Bendamustine impairs humoral but not cellular immunity to SARS-CoV-2 vaccination in rituximab-treated B-cell lymphoma-affected patients.

Background: Patients with B-cell lymphoma are a fragile category of subjects, particularly exposed to infections and characterized by an impaired vaccination response due to the disease itself and, even more, to the chemotherapy regimen. For this reason, extensive knowledge of the immune response status of these subjects is of fundamental importance to obtain possible indications for a tailored immunization strategy. Methods: We enrolled two cohorts of patients with B-cell lymphoma under rituximab treatment or 3-24 months after treatment. In all patients, we evaluated both humoral and cellular immunological memory toward SARS-CoV-2, after standard vaccination and upon one booster dose. Results: We observed no Spike-specific IgG production in patients (n = 25) under anti-CD20 treatment, whereas patients (n = 16) vaccinated after the completion of chemotherapy showed a higher humoral response. Evaluating SARS-CoV-2-specific T-cell response, we found that patients in both cohorts had developed robust cellular immunity after vaccination. Of the 21 patients (51%) that experienced a breakthrough SARS-CoV-2 infection, only six patients developed severe disease. Interestingly, these six patients had all been treated with rituximab plus bendamustine. Notably, we observed that Spike-specific IgG levels in patients treated with rituximab plus bendamustine were absent or lower compared with those in patients treated with rituximab plus other chemotherapy, whereas Spike-specific T-cell response was not different based on chemotherapy regiment. Discussion: Our results show that, in patients with B-cell lymphoma under rituximab therapy, anti-SARS-CoV-2 mRNA vaccination induces a weak or absent humoral response but a consistent T-cell response. In addition, chemotherapy regimens with bendamustine further reduce patients' ability to mount a Spike-specific humoral response even after a long time period from chemotherapy discontinuation. These results provide evidence that different chemotherapeutics display different immunosuppressive properties that could be taken in to account in the choice of the right drug regimen for the right patient. Moreover, they question whether immunocompromised patients, particularly those treated with bendamustine, need interventions to improve vaccine-induced immune response.

Clonally expanded PD-1-expressing T cells are enriched in synovial fluid of juvenile idiopathic arthritis patients.

Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic condition in childhood. The disease etiology remains largely unknown; however, a key role in JIA pathogenesis is surely mediated by T cells. T-lymphocytes activity is controlled via signals, known as immune checkpoints. Delivering an inhibitory signal or blocking a stimulatory signal to achieve immune suppression is critical in autoimmune diseases. However, the role of immune checkpoints in chronic inflammation and autoimmunity must still be deciphered. In this study, we investigated at the single-cell level the feature of T cells in JIA chronic inflammation, both at the transcriptome level via single-cell RNA sequencing and at the protein level by flow cytometry. We found that despite the heterogeneity in the composition of synovial CD4+ and CD8+ T cells, those characterized by PD-1 expression were clonally expanded tissue-resident memory (Trm)-like cells and displayed the highest proinflammatory capacity, suggesting their active contribution in sustaining chronic inflammation in situ. Our data support the concept that novel therapeutic strategies targeting PD-1 may be effective in the treatment of JIA. With this approach, it may become possible to target overactive T cells regardless of their cytokine production profile.

Musculin does not modulate the disease course of Experimental Autoimmune Encephalomyelitis and DSS colitis.

Previous evidences show that Musculin (Msc), a repressor member of basic helix-loop-helix transcription factors, is responsible in vitro for the low responsiveness of human Th17 cells to the growth factor IL-2, providing an explanation for Th17 cells rarity in inflammatory tissue. However, how and to what extent Musculin gene can regulate the immune response in vivo in an inflammatory context is still unknown. Here, exploiting two animal models of inflammatory diseases, the Experimental Autoimmune Encephalomyelitis (EAE) and the dextran sodium sulfate (DSS)-induced colitis, we evaluated the effect of Musculin gene knock-out on clinical course, performing also a deep immune phenotypical analysis on T cells compartment and an extended microbiota analysis in colitis-sick mice. We found that, at least during the early phase, Musculin gene has a very marginal role in modulating both the diseases. Indeed, the clinical course and the histological analysis showed no differences between wild type and Msc knock-out mice, whereas immune system appeared to give rise to a regulatory milieu in lymph nodes of EAE mice and in the spleen of DSS colitis-sick mice. Moreover, in the microbiota analysis, we found irrelevant differences between wild type and Musculin knock-out colitis-sick mice, with a similar bacterial strains' frequency and diversity after the DSS treatment. This work strengthened the idea of a negligible Msc gene involvement in these models.

Fourth Dose of mRNA COVID-19 Vaccine Transiently Reactivates Spike-Specific Immunological Memory in People Living with HIV (PLWH).

Background: People Living With HIV (PLWH), with advanced disease, lower CD4+ T cell counts or an unsuppressed HIV viral load can have a suboptimal vaccine response. For this reason, in the current COVID-19 pandemic, they represent a prioritized population for the SARS-CoV-2 fourth (or second booster) vaccine dose. This work aims to investigate the effects of a second booster on the reactivation of the spike-specific humoral and cell-mediated immune responses in PLWH. Methods: A total of eight PLWH, who received a fourth dose of the original mRNA vaccines were enrolled. They were evaluated before and then 7 days, 1 month and 2 months after the injection. The humoral response was assessed via a chemiluminescent immunoassay. Immunophenotyping and the functional evaluation of the SARS-CoV-2-specific cellular immune responses were performed via flow cytometry. Results: Anti-spike IgG levels were above the cut-off value for all subjects at all timepoints. The spike-specific CD4+ T cell response was reactivated one week after the fourth vaccine dose, and on average declined at two months post-vaccination. A similar trend was observed for the spike-specific B cells. A low percentage of spike-specific CD4+ T cells was activated by the B.1.1.529 BA.1 Omicron-spike mutated peptides, and the majority of these cells were reactive to the conserved portions of the spike protein. Similarly, the majority of the spike-specific memory B cells were able to bind both Wuhan and Omicron-spike entire protein. Conclusions: Spike-specific adaptive immune responses are transiently reactivated in PLWH following the fourth mRNA vaccine dose. The breadth of the immune responses to the mutated spike protein provides insight on the possible cross-reactivity for the SARS-CoV-2 variants of concern (VOCs).

Clinical and Immunological Features of SARS-CoV-2 Breakthrough Infections in Vaccinated Individuals Requiring Hospitalization.

BACKGROUND AND PURPOSE: Waning immunity and the surge of SARS-CoV-2 variants are responsible for breakthrough infections, i.e., infections in fully vaccinated individuals. Although the majority of vaccinated infected subjects report mild or no symptoms, some others require hospitalization. The clinical and immunological features of vaccinated hospitalized COVID-19 patients are currently unknown. METHODS: Twenty-nine unvaccinated and 36 vaccinated hospitalized COVID-19 patients were prospectively enrolled and clinical and laboratory data were gathered. Immunophenotyping of leukocytes' subsets, T and B cell SARS-CoV-2-specific responses were evaluated via flow cytometry. Anti-IFN-α autoantibodies were measured via ELISA. RESULTS: Despite vaccinated patients were older and with more comorbidities, unvaccinated subjects showed higher levels of pro-inflammatory markers, more severe disease, and increased mortality rate. Accordingly, they presented significant alterations in the circulating leukocyte composition, typical of severe COVID-19. Vaccinated patients displayed higher levels of anti-Spike IgGs and Spike-specific B cells. Of all participants, survivors showed higher levels of anti-Spike IgGs and Spike-specific CD4+ T cells than non-survivors. At hospital admission, 6 out of 65 patients (9.2%) displayed high serum concentrations of autoantibodies targeting IFN-α. Remarkably, 3 were unvaccinated and eventually died, while the other 3 were vaccinated and survived. CONCLUSION: Despite more severe pre-existing clinical conditions, vaccinated patients have good outcome. A rapid activation of anti-SARS-CoV-2-specific immunity is fundamental for the resolution of the infection. Therefore, prior immunization through vaccination provides a significant contribution to prevention of disease worsening and can even overcome the presence of high-risk factors (i.e., older age, comorbidities, anti-IFN-α autoantibodies).

SARS-CoV-2 Spike-Specific CD4+ T Cell Response Is Conserved Against Variants of Concern, Including Omicron.

Although accumulating data have investigated the effect of SARS-CoV-2 mutations on antibody neutralizing activity, less is known about T cell immunity. In this work, we found that the ancestral (Wuhan strain) Spike protein can efficaciously reactivate CD4+ T cell memory in subjects with previous Alpha variant infection. This finding has practical implications, as in many countries only one vaccine dose is currently administered to individuals with previous COVID-19, independently of which SARS-CoV-2 variant was responsible of the infection. We also found that only a minority of Spike-specific CD4+ T cells targets regions mutated in Alpha, Beta and Delta variants, both after natural infection and vaccination. Finally, we found that the vast majority of Spike-specific CD4+ T cell memory response induced by natural infection or mRNA vaccination is conserved also against Omicron variant. This is of importance, as this newly emerged strain is responsible for a sudden rise in COVID-19 cases worldwide due to its increased transmissibility and ability to evade antibody neutralization. Collectively, these observations suggest that most of the memory CD4+ T cell response is conserved against SARS-CoV-2 variants of concern, providing an efficacious line of defense that can protect from the development of severe forms of COVID-19.

SARS-CoV-2 infection and vaccination trigger long-lived B and CD4+ T lymphocytes with implications for booster strategies.

BACKGROUNDImmunization against SARS-CoV-2, the causative agent of COVID-19, occurs via natural infection or vaccination. However, it is currently unknown how long infection- or vaccination-induced immunological memory will last.METHODSWe performed a longitudinal evaluation of immunological memory to SARS-CoV-2 up to 1 year after infection and following mRNA vaccination in naive individuals and individuals recovered from COVID-19 infection.RESULTSWe found that memory cells are still detectable 8 months after vaccination, while antibody levels decline significantly, especially in naive individuals. We also found that a booster injection is efficacious in reactivating immunological memory to spike protein in naive individuals, whereas it was ineffective in previously SARS-CoV-2-infected individuals. Finally, we observed a similar kinetics of decay of humoral and cellular immunity to SARS-CoV-2 up to 1 year following natural infection in a cohort of unvaccinated individuals.CONCLUSIONShort-term persistence of humoral immunity, together with the reduced neutralization capacity versus the currently prevailing SARS-CoV-2 variants, may account for reinfections and breakthrough infections. Long-lived memory B and CD4+ T cells may protect from severe disease development. In naive individuals, a booster dose restored optimal anti-spike immunity, whereas the needs for vaccinated individuals who have recovered from COVID-19 have yet to be defined.FUNDINGThis study was supported by funds to the Department of Experimental and Clinical Medicine, University of Florence (Project Excellence Departments 2018-2022), the University of Florence (project RICTD2122), the Italian Ministry of Health (COVID-2020-12371849), and the region of Tuscany (TagSARS CoV 2).

IL4I1 Is Expressed by Head-Neck Cancer-Derived Mesenchymal Stromal Cells and Contributes to Suppress T Cell Proliferation.

Amino acids have a primary role in cancer metabolism. Beyond their primary biosynthetic role, they represent also an alternative fuel while their catabolites can influence the epigenetic control of gene expression and suppress anti-tumor immune responses. The accumulation of amino-acid derivatives in the tumor microenvironment depends not only on the activity of tumor cells, but also on stromal cells. In this study, we show that mesenchymal stromal cells derived from head-neck cancer express the amino acid oxidase IL4I1 that has been detected in different types of tumor cells. The catabolic products of IL4I1, H2O2, and kynurenines are known to suppress T cell response. We found that neutralization of IL4I1 activity can restore T cell proliferation. Thus, therapeutical strategies targeting enzymes involved in amino-acid catabolism may be helpful to contemporary block tumor cell migration and restore an efficacious anti-tumor immunity.

Heterogeneous magnitude of immunological memory to SARS-CoV-2 in recovered individuals.

OBJECTIVE: Although the adaptive immune response to SARS-CoV-2 has been characterised in the acute and early convalescent phase of the disease, few studies explore whether natural infection elicits long-lasting immunological memory in recovered individuals. In this work, we aimed to assess the maintenance of immunological memory to SARS-CoV-2. METHODS: We evaluated the long-term virus-specific cellular and humoral immune response in the members of an Italian Serie A football team, who experienced a cluster of COVID-19 in March 2020, which was strictly evaluated in the following months. RESULTS: Our results highlight a heterogeneous magnitude of immunological memory at 5 months after infection. Indeed, 20% of the subjects displayed a weak cellular and humoral memory to SARS-CoV-2, suggesting that they may be at higher risk of reinfection. In addition, a history of symptomatic COVID-19 was associated with higher levels of SARS-CoV-2-reactive CD4+ T cells and specific antibody levels than in asymptomatic individuals. CONCLUSION: Collectively, these data demonstrate that immunity to SARS-CoV-2 is maintained five months postinfection even if the magnitude of response is heterogeneous among individuals. This finding suggests that some COVID-19-recovered subjects may benefit from vaccination.

First-dose mRNA vaccination is sufficient to reactivate immunological memory to SARS-CoV-2 in subjects who have recovered from COVID-19.

The characterization of the adaptive immune response to COVID-19 vaccination in individuals who recovered from SARS-CoV-2 infection may define current and future clinical practice. To determine the effect of the 2-dose BNT162b2 mRNA COVID-19 vaccination schedule in individuals who recovered from COVID-19 (COVID-19-recovered subjects) compared with naive subjects, we evaluated SARS-CoV-2 Spike-specific T and B cell responses, as well as specific IgA, IgG, IgM, and neutralizing antibodies titers in 22 individuals who received the BNT162b2 mRNA COVID-19 vaccine, 11 of whom had a previous history of SARS-CoV-2 infection. Evaluations were performed before vaccination and then weekly until 7 days after second injection. Data obtained clearly showed that one vaccine dose is sufficient to increase both cellular and humoral immune response in COVID-19-recovered subjects without any additional improvement after the second dose. On the contrary, the second dose proved mandatory in naive subjects to further enhance the immune response. These findings were further confirmed at the serological level in a larger cohort of naive (n = 68) and COVID-19-recovered (n = 29) subjects, tested up to 50 days after vaccination. These results question whether a second vaccine injection in COVID-19-recovered subjects is required, and indicate that millions of vaccine doses may be redirected to naive individuals, thus shortening the time to reach herd immunity.

Human and Murine T-Helper Cell Recovery from Organs and Tissues.

Isolation of cells from organs and tissues represents a challenge for many researchers. Cell yield, purity, and cell death are common problems associated with it, which greatly affect experimental results in terms of reproducibility and biological observations. Here, we describe state-of-the-art protocols of how to isolate CD4+ T cells from both human and murine organs and tissues, reducing at minimum cell death, while increasing at the same time cell yield and purity.

Metabolomic/lipidomic profiling of COVID-19 and individual response to tocilizumab.

The current pandemic emergence of novel coronavirus disease (COVID-19) poses a relevant threat to global health. SARS-CoV-2 infection is characterized by a wide range of clinical manifestations, ranging from absence of symptoms to severe forms that need intensive care treatment. Here, plasma-EDTA samples of 30 patients compared with age- and sex-matched controls were analyzed via untargeted nuclear magnetic resonance (NMR)-based metabolomics and lipidomics. With the same approach, the effect of tocilizumab administration was evaluated in a subset of patients. Despite the heterogeneity of the clinical symptoms, COVID-19 patients are characterized by common plasma metabolomic and lipidomic signatures (91.7% and 87.5% accuracy, respectively, when compared to controls). Tocilizumab treatment resulted in at least partial reversion of the metabolic alterations due to SARS-CoV-2 infection. In conclusion, NMR-based metabolomic and lipidomic profiling provides novel insights into the pathophysiological mechanism of human response to SARS-CoV-2 infection and to monitor treatment outcomes.

Cell-mediated and humoral adaptive immune responses to SARS-CoV-2 are lower in asymptomatic than symptomatic COVID-19 patients.

The characterization of cell-mediated and humoral adaptive immune responses to SARS-CoV-2 is fundamental to understand COVID-19 progression and the development of immunological memory to the virus. In this study, we detected T-cells reactive to SARS-CoV-2 proteins M, S, and N, as well as serum virus-specific IgM, IgA, IgG, in nearly all SARS-CoV-2 infected individuals, but not in healthy donors. Virus-reactive T cells exhibited signs of in vivo activation, as suggested by the surface expression of immune-checkpoint molecules PD1 and TIGIT. Of note, we detected antigen-specific adaptive immune response both in asymptomatic and symptomatic SARS-CoV-2 infected subjects. More importantly, symptomatic patients displayed a significantly higher magnitude of both cell-mediated and humoral adaptive immune response to the virus, as compared to asymptomatic individuals. These findings suggest that an uncontrolled adaptive immune response contribute to the development of the life-threatening inflammatory phase of the disease. Finally, this study might open the way to develop effective vaccination strategies.

Quantitative and qualitative alterations of circulating myeloid cells and plasmacytoid DC in SARS-CoV-2 infection.

SARS-CoV-2 is responsible for a new infectious disease (COVID-19) in which individuals can either remain asymptomatic or progress from mild to severe clinical conditions including acute respiratory distress syndrome and multiple organ failure. The immune mechanisms that potentially orchestrate the pathology in SARS-CoV-2 infection are complex and only partially understood. There is still paucity of data on the features of myeloid cells involved in this viral infection. For this reason, we investigated the different activation status profiles and the subset distribution of myeloid cells and their correlation with disease progression in 40 COVID-19 patients at different stages of disease. COVID-19 patients showed a decrease in the absolute number of plasmacytoid and myeloid dendritic cells, different subset distribution of monocytes and different activation patterns of both monocytes and neutrophils, coupled to a significant reduction of HLA-DR monocyte levels. We found that some of these alterations are typical of all COVID-19 patients, while some others vary at different stages of the disease and correlate with biochemical parameters of inflammation. Collectively, these data suggest that not only the lymphoid, but also the myeloid compartment, is severely affected by SARS-CoV-2 infection.

Human T cells interacting with HNSCC-derived mesenchymal stromal cells acquire tissue-resident memory like properties.

Tissue-resident memory (Trm) cells are specialized components of both CD4+ and CD8+ T cell subsets that persist in peripheral nonlymphoid tissues following infections and provide fast response in case of a secondary invasion by the same pathogen. Trm cells express the surface markers CD69, CD103, and the immune checkpoint molecule PD-1. Trm cells develop not only in the context of infections but also in tumors, where they can provide a line of defense as suggested by the positive correlation between the frequency of tumor-infiltrating Trm cells and patients' survival. Trm cells persistence in peripheral tissues depends on their adaptation to the local microenvironment and the presence of survival factors, mainly IL-7, IL-15, and Notch ligands. However, the cell sources of these factors are largely unknown, especially in the context of tumors. Here, we show that head-neck squamous cell carcinoma (HNSCC) is enriched in CD4+ and CD8+ T cells with a Trm phenotype. Moreover, we show that mesenchymal stromal cells that accumulate in HNSCC are a source of survival factors and allow proper expression of Trm-typical markers in a VCAM1-dependent manner.

Impaired immune cell cytotoxicity in severe COVID-19 is IL-6 dependent.

BACKGROUNDCoronavirus disease 19 (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Antiviral immune response is crucial to achieve pathogen clearance; however, in some patients an excessive and aberrant host immune response can lead to an acute respiratory distress syndrome. The comprehension of the mechanisms that regulate pathogen elimination, immunity, and pathology is essential to better characterize disease progression and widen the spectrum of therapeutic options.METHODSWe performed a flow cytometric characterization of immune cell subsets from 30 patients with COVID-19 and correlated these data with clinical outcomes.RESULTSPatients with COVID-19 showed decreased numbers of circulating T, B, and NK cells and exhibited a skewing of CD8+ T cells toward a terminally differentiated/senescent phenotype. In agreement, CD4+ T and CD8+ T, but also NK cells, displayed reduced antiviral cytokine production capability. Moreover, a reduced cytotoxic potential was identified in patients with COVID-19, particularly in those who required intensive care. The latter group of patients also showed increased serum IL-6 levels that inversely correlated to the frequency of granzyme A-expressing NK cells. Off-label treatment with tocilizumab restored the cytotoxic potential of NK cells.CONCLUSIONThe association between IL-6 serum levels and the impairment of cytotoxic activity suggests the possibility that targeting this cytokine may restore antiviral mechanisms.FUNDINGThis study was supported by funds from the Department of Experimental and Clinical Medicine of University of Florence (the ex-60% fund and the "Excellence Departments 2018-2022 Project") derived from Ministero dell'Istruzione, dell'Università e della Ricerca (Italy).