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HIV-1 infection cannot be cured due to long-lived viral reservoirs formed by latently infected CD4 + T cells. "Shock and Kill" strategy has been considered to eliminate the viral reservoir and achieve a functional cure but the stimulation of cytotoxic immunity is necessary. Ponatinib is a tyrosine kinase inhibitor (TKI) clinically used against chronic myeloid leukemia (CML) that has demonstrated to be effective against HIV-1 infection in vitro . Several TKIs may induce a potent cytotoxic response against cancer cells that makes possible to discontinue treatment in people with CML who present long-term deep molecular response. In this longitudinal study, we analyzed the capacity of ponatinib to induce an antiviral response against HIV-1 infection in peripheral blood mononuclear cells (PBMCs) obtained from people with CML previously treated with imatinib for a median of 10 years who changed to ponatinib for 12 months to boost the anticancer response before discontinuing any TKI as part of the clinical trial NCT04043676. Participants were followed-up for an additional 12 months in the absence of treatment. PBMCs were obtained at different time points and then infected in vitro with HIV-1. The rate of infection was determined by quantifying the intracellular levels of p24-gag in CD4 + T cells. The levels of p24-gag+ CD4 + T-cells were lower when these cells were obtained during and after treatment with ponatinib in comparison with those obtained during treatment with imatinib. Cytotoxicity of PBMCs against HIV-infected target cells was significantly higher during treatment with ponatinib than during treatment with imatinib, and it was maintained at least 12 months after discontinuation. There was a significant negative correlation between the lower levels of p24-gag+ CD4 + T-cells and the higher cytotoxicity induced by PBMCs when cells were obtained during and after treatment with ponatinib. This cytotoxic immunity was mostly based on higher levels of Natural Killer and Tγδ cells seemingly boosted by ponatinib. In conclusion, transient treatment with immunomodulators like ponatinib along with ART could be explored to boost the antiviral activity of cytotoxic cells and contribute to the elimination of HIV-1 reservoir., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Manzanares, Ramos-Martín, Rodríguez-Mora, Casado-Fernández, Sánchez-Menéndez, Simón-Rueda, Mateos, Cervero, Spivak, Planelles, Torres, García-Gutiérrez and Coiras.)

HIV-1 infection is efficiently controlled by the antiretroviral treatment (ART) but viral persistence in long-lived reservoirs formed by CD4 + T cells and macrophages impedes viral eradication and creates a chronic inflammatory environment. Dasatinib is a tyrosine kinase inhibitor clinically used against chronic myeloid leukemia (CML) that has also showed an anti-inflammatory potential. We previously reported that dasatinib is very efficient at interfering with HIV-1 infection of CD4 + T cells by preserving the antiviral activity of SAMHD1, an innate immune factor that blocks T-cell activation and proliferation and that is inactivated by phosphorylation at T592 (pSAMHD1). We observed that short-term treatment in vitro with dasatinib significantly reduced pSAMHD1 in monocyte-derived macrophages (MDMs) isolated from people with HIV (PWH) and healthy donors, interfering with HIV-1 infection. This inhibition was based on low levels of 2-LTR circles and proviral integration, while viral reverse transcription was not affected. MDMs isolated from people with CML on long-term treatment with dasatinib also showed low levels of pSAMHD1 and were resistant to HIV-1 infection. In addition, dasatinib decreased the inflammatory potential of MDMs by reducing the release of M1-related cytokines like TNFα, IL-1β, IL-6, CXCL8, and CXCL9, but preserving the antiviral activity through normal levels of IL-12 and IFNγ. Due to the production of M2-related anti-inflammatory cytokines like IL-1RA and IL-10 was also impaired, dasatinib appeared to interfere with MDMs differentiation. The use of dasatinib along with ART could be used against HIV-1 reservoir in CD4 and macrophages and to alleviate the chronic inflammation characteristic of PWH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Individuals with chronic myeloid leukemia (CML) constitute a unique group within individuals with oncohematological disease (OHD). They receive treatment with tyrosine kinase inhibitors (TKIs) that present immunomodulatory properties, and they may eventually be candidates for treatment discontinuation under certain conditions despite the chronic nature of the disease. In addition, these individuals present a lower risk of infection than other immunocompromised patients. For this study, we recruited a cohort of 29 individuals with CML in deep molecular response who were on treatment with TKIs (n = 23) or were on treatment-free remission (TFR) (n = 6), and compared both humoral and cellular immune responses with 20 healthy donors after receiving the complete vaccination schedule against SARS-CoV-2. All participants were followed up for 17 months to record the development of COVID-19 due to breakthrough infections. All CML individuals developed an increased humoral response, with similar seroconversion rates and neutralizing titers to healthy donors, despite the presence of high levels of immature B cells. On the whole, the cellular immune response was also comparable to that of healthy donors, although the antibody dependent cytotoxic activity (ADCC) was significantly reduced. Similar rates of mild breakthrough infections were observed between groups, although the proportion was higher in the CML individuals on TFR, most likely due to the immunomodulatory effect of these drugs. In conclusion, as with the healthy donors, the vaccination did not impede breakthrough infections completely in individuals with CML, although it prevented the development of severe or critical illness in this special population of individuals with OHD.

The high morbimortality due to SARS-CoV-2 infection in oncohematological diseases (OHD) and hematopoietic stem cell transplant (HSCT) recipients in the pre-vaccine era has made vaccination a priority in this group. After HSCT, the immune responses against common vaccines such as tetanus, varicella, rubella, and polio may be lost. However, the loss of immunity developed by COVID-19 vaccination after HSCT has not been completely defined. In this study, both humoral and cellular immunity against SARS-CoV-2 were analyzed in 29 individuals with OHD who were vaccinated before receiving allogeneic ( n = 11) or autologous (n = 18) HSCT. All participants had low but protective levels of neutralizing IgGs against SARS-CoV-2 after HSCT despite B-cell lymphopenia and immaturity. Although antibody-dependent cellular cytotoxicity was impaired, direct cellular cytotoxicity was similar to healthy donors in participants with autologous-HSCT, in contrast to individuals with allogeneic-HSCT, which severely deteriorated. No significant changes were observed in the immune response before and after HSCT. During follow-up, all reported post-HSCT SARS-CoV-2 infections were mild. This data emphasizes that COVID-19 vaccination is effective, necessary, and safe for individuals with OHD and also supports the persistence of some degree of immune protection after HSCT, at least in the short term, when patients cannot yet be revaccinated.

The main objective of this study was to determine the influence of the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) on the outcome of unvaccinated individuals with critical COVID-19 admitted to the ICU. Blood samples from 23 individuals were collected upon admission and then every 2 weeks for 13 weeks until death (Exitus group) ( n = 13) or discharge (Survival group) ( n = 10). We did not find significant differences between groups in sociodemographic, clinical, or biochemical data that may influence the fatal outcome. However, direct cellular cytotoxicity of PBMCs from individuals of the Exitus group against pseudotyped SARS-CoV-2-infected Vero E6 cells was significantly reduced upon admission (-2.69-fold; p = 0.0234) and after 4 weeks at the ICU (-5.58-fold; p = 0.0290), in comparison with individuals who survived, and it did not improve during hospitalization. In vitro treatment with IL-15 of these cells did not restore an effective cytotoxicity at any time point until the fatal outcome, and an increased expression of immune exhaustion markers was observed in NKT, CD4+, and CD8+ T cells. However, IL-15 treatment of PBMCs from individuals of the Survival group significantly increased cytotoxicity at Week 4 (6.18-fold; p = 0.0303). Consequently, immunomodulatory treatments that may overcome immune exhaustion and induce sustained, efficient cytotoxic activity could be essential for survival during hospitalization due to critical COVID-19.

The humoral immune response developed after receiving the full vaccination schedule against COVID-19 is impaired in individuals who received anti-CD20 therapy 6-9 months before vaccination. However, there is little information about the cellular immune responses elicited in these individuals. In this study, we analyzed the humoral and cellular immune responses in 18 individuals with hematological disease who received the last dose of rituximab 13.8 months (IQR 9.4-19) before the booster dose. One month after receiving the booster dose, the seroconversion rate in the rituximab-treated cohort increased from 83.3% to 88.9% and titers of specific IgGs against SARS-CoV-2 increased 1.53-fold ( p = 0.0098), while the levels of neutralizing antibodies increased 3.03-fold ( p = 0.0381). However, the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) from rituximab-treated individuals remained unchanged, and both antibody-dependent cellular cytotoxicity (ADCC) and direct cellular cytotoxicity (CDD) were reduced 1.7-fold ( p = 0.0047) and 2.0-fold ( p = 0.0086), respectively, in comparison with healthy donors. Breakthrough infections rate was higher in our cohort of rituximab-treated individuals (33.33%), although most of the infected patients (83.4%) developed a mild form of COVID-19. In conclusion, our findings confirm a benefit in the humoral, but not in the cellular, immune response in rituximab-treated individuals after receiving a booster dose of an mRNA-based vaccine against COVID-19.

The clinical presentations of COVID-19 may range from an asymptomatic or mild infection to a critical or fatal disease. Several host factors such as elderly age, male gender, and previous comorbidities seem to be involved in the most severe outcomes, but also an impaired immune response that causes a hyperinflammatory state but is unable to clear the infection. In order to get further understanding about this impaired immune response, we aimed to determine the association of specific HLA alleles with different clinical presentations of COVID-19. Therefore, we analyzed HLA Class I and II, as well as KIR gene sequences, in 72 individuals with Spanish Mediterranean Caucasian ethnicity who presented mild, severe, or critical COVID-19, according to their clinical characteristics and management. This cohort was recruited in Madrid (Spain) during the first and second pandemic waves between April and October 2020. There were no significant differences in HLA-A or HLA-B alleles among groups. However, despite the small sample size, we found that HLA-C alleles from group C1 HLA-C*08:02, -C*12:03, or -C*16:01 were more frequently associated in individuals with mild COVID-19 (43.8%) than in individuals with severe (8.3%; p = 0.0030; pc = 0.033) and critical (16.1%; p = 0.0014; pc = 0.0154) disease. C1 alleles are supposed to be highly efficient to present peptides to T cells, and HLA-C*12:03 may present a high number of verified epitopes from abundant SARS-CoV-2 proteins M, N, and S, thereby being allegedly able to trigger an efficient antiviral response. On the contrary, C2 alleles are usually poorly expressed on the cell surface due to low association with β2-microglobulin (β2M) and peptides, which may impede the adequate formation of stable HLA-C/β2M/peptide heterotrimers. Consequently, this pilot study described significant differences in the presence of specific HLA-C1 alleles in individuals with different clinical presentations of COVID-19, thereby suggesting that HLA haplotyping could be valuable to get further understanding in the underlying mechanisms of the impaired immune response during critical COVID-19., Competing Interests: The authors have declared that no competing interests exist.

There is now sufficient evidence to support that vitamin D deficiency may predispose to SARS-CoV-2 infection and increase COVID-19 severity and mortality. It has been suggested that vitamin D 3 supplementation may be used prophylactically as an affordable and safe strategy that could be added to the existing COVID-19 standard treatment. This multicenter, single-blinded, prospective randomized pilot clinical trial aimed to evaluate the safety, tolerability, and effectiveness of 10,000 IU/day in comparison with 2000 IU/day of cholecalciferol supplementation for 14 days to reduce the duration and severity of COVID-19 in 85 hospitalized individuals. The median age of the participants was 65 years (Interquartile range (IQR): 53-74), most of them (71%) were men and the mean baseline of 25-hydroxyvitamin D (25(OH)D) in serum was 15 ng/ml (standard deviation (SD):6). After 14 days of supplementation, serum 25(OH)D levels were significantly increased in the group who received 10,000IU/day ( p < 0.0001) ( n = 44) in comparison with the 2,000IU/day group ( n = 41), especially in overweight and obese participants, and the higher dose was well tolerated. A fraction of the individuals in our cohort (10/85) developed acute respiratory distress syndrome (ARDS). The median length of hospital stay in these patients with ARDS was significantly different in the participants assigned to the 10,000IU/day group ( n = 4; 7 days; IQR: 4-13) and the 2,000IU/day group ( n = 6; 27 days; IQR: 12-45) ( p = 0.04). Moreover, the inspired oxygen fraction was reduced 7.6-fold in the high dose group ( p = 0.049). In terms of blood parameters, we did not identify overall significant improvements, although the platelet count showed a modest but significant difference in those patients who were supplemented with the higher dose ( p = 0.0492). In conclusion, the administration of 10,000IU/day of vitamin D 3 for 14 days in association with the standard clinical care during hospitalization for COVID-19 was safe, tolerable, and beneficial, thereby helping to improve the prognosis during the recovery process., Competing Interests: Drug Cholecalciferol (vitamin D) used in this study was donated by Italfarmaco Group (Cholecalciferol 25,000IU/2.5 ml oral solution). Italfarmaco Group had no role in the design and conduct of the study, in the collection, management, analysis, and interpretation of the data, or the preparation, review, or approval of the manuscript., (Copyright © 2022 Cervero, López-Wolf, Casado, Novella-Mena, Ryan-Murua, Taboada-Martínez, Rodríguez-Mora, Vigón, Coiras and Torres.)

Main cause of severe illness and death in COVID-19 patients appears to be an excessive but ineffectual inflammatory immune response that may cause severe acute respiratory distress syndrome (ARDS). Vitamin D may favour an anti-inflammatory environment and improve cytotoxic response against some infectious diseases. A multicenter, single-blind, prospective, randomized clinical trial was approved in patients with COVID-19 pneumonia and levels of 25-hydroxyvitamin D (25(OH)D) of 14.8 ng/ml (SD: 6.18) to test antiviral efficacy, tolerance and safety of 10,000 IU/day of cholecalciferol (vitamin D 3 ) for 14 days, in comparison with 2000 IU/day. After supplementation, mean serum 25(OH)D levels increased to 19 ng/ml on average in 2000 IU/day versus 29 ng/ml in 10,000 IU/day group (p < 0.0001). Although levels of inflammatory cytokines were not modified by treatment with 10,000 IU/day, there was an increase of anti-inflammatory cytokine IL-10 and higher levels of CD4+ T cells, with predominance of T central memory subpopulation. Cytotoxic response against pseudotyped SARS-CoV-2 infected cells was increased more than 4-fold in patients who received 10,000 IU/day. Moreover, levels of IFNγ were significantly higher in this group. Beneficial effect of supplementation with 10,000 IU/day was also observed in participants who developed ARDS and stayed at the hospital for 8.0 days, whereas those who received 2000 IU/day stayed for 29.2 days (p = 0.0381). Administration of high doses of vitamin D 3 as adjuvant of the standard care treatment during hospitalization for COVID-19 may improve the inflammatory environment and cytotoxic response against pseudotyped SARS-CoV-2 infected cells, shortening the hospital stay and, possibly, improving the prognosis., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Individuals with oncohematological diseases (OHD) may develop an impaired immune response against vaccines due to the characteristics of the disease or to its treatment. Humoral response against SARS-CoV-2 has been described to be suboptimal in these patients, but the quality and efficiency of the cellular immune response has not been yet completely characterized. In this study, we analyzed the early humoral and cellular immune responses in individuals with different OHD after receiving one dose of an authorized vaccine against SARS-CoV-2. Humoral response, determined by antibodies titers and neutralizing capacity, was overall impaired in individuals with OHD, except for the cohort of chronic myeloid leukemia (CML), which showed higher levels of specific IgGs than healthy donors. Conversely, the specific direct cytotoxic cellular immunity response (DCC) against SARS-CoV-2, appeared to be enhanced, especially in individuals with CML and chronic lymphocytic leukemia (CLL). This increased cellular immune response, developed earlier than in healthy donors, showed a modest cytotoxic activity that was compensated by significantly increased numbers, likely due to the disease or its treatment. The analysis of the immune response through subsequent vaccine doses will help establish the real efficacy of COVID-19 vaccines in individuals with OHD.

LGMDD2 is a rare form of muscular dystrophy characterized by one of the three heterozygous deletions described within the TNPO3 gene that result in the addition of a 15-amino acid tail in the C-terminus.TNPO3 is involved in the nuclear import of splicing factors and acts as a host cofactor for HIV-1 infection by mechanisms not yet deciphered. Further characterization of the crosstalk between HIV-1 infection and LGMDD2 disease may contribute to a better understanding of both the cellular alterations occurring in LGMDD2 patients and the role of TNPO3 in the HIV-1 cycle. To this regard, transcriptome profiling of PBMCs from LGMDD2 patients carrying the deletion c.2771delA in the TNPO3 gene was compared to healthy controls. A total of 545 differentially expressed genes were detected between LGMDD2 patients and healthy controls, with a high representation of G protein-coupled receptor binding chemokines and metallopeptidases among the most upregulated genes in LGMDD2 patients. Plasma levels of IFN-β and IFN-γ were 4.7- and 2.7-fold higher in LGMDD2 patients, respectively. An increase of 2.3-fold in the expression of the interferon-stimulated gene MxA was observed in activated PBMCs from LGMDD2 patients after ex vivo HIV-1 pseudovirus infection. Thus, the analysis suggests a pro-inflammatory state in LGMDD2 patients also described for other muscular dystrophies, that is characterized by the alteration of IL-17 signaling pathway and the consequent increase of metallopeptidases activity and TNF response. In summary, the increase in interferons and inflammatory mediators suggests an antiviral environment and resistance to HIV-1 infection but that could also impair muscular function in LGMDD2 patients, worsening disease evolution. Biomarkers of disease progression and therapeutic strategies based on these genes and mechanisms should be further investigated for this type of muscular dystrophy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Diez-Fuertes, López-Huertas, García-Pérez, Calonge, Bermejo, Mateos, Martí, Muelas, Vílchez, Coiras, Alcamí and Rodríguez-Mora.)

Oncohematological patients show a low immune response against SARS-CoV-2, both to natural infection and after vaccination. Most studies are focused on the analysis of the humoral response; therefore, the information available about the cellular immune response is limited. In this study, we analyzed the humoral and cellular immune responses in nine individuals who received chemotherapy for their oncohematological diseases, as well as consolidation with autologous stem cell transplantation (ASCT), after being naturally infected with SARS-CoV-2. All individuals had asymptomatic or mild COVID-19 and were not vaccinated against SARS-CoV-2. These results were compared with matched healthy individuals who also had mild COVID-19. The humoral response against SARS-CoV-2 was not detected in 6 of 9 oncohematological individuals prior to ASCT. The levels of antibodies and their neutralization capacity decreased after ASCT. Conversely, an enhanced cytotoxic activity against SARS-CoV-2-infected cells was observed after chemotherapy plus ASCT, mostly based on high levels of NK, NKT, and CD8+TCRγδ+ cell populations that were able to produce IFNγ and TNFα. These results highlight the importance of performing analyses not only to evaluate the levels of IgGs against SARS-CoV-2, but also to determine the quality of the cellular immune response developed during the immune reconstitution after ASCT.

Long-COVID is a new emerging syndrome worldwide that is characterized by the persistence of unresolved signs and symptoms of COVID-19 more than 4 weeks after the infection and even after more than 12 weeks. The underlying mechanisms for Long-COVID are still undefined, but a sustained inflammatory response caused by the persistence of SARS-CoV-2 in organ and tissue sanctuaries or resemblance with an autoimmune disease are within the most considered hypotheses. In this study, we analyzed the usefulness of several demographic, clinical, and immunological parameters as diagnostic biomarkers of Long-COVID in one cohort of Spanish individuals who presented signs and symptoms of this syndrome after 49 weeks post-infection, in comparison with individuals who recovered completely in the first 12 weeks after the infection. We determined that individuals with Long-COVID showed significantly increased levels of functional memory cells with high antiviral cytotoxic activity such as CD8 + TEMRA cells, CD8 ± TCRγδ + cells, and NK cells with CD56 + CD57 + NKG2C + phenotype. The persistence of these long-lasting cytotoxic populations was supported by enhanced levels of CD4 + Tregs and the expression of the exhaustion marker PD-1 on the surface of CD3 + T lymphocytes. With the use of these immune parameters and significant clinical features such as lethargy, pleuritic chest pain, and dermatological injuries, as well as demographic factors such as female gender and O + blood type, a Random Forest algorithm predicted the assignment of the participants in the Long-COVID group with 100% accuracy. The definition of the most accurate diagnostic biomarkers could be helpful to detect the development of Long-COVID and to improve the clinical management of these patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Galán, Vigón, Fuertes, Murciano-Antón, Casado-Fernández, Domínguez-Mateos, Mateos, Ramos-Martín, Planelles, Torres, Rodríguez-Mora, López-Huertas and Coiras.)

The latent viral reservoir formed by HIV-1, mainly in CD4 + T cells, is responsible for the failure of antiretroviral therapy (ART) to achieve a complete elimination of the virus in infected individuals. We previously determined that CD4 + T cells from individuals with chronic myeloid leukemia (CML) on treatment with dasatinib are resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral effect is the preservation of SAMHD1 activity. In this study, we aimed to evaluate the impact of dasatinib on the viral reservoir of HIV-infected individuals with CML who were on simultaneous treatment with ART and dasatinib. Due to the low estimated incidence of HIV-1 infection and CML (1:65,000), three male individuals were recruited in Spain and Germany. These individuals had been on treatment with standard ART and dasatinib for median 1.3 years (IQR 1.3-5.3 years). Reservoir size and composition in PBMCs from these individuals was analyzed in comparison with HIV-infected individuals on triple ART regimen and undetectable viremia. The frequency of latently infected cells was reduced more than 5-fold in these individuals. The reactivation of proviruses from these cells was reduced more than 4-fold and, upon activation, SAMHD1 phosphorylation was reduced 40-fold. Plasma levels of the homeostatic cytokine IL-7 and CD4 effector subpopulations TEM and TEMRA in peripheral blood were also reduced. Therefore, treatment of HIV-infected individuals with dasatinib as adjuvant of ART could disturb the reservoir reactivation and reseeding, which might have a beneficial impact to reduce its size., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

SARS-CoV-2 infection causes COVID-19, ranging from mild to critical disease in symptomatic subjects. It is essential to better understand the immunologic responses occurring in patients with the most severe outcomes. In this study, parameters related to the humoral immune response elicited against SARS-CoV-2 were analysed in 61 patients with different presentations of COVID-19 who were recruited in Hospitals and Primary Healthcare Centres in Madrid, Spain, during the first pandemic peak between April and June 2020. Subjects were allocated as mild patients without hospitalization, severe patients hospitalized or critical patients requiring ICU assistance. Critical patients showed significantly enhanced levels of B cells with memory and plasmablast phenotypes, as well as higher levels of antibodies against SARS-CoV-2 with neutralization ability, which were particularly increased in male gender. Despite all this, antibody-dependent cell-mediated cytotoxicity was defective in these individuals. Besides, patients with critical COVID-19 also showed increased IgG levels against herpesvirus such as CMV, EBV, HSV-1 and VZV, as well as detectable CMV and EBV viremia in plasma. Altogether, these results suggest an enhanced but ineffectual immune response in patients with critical COVID-19 that allowed latent herpesvirus reactivation. These findings should be considered during the clinical management of these patients due to the potential contribution to the most severe disease during SARS-CoV-2 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vigón, García-Pérez, Rodríguez-Mora, Torres, Mateos, Castillo de la Osa, Cervero, Malo De Molina, Navarro, Murciano-Antón, García-Gutiérrez, Planelles, Alcamí, Pérez-Olmeda, Coiras and López-Huertas.)

Infection by novel coronavirus SARS-CoV-2 causes different presentations of COVID-19 and some patients may progress to a critical, fatal form of the disease that requires their admission to ICU and invasive mechanical ventilation. In order to predict in advance which patients could be more susceptible to develop a critical form of COVID-19, it is essential to define the most adequate biomarkers. In this study, we analyzed several parameters related to the cellular immune response in blood samples from 109 patients with different presentations of COVID-19 who were recruited in Hospitals and Primary Healthcare Centers in Madrid, Spain, during the first pandemic peak between April and June 2020. Hospitalized patients with the most severe forms of COVID-19 showed a potent inflammatory response that was not translated into an efficient immune response. Despite the high levels of effector cytotoxic cell populations such as NK, NKT and CD8+ T cells, they displayed immune exhaustion markers and poor cytotoxic functionality against target cells infected with pseudotyped SARS-CoV-2 or cells lacking MHC class I molecules. Moreover, patients with critical COVID-19 showed low levels of the highly cytotoxic TCRγδ+ CD8+ T cell subpopulation. Conversely, CD4 count was greatly reduced in association to high levels of Tregs, low plasma IL-2 and impaired Th1 differentiation. The relative importance of these immunological parameters to predict COVID-19 severity was analyzed by Random Forest algorithm and we concluded that the most important features were related to an efficient cytotoxic response. Therefore, efforts to fight against SARS-CoV-2 infection should be focused not only to decrease the disproportionate inflammatory response, but also to elicit an efficient cytotoxic response against the infected cells and to reduce viral replication., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vigón, Fuertes, García-Pérez, Torres, Rodríguez-Mora, Mateos, Corona, Saez-Marín, Malo, Navarro, Murciano-Antón, Cervero, Alcamí, García-Gutiérrez, Planelles, López-Huertas and Coiras.)

BCR-ABL is an aberrant tyrosine kinase responsible for chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKIs) induce a potent antileukemic response mostly based on the inhibition of BCR-ABL, but they also increase the activity of Natural Killer (NK) and CD8+ T cells. After several years, patients may interrupt treatment due to sustained, deep molecular response. By unknown reasons, half of the patients relapse during treatment interruption, whereas others maintain a potent control of the residual leukemic cells for several years. In this study, several immunological parameters related to sustained antileukemic control were analyzed. According to our results, the features more related to poor antileukemic control were as follows: low levels of cytotoxic cells such as NK, (Natural Killer T) NKT and CD8±TCRγβ+ T cells; low expression of activating receptors on the surface of NK and NKT cells; impaired synthesis of proinflammatory cytokines or proteases from NK cells; and HLA-E*0103 homozygosis and KIR haplotype BX. A Random Forest algorithm predicted 90% of the accuracy for the classification of CML patients in groups of relapse or non-relapse according to these parameters. Consequently, these features may be useful as biomarkers predictive of CML relapse in patients that are candidates to initiate treatment discontinuation.

Tyrosine kinase inhibitors (TKIs) are successfully used in clinic to treat chronic myeloid leukemia (CML). Our group previously described that CD4+ T cells from patients with CML on treatment with TKIs such as dasatinib were resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral activity was primarily based on the inhibition of SAMHD1 phosphorylation, which preserves the activity against HIV-1 of this innate immune factor. Approximately 50% CML patients who achieved a deep molecular response (DMR) may safely withdraw TKI treatment without molecular recurrence. Therefore, it has been speculated that TKIs may induce a potent antileukemic response that is maintained in most patients even one year after treatment interruption (TI). Subsequent to in vitro T-cell activation, we observed that SAMHD1 was phosphorylated in CD4+ T cells from CML patients who withdrew TKI treatment more than one year earlier, which indicated that these cells were now susceptible to HIV-1 infection. Importantly, these patients were seronegative for HIV-1 and seropositive for cytomegalovirus (CMV), but without CMV viremia. Although activated CD4+ T cells from CML patients on TI were apparently permissive to HIV-1 infection ex vivo, the frequency of proviral integration was reduced more than 12-fold on average when these cells were infected ex vivo in comparison with cells isolated from untreated, healthy donors. This reduced susceptibility to infection could be related to an enhanced NK-dependent cytotoxic activity, which was increased 8-fold on average when CD4+ T cells were infected ex vivo with HIV-1 in the presence of autologous NK cells. Enhanced cytotoxic activity was also observed in CD8 + T cells from these patients, which showed 8-fold increased expression of TCRγδ and more than 18-fold increased production of IFNγ upon activation with CMV peptides. In conclusion, treatment with TKIs induced a potent antileukemic response that may also have antiviral effects against HIV-1 and CMV, suggesting that transient use of TKIs in HIV-infected patients could develop a sustained antiviral response that would potentially interfere with HIV-1 reservoir dynamics., (Copyright © 2020. Published by Elsevier Inc.)

Purpose of Review: HIV-1 infection is incurable due to the existence of latent reservoirs that persist in the face of cART. In this review, we describe the existence of multiple HIV-1 reservoirs, the mechanisms that support their persistence, and the potential use of tyrosine kinase inhibitors (TKIs) to block several pathogenic processes secondary to HIV-1 infection., Recent Findings: Dasatinib interferes in vitro with HIV-1 persistence by two independent mechanisms. First, dasatinib blocks infection and potential expansion of the latent reservoir by interfering with the inactivating phosphorylation of SAMHD1. Secondly, dasatinib inhibits the homeostatic proliferation induced by γc-cytokines. Since homeostatic proliferation is thought to be the main mechanism behind the maintenance of the latent reservoir, we propose that blocking this process will gradually reduce the size of the reservoir. TKIs together with cART will interfere with HIV-1 latent reservoir persistence, favoring the prospect for viral eradication.

Current antiretroviral treatment (ART) may control HIV-1 replication but it cannot cure the infection due to the formation of a reservoir of latently infected cells. CD4+ T cell activation during HIV-1 infection eliminates the antiviral function of the restriction factor SAMHD1, allowing proviral integration and the reservoir establishment. The role of tyrosine kinases during T-cell activation is essential for these processes. Therefore, the inhibition of tyrosine kinases could control HIV-1 infection and restrict the formation of the reservoir. A family of tyrosine kinase inhibitors (TKIs) is successfully used in clinic for treating chronic myeloid leukemia (CML). The safety and efficacy against HIV-1 infection of five TKIs was assayed in PBMCs isolated from CML patients on prolonged treatment with these drugs that were infected ex vivo with HIV-1. We determined that the most potent and safe TKI against HIV-1 infection was dasatinib, which preserved SAMHD1 antiviral function and avoid T-cell activation through TCR engagement and homeostatic cytokines. Imatinib and nilotinib showed lower potency and bosutinib was quite toxic in vitro. Ponatinib presented similar profile to dasatinib but as it has been associated with higher incidence of arterial ischemic events, dasatinib would be the better choice of TKI to be used as adjuvant of ART in order to avoid the establishment and replenishment of HIV-1 reservoir and move forward towards an HIV cure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

HIV-1 induces changes in the miRNA expression profile of infected CD4+ T cells that could improve viral replication. HIV-1 regulator Tat modifies the cellular gene expression and has been appointed as an RNA silencing suppressor. Tat is a 101-residue protein codified by two exons that regulates the elongation of viral transcripts. The first exon of Tat (amino acids 1-72) forms the transcriptionally active protein Tat72, but the presence of the second exon (amino acids 73-101) results in a more competent regulatory protein (Tat101) with additional functions. Intracellular, full-length Tat101 induces functional and morphological changes in CD4+ T cells that contribute to HIV-1 pathogenesis such as delay in T-cell proliferation and protection against FasL-mediated apoptosis. But the precise mechanism by which Tat produces these changes remains unknown. We analyzed how the stable expression of intracellular Tat101 and Tat72 modified the miRNA expression profile in Jurkat cells and if this correlated with changes in apoptotic pathways and cell cycle observed in Tat-expressing cells. Specifically, the enhanced expression of hsa-miR-21 and hsa-miR-222 in Jurkat-Tat101 cells was associated with the reduced expression of target mRNAs encoding proteins related to apoptosis and cell cycle such as PTEN, PDCD4 and CDKN1B. We developed Jurkat cells with stable expression of hsa-miR-21 or hsa-miR-222 and observed a similar pattern to Jurkat-Tat101 in resistance to FasL-mediated apoptosis, cell cycle arrest in G2/M and altered cell morphology. Consequently, upregulation of hsa-miR-21 and hsa-miR-222 by Tat may contribute to protect against apoptosis and to anergy observed in HIV-infected CD4+ T cells.

A potential strategy to cure HIV-1 infection is to use latency reversing agents (LRAs) to eliminate latent reservoirs established in resting CD4+ T (rCD4+) cells. As no drug has been shown to be completely effective, finding new drugs and combinations are of increasing importance. We studied the effect of Maraviroc (MVC), a CCR5 antagonist that activates NF-κB, on HIV-1 replication from latency. HIV-1-latency models based on CCL19 or IL7 treatment, before HIV-1 infection were used. Latently infected primary rCD4+ or central memory T cells were stimulated with MVC alone or in combination with Bryostatin-1, a PKC agonist known to reverse HIV-1 latency. MVC 5 μM and 0.31 μM were chosen for further studies although other concentrations of MVC also increased HIV-1 replication. MVC was as efficient as Bryostatin-1 in reactivating X4 and R5-tropic HIV-1. However, the combination of MVC and Bryostatin-1 was antagonistic, probably because Bryostatin-1 reduced CCR5 expression levels. Although HIV-1 reactivation had the same tendency in both latency models, statistical significance was only achieved in IL7-treated cells. These data suggest that MVC should be regarded as a new LRA with potency similar as Bryostatin-1. Further studies are required to describe the synergistic effect of MVC with other LRAs.

Massive activation of infected CD4+ T cells during acute HIV-1 infection leads to reservoir seeding and T-cell destruction. During T-cell activation, the antiviral effect of the innate factor SAMHD1 is neutralized through phosphorylation at T592, allowing HIV-1 infection. Dasatinib, a tyrosine kinase inhibitor currently used for treating chronic myeloid leukemia, has been described to control HIV-1 replication through its negative effect on T-cell proliferation and viral entry. We demonstrate that Dasatinib can actually interfere with SAMHD1 phosphorylation in human peripheral blood lymphocytes, preserving its antiviral activity against HIV-1. Dasatinib prevented SAMHD1 phosphorylation in vitro and ex vivo, impairing HIV-1 reverse transcription and proviral integration. This was the major mechanism of action because the presence of Vpx, which degrades SAMHD1, in HIV-1 virions impeded the inhibitory effect of Dasatinib on HIV-1 replication. In fact, infection with VSV-pseudotyped HIV-1 virions and fusion of BlaM-Vpr-containing HIV-1 viruses with activated PBMCs in the presence of Dasatinib suggested that Dasatinib was not acting at fusion level. Finally, PBMCs from patients on chronic treatment with Dasatinib showed a lower level of SAMHD1 phosphorylation in response to activating stimuli and low susceptibility to HIV-1 infection ex vivo. Consequently, Dasatinib is a compound currently used in clinic that preserves the antiviral function of SAMHD1. Using Dasatinib as adjuvant of antiretroviral therapy during early primary HIV-1 infection would contribute to reduce viral replication and spread, prevent reservoir seeding, and preserve CD4 counts and CTL responses. These events would create a more favorable virologic and immunologic environment for future interventional studies aiming at HIV-1 eradication., (Copyright © 2016 Elsevier Inc. All rights reserved.)

PKCθ is essential for the activation of CD4(+) T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4(+) T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T(538) residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4(+) T cells and could be used as a therapeutic target.

Background: HIV-1 replication results in mitochondrial damage that is enhanced during antiretroviral therapy (ART). The onset of HIV-1 replication is regulated by viral protein Tat, a 101-residue protein codified by two exons that elongates viral transcripts. Although the first exon of Tat (aa 1-72) forms itself an active protein, the presence of the second exon (aa 73-101) results in a more competent transcriptional protein with additional functions., Results: Mitochondrial overall functions were analyzed in Jurkat cells stably expressing full-length Tat (Tat101) or one-exon Tat (Tat72). Representative results were confirmed in PBLs transiently expressing Tat101 and in HIV-infected Jurkat cells. The intracellular expression of Tat101 induced the deregulation of metabolism and cytoskeletal proteins which remodeled the function and distribution of mitochondria. Tat101 reduced the transcription of the mtDNA, resulting in low ATP production. The total amount of mitochondria increased likely to counteract their functional impairment. These effects were enhanced when Tat second exon was expressed., Conclusions: Intracellular Tat altered mtDNA transcription, mitochondrial content and distribution in CD4+ T cells. The importance of Tat second exon in non-transcriptional functions was confirmed. Tat101 may be responsible for mitochondrial dysfunctions found in HIV-1 infected patients.

HIV-1 infection cannot be cured due to reservoirs formed early after infection. Decreasing the massive CD4+ T cell activation that occurs at the beginning of the disease would delay reservoir seeding, providing a better prognosis for patients. CD4+ T cell activation is mediated by protein kinase C (PKC) theta (θ), which is involved in T-cell proliferation, as well as NF-κB, NF-AT, and AP-1 activation. We found that PKCθ activity increased viral replication, but also that HIV-1 induced higher activation of PKCθ in infected CD4+ T cells, creating a feedback loop. Therefore, specific inhibition of PKCθ activity could contribute to control HIV-1 replication. We tested the efficacy of seven PKCθ specific inhibitors to control HIV-1 replication in CD4+ T cells and selected two of the more potent and safer: CGX1079 and CGX0471. They reduced PKCθ phosphorylation at T538 and its translocation to the plasma membrane, which correlated with decreased HIV-1 retrotranscription through partial inhibition of SAMHD1 antiviral activity, rendering lower proviral integration. CGX1079 and CGX0471 also interfered with viral transcription, which would reduce the production of new virions, as well as the subsequent spread and infection of new targets that would increase the reservoir size. CGX1079 and CGX0471 did not completely abrogate T-cell functions such as proliferation and CD8-mediated release of IFN-γ in PBMCs from HIV-infected patients, thereby avoiding general immunosuppresion. Consequently, using PKCθ inhibitors as adjuvant of antiretroviral therapy in recently infected patients would decrease the pool of activated CD4+ T cells, thwarting proviral integration and reducing the reservoir size., (Copyright © 2015 Elsevier Inc. All rights reserved.)

HIV-1 replication is efficiently controlled by the regulator protein Tat (101 amino acids) and codified by two exons, although the first exon (1-72 amino acids) is sufficient for this process. Tat can be released to the extracellular medium, acting as a soluble pro-apoptotic factor in neighboring cells. However, HIV-1-infected CD4(+) T lymphocytes show a higher resistance to apoptosis. We observed that the intracellular expression of Tat delayed FasL-mediated apoptosis in both peripheral blood lymphocytes and Jurkat cells, as it is an essential pathway to control T cell homeostasis during immune activation. Jurkat-Tat cells showed impairment in the activation of caspase-8, deficient release of mitochondrial cytochrome c, and delayed activation of both caspase-9 and -3. This protection was due to a profound deregulation of proteins that stabilized the mitochondrial membrane integrity, such as heat shock proteins, prohibitin, or nucleophosmin, as well as to the up-regulation of NF-κB-dependent anti-apoptotic proteins, such as BCL2, c-FLIPS, XIAP, and C-IAP2. These effects were observed in Jurkat expressing full-length Tat (Jurkat-Tat101) but not in Jurkat expressing the first exon of Tat (Jurkat-Tat72), proving that the second exon, and particularly the NF-κB-related motif ESKKKVE, was necessary for Tat-mediated protection against FasL apoptosis. Accordingly, the protection exerted by Tat was independent of its function as a regulator of both viral transcription and elongation. Moreover, these data proved that HIV-1 could have developed strategies to delay FasL-mediated apoptosis in infected CD4(+) T lymphocytes through the expression of Tat, thus favoring the persistent replication of HIV-1 in infected T cells.