed at prevention or pre-emptive treatment of reactivation of CMV and EBV post aSCT. Major inclusion criteria were male or female HLA matched (10/10) unrelated or related donor seropositive for CMV and EBV, patient age between 18 and 75, the use of PBSC as stem cell source and the presence of on of the following HLA alleles: A*01:01, A*02:01, B07:02, B*08:01, B*35:01, C*07:02. CMV/EBV specific T cells were generated from a fraction of the allogeneic stem cell graft by stimulation with peptide pools for both viruses developed at the University Hospital Erlangen. Three doses of 5x10E4 CD3+ T cells per kilogram body weight were applied with an interval of at least 30 days without dose escalation starting earliest at day 30 after aSCT. Observation period was 204 days after aSCT. Primary endpoint of the study was toxicity of the T-cell-product assessed as acute transfusion reaction and acute GvHD due to the infusion of activated T cells. Results: 33 patients were screened and 29 patients were randomized. 16 patients were randomized into the treatment group of who 3 patients crossed over to the control arm due to failure of manufacturing the product or due to not meeting release criteria. Four patients were excluded from the study before receiving the first dose due to transplant related toxicity or withdrawal of consent. In total 9 patients were treated with 25 doses of the T cell product (dose range 2.1-4.1x10E4/kg CD3+ T cells). The earliest infusion was on day 30 (range: 30-69) post aSCT. There was no statistical difference between the groups regarding disease, patient age, donor type, performance status and comorbidities. The median follow up for both groups was 930 days. None of the patients treated experienced any acute transfusion reaction. Two patients developed acute GvHD after the second infusion and did not receive the third dose. Overall there was no statistical significant difference regarding survival (A) (treatment: 55.9% vs. control 69.2%, p=0.44), transplant related mortality (B) (22.2% vs. 7.1%, p=0.28), relapse (C) (11.1% vs. 14.3%) and acute GvHD (D,E) (33.3% vs. 46.2%, p=0.44). Number of AE per patient (27.1±24.6 vs. 24.4±11.2, p=0.60) and severity of AE (1.60±0.39 vs. 1.64±0.24) were similar in both groups. The incidence of reactivation of CMV (44.4% vs. 64.3%, p=0.16) and EBV (77.8% vs. 44.4%, p=0.12) was not statistically different and not associated with the treatment as several reactivations of occurred before application of the first dose. Within the treatment group we observed in several cases substantial expansion of CMV and EBV specific T cells in the peripheral blood which could not attributed to the presence viral antigens in case of reactivation. Conclusion: The study met its primary endpoint as there was no toxicity of the T-cell product observed. The cumulative incidence of acute GvHD as the primary endpoint was not statistically different in both groups and there was no acute toxicity related to the product. The study suffered from slow recruitment due to the market approval of Letermovir and low patient numbers did not allow for conclusive analysis of secondary endpoints such as the use of antiviral therapy.

Characteristics of Tuberculous Meningitis in HIV-Positive Patients from Northeast Romania.

One of the most severe forms of extrapulmonary tuberculosis (EPTB) is tuberculous meningitis (TBM), which is linked to significant morbidity and high mortality. It is well recognized that human immunodeficiency virus (HIV)-positive people are more likely to develop EPTB, including TBM, especially if they have severe immunodeficiencies. We aim to highlight the profile and the characteristics of TBM in HIV-infected patients. We conducted a retrospective clinical study based on hospital medical records of patients diagnosed with HIV/AIDS (acquired immunodeficiency syndrome) and TBM in Northeast Romania, hospitalized at "St. Parascheva" Clinical Hospital of Infectious Diseases of Iasi from 1 January 2010 to 1 December 2022. From a total number of 1692 patients on record in our center, 195 had a HIV-tuberculosis (TB) coinfection, and 19 cases were HIV-TBM coinfected. Six cases were newly HIV-diagnosed late presenters, and thirteen patients' names were already found in the center's records with deficient immunological viral status (median CD4 lymphocyte level 47/mm3). The average age in the study group was 27 years old. The clinical manifestations and cerebrospinal fluid (CSF) variables were typical in most cases, despite the severe immunodepression of the patients. The Thwaites scoring system correctly identified 89.5% of the patients. The median admission period was 18 days; the lethality rate was 31.6%, despite access to ART and anti-TB drugs, and was associated with a more severe immunosuppression. No rifampicin resistance was detected. TBM appeared in a minority of our HIV cohort and affected severely immunodepressed patients; the clinical and CSF variables had a typical aspect in most cases, and the Thwaites scoring system performed well for this type of patient. The lethality rate was high and was correlated with a more severe immunodepression.