The Impact of Cytomegalovirus Infection on Natural Killer and CD8+ T Cell Phenotype in Multiple Sclerosis.

Multiple sclerosis (MS) is a debilitating neurological disease that has been classified as an immune-mediated attack on myelin, the protective sheath of nerves. Some aspects of its pathogenesis are still unclear; nevertheless, it is generally established that viral infections influence the course of the disease. Cytomegalovirus (CMV) is a major pathogen involved in alterations of the immune system, including the expansion of highly differentiated cytotoxic CD8+ T cells and the accumulation of adaptive natural killer (NK) cells expressing high levels of the NKG2C receptor. In this study, we evaluated the impact of latent CMV infection on MS patients through the characterization of peripheral NK cells, CD8+ T cells, and NKT-like cells using flow cytometry. We evaluated the associations between immune cell profiles and clinical features such as MS duration and MS progression, evaluated using the Expanded Disability Status Scale (EDSS). We showed that NK cells, CD8+ T cells, and NKT-like cells had an altered phenotype in CMV-infected MS patients and displayed high levels of the NKG2C receptor. Moreover, in MS patients, increased NKG2C expression levels were found to be associated with higher EDSS scores. Overall, these results support the hypothesis that CMV infection imprints the immune system by modifying the phenotype and receptor repertoire of NK and CD8+ T cells, suggesting a detrimental role of CMV on MS progression.

Evaluation of BAFF, APRIL and CD40L in Ocrelizumab-Treated pwMS and Infectious Risk.

BACKGROUND: The anti-CD20 monoclonal antibody ocrelizumab has been widely employed in the treatment of people with multiple sclerosis (pwMS). However, its B-cell-depleting effect may induce a higher risk of infectious events and alterations in the secretion of B-cell-activating factors, such as BAFF, APRIL and CD40L. METHODS: The aim of this study was to investigate plasma BAFF, APRIL and CD40L levels and their relationship with infectious risk in ocrelizumab-treated pwMS at baseline (T0), at 6 months (T6) and at 12 months (T12) after starting the treatment. As a control group, healthy donors (HD) were enrolled too. RESULTS: A total of 38 pwMS and 26 HD were enrolled. At baseline, pwMS showed higher plasma BAFF (p < 0.0001), APRIL (p = 0.0223) and CD40L (p < 0.0001) levels compared to HD. Compared to T0, plasma BAFF levels were significantly increased at both T6 and T12 (p < 0.0001 and p < 0.0001, respectively). Whereas plasma APRIL and CD40L levels were decreased at T12 (p = 0.0003 and p < 0.0001, respectively). When stratifying pwMS according to the development of an infectious event during the 12-month follow-up period in two groups-with (14) and without an infectious event (24)-higher plasma BAFF levels were observed at all time-points; significantly, in the group with an infectious event compared to the group without an infectious event (T0: p < 0.0001, T6: p = 0.0056 and T12: p = 0.0400). Conclusions: BAFF may have a role as a marker of immune dysfunction and of infectious risk.

Breakthrough infections after COVID-19 vaccinations do not elicit platelet hyperactivation and are associated with high platelet-lymphocyte and low platelet-neutrophil aggregates.

Background: Severe COVID-19 is associated with an excessive immunothrombotic response and thromboinflammatory complications. Vaccinations effectively reduce the risk of severe clinical outcomes in patients with COVID-19, but their impact on platelet activation and immunothrombosis during breakthrough infections is not known. Objectives: To investigate how preemptive vaccinations modify the platelet-immune crosstalk during COVID-19 infections. Methods: Cross-sectional flow cytometry study of the phenotype and interactions of platelets circulating in vaccinated (n = 21) and unvaccinated patients with COVID-19, either admitted to the intensive care unit (ICU, n = 36) or not (non-ICU, n = 38), in comparison to matched SARS-CoV-2-negative patients (n = 48), was performed. Results: In the circulation of unvaccinated non-ICU patients with COVID-19, we detected hyperactive and hyperresponsive platelets and platelet aggregates with adaptive and innate immune cells. In unvaccinated ICU patients with COVID-19, most of whom had severe acute respiratory distress syndrome, platelets had high P-selectin and phosphatidylserine exposure but low capacity to activate integrin αIIbß3, dysfunctional mitochondria, and reduced surface glycoproteins. In addition, in the circulation of ICU patients, we detected microthrombi and platelet aggregates with innate, but not with adaptive, immune cells. In vaccinated patients with COVID-19, who had no acute respiratory distress syndrome, platelets had surface receptor levels comparable to those in controls and did not form microthrombi or platelet-granulocyte aggregates but aggregated avidly with adaptive immune cells. Conclusion: Our study provides evidence that vaccinated patients with COVID-19 are not associated with platelet hyperactivation and are characterized by platelet-leukocyte aggregates that foster immune protection but not excessive immunothrombosis. These findings advocate for the importance of vaccination in preventing severe COVID-19.

Multiple sclerosis-disease modifying therapies affect humoral and T-cell response to mRNA COVID-19 vaccine.

Background: The mRNA vaccines help protect from COVID-19 severity, however multiple sclerosis (MS) disease modifying therapies (DMTs) might affect the development of humoral and T-cell specific response to vaccination. Methods: The aim of the study was to evaluate humoral and specific T-cell response, as well as B-cell activation and survival factors, in people with MS (pwMS) under DMTs before (T0) and after two months (T1) from the third dose of vaccine, comparing the obtained findings to healthy donors (HD). All possible combinations of intracellular IFNγ, IL2 and TNFα T-cell production were evaluated, and T-cells were labelled "responding T-cells", those cells that produced at least one of the three cytokines of interest, and "triple positive T-cells", those cells that produced simultaneously all the three cytokines. Results: The cross-sectional evaluation showed no significant differences in anti-S antibody titers between pwMS and HD at both time-points. In pwMS, lower percentages of responding T-cells at T0 (CD4: p=0.0165; CD8: p=0.0022) and triple positive T-cells at both time-points compared to HD were observed (at T0, CD4: p=0.0007 and CD8: p=0.0703; at T1, CD4: p=0.0422 and CD8: p=0.0535). At T0, pwMS showed higher plasma levels of APRIL, BAFF and CD40L compared to HD (p<0.0001, p<0.0001 and p<0.0001, respectively) and at T1, plasma levels of BAFF were still higher in pwMS compared to HD (p=0.0022).According to DMTs, at both T0 and T1, lower anti-S antibody titers in the depleting/sequestering-out compared to the enriching-in pwMS subgroup were found (p=0.0410 and p=0.0047, respectively) as well as lower percentages of responding CD4+ T-cells (CD4: p=0.0394 and p=0.0004, respectively). Moreover, the depleting/sequestering-out subgroup showed higher percentages of IFNγ-IL2-TNFα+ T-cells at both time-points, compared to the enriching-in subgroup in which a more heterogeneous cytokine profile was observed (at T0 CD4: p=0.0187; at T0 and T1 CD8: p =0.0007 and p =0.0077, respectively). Conclusion: In pwMS, humoral and T-cell response to vaccination seems to be influenced by the different DMTs. pwMS under depleting/sequestering-out treatment can mount cellular responses even in the presence of a low positive humoral response, although the cellular response seems qualitatively inferior compared to HD. An understanding of T-cell quality dynamic is needed to determine the best vaccination strategy and in general the capability of immune response in pwMS under different DMT.

Infectious risk in multiple sclerosis patients treated with disease-modifying therapies: A three-year observational cohort study.

BACKGROUND: The disease-modifying therapies (DMTs) largely used in multiple sclerosis (MS) may result in higher infectious risk. OBJECTIVE: We aimed to investigate the infectious risk in DMT-treated MS patients. METHODS: MS patients were evaluated for infectious risk before starting, switching or during DMT. RESULTS: In this three-year observational cohort study 174 MS patients were enrolled. Among them, 18 patients were anti-HBc + and 19 patients were QuantiFERON®-TB Gold In-Tube (QFT) + . No patients with anti-HBc + showed a detectable HBV-DNA and all started DMT. Among QTB + patients, 17 latent TB infections (LTBIs) and 2 active TB infections (TBIs) were identified. After one month of LTBI prophylaxis or TB treatment, respectively, all patients started DMTs.Overall, 149 started DMTs. During DMTs, one ocrelizumab-treated patient with anti-HBc + developed HBV reactivation and six patients (3 on natalizumab, 2 on ocrelizumab and 1 on IFN-ß) showed reactivation of HSV-1, with detectable plasma DNA. Finally, 1 cladribine-treated patient experienced VZV reactivation. All the reactivations of latent infections have been successfully treated. CONCLUSION: Screening of infectious diseases in DMT candidate MS patients helps to mitigate the infectious risk. During DMTs, a regular assessment of infectious risk allows to avoid discontinuing MS therapy and guarantees a higher degree of safety.

Early IgG / IgA response in hospitalized COVID-19 patients is associated with a less severe disease.

We determined the kinetics of anti-SARS-CoV-2 antibody response in fifteen hospitalized COVID-19 patients. Patients were divided into mild/moderate (mild, n = 1; moderate, n = 4) or severe (n = 10) and virus-specific anti-Nucleocapsid IgM, anti-Spike IgG and anti-Spike IgA were measured in serial serum samples collected 0 to 15 days after hospital admission. Surrogate neutralization assays were performed by testing inhibition of ACE-2 binding to Spike. In 3 patients (2 severe and 1 moderate case), serum antibodies and T-cell memory were monitored 6 months after baseline. Although IgM response tended to appear first, patients affected by less severe disease were more prone to an early IgG/IgA response. Neutralization of Spike binding to ACE2 correlated with anti-Spike IgG and IgA. IgG and IgA antibody response persisted at the 6 months follow-up. A recall T-cell response to the Spike antigen was observed in 2 out of 3 patients, not related to disease severity.

Chest computed tomography score, cycle threshold values and secondary infection in predicting COVID-19 mortality.

This retrospective and observational cohort study investigated chest computed tomography (CT) findings, cycle threshold (Ct) values in RT-PCR of SARS-CoV-2 and secondary infection occurrence to predict prognosis in COVID-19 patients. At hospital admission, CT findings and Ct values were collected. Microbiology tests performed after 48 hours from hospitalization were reviewed. According to in-hospital mortality, patients were grouped into non-survivors and survivors. Among 283 patients evaluated, in-hospital mortality rate was 13.8% (39/283). Secondary infection occurrence was 15.2% (43/283). Cut-off values for CT score >13.5 (AUC=0.682 p=0.0009) and for Ct <23.4 (AUC=0.749, p<0.0001) were predictive of death. Super-additive and synergic effects between high CT score plus secondary infection occurrence as well as between high CT score plus low Ct values affecting patient's outcome were observed. Chest CT score and Ct values in RT-PCR of SARS-CoV-2 could have a combination role for severity stratification of COVID-19 patients.

Early IgG / IgA response in hospitalized COVID-19 patients is associated with a less severe disease.

We determined the kinetics of anti-SARS-CoV-2 antibody response in fifteen hospitalized COVID-19 patients. Patients were divided into mild/moderate (mild, n = 1; moderate, n = 4) or severe (n = 10) and virus-specific anti-Nucleocapsid IgM, anti-Spike IgG and anti-Spike IgA were measured in serial serum samples collected 0 to 15 days after hospital admission. Surrogate neutralization assays were performed by testing inhibition of ACE-2 binding to Spike. In 3 patients (2 severe and 1 moderate case), serum antibodies and T-cell memory were monitored 6 months after baseline. Although IgM response tended to appear first, patients affected by less severe disease were more prone to an early IgG/IgA response. Neutralization of Spike binding to ACE2 correlated with anti-Spike IgG and IgA. IgG and IgA antibody response persisted at the 6 months follow-up. A recall T-cell response to the Spike antigen was observed in 2 out of 3 patients, not related to disease severity.

Real-life use of tocilizumab with or without corticosteroid in hospitalized patients with moderate-to-severe COVID-19 pneumonia: A retrospective cohort study.

OBJECTIVE: To evaluate the effectiveness of Tocilizumab (with or without corticosteroids) in a real-life context among moderate-to-severe COVID-19 patients hospitalized at the Infectious Diseases ward of two hospitals in Lazio region, Italy, during the first wave of SARS-CoV-2 pandemic. METHOD: We conducted a retrospective cohort study among moderate-to-severe COVID-19 pneumonia to assess the influence of tocilizumab (with or without corticosteroids) on: 1) primary composite outcome: risk for death/invasive mechanical ventilation/ICU-transfer at 14 days from hospital admission; 2) secondary outcome: COVID-related death only. Both outcomes were also assessed at 28 days and restricted to baseline more severe cases. We also evaluated the safety of tocilizumab. RESULTS: Overall, 412 patients were recruited, being affected by mild (6.8%), moderate (66.3%) or severe (26.9%) COVID-19 at baseline. The median participant' age was 63 years, 56.5% were men, the sum of comorbidities was 1.34 (±1.44), and the median time from symptom onset to hospital admission was 7 [3-10] days. Patients were subdivided in 4 treatment groups: standard of care (SoC) only (n = 172), SoC plus corticosteroid (n = 65), SoC plus tocilizumab (n = 50), SoC plus tocilizumab and corticosteroid (n = 125). Twenty-six (6.3%) patients underwent intubation, and 37 (9%) COVID-related deaths were recorded. After adjusting for several factors, multivariate analysis showed that tocilizumab (with or without corticosteroids) was associated to improved primary and secondary outcomes at 14 days, and at 28-days only when tocilizumab administered without corticosteroid. Among more severe cases the protective effect of tocilizumab (± corticosteroids) was observed at both time-points. No safety concerns were recorded. CONCLUSION: Although contrasting results from randomized clinical trials to date, in our experience tocilizumab was a safe and efficacious therapeutic option for patients with moderate-to-severe COVID-19 pneumonia. Its efficacy was improved by the concomitant administration of corticosteroids in patients affected by severe-COVID-19 pneumonia at baseline.

Increased sCD163 and sCD14 Plasmatic Levels and Depletion of Peripheral Blood Pro-Inflammatory Monocytes, Myeloid and Plasmacytoid Dendritic Cells in Patients With Severe COVID-19 Pneumonia.

Background: Emerging evidence argues that monocytes, circulating innate immune cells, are principal players in COVID-19 pneumonia. The study aimed to investigate the role of soluble (s)CD163 and sCD14 plasmatic levels in predicting disease severity and characterize peripheral blood monocytes and dendritic cells (DCs), in patients with COVID-19 pneumonia (COVID-19 subjects). Methods: On admission, in COVID-19 subjects sCD163 and sCD14 plasmatic levels, and peripheral blood monocyte and DC subsets were compared to healthy donors (HDs). According to clinical outcome, COVID-19 subjects were divided into ARDS and non-ARDS groups. Results: Compared to HDs, COVID-19 subjects showed higher sCD163 (p<0.0001) and sCD14 (p<0.0001) plasmatic levels. We observed higher sCD163 plasmatic levels in the ARDS group compared to the non-ARDS one (p=0.002). The cut-off for sCD163 plasmatic level greater than 2032 ng/ml was predictive of disease severity (AUC: 0.6786, p=0.0022; sensitivity 56.7% [CI: 44.1-68.4] specificity 73.8% [CI: 58.9-84.7]). Positive correlation between plasmatic levels of sCD163, LDH and IL-6 and between plasmatic levels of sCD14, D-dimer and ferritin were found. Compared to HDs, COVID-19 subjects showed lower percentages of non-classical (p=0.0012) and intermediate monocytes (p=0.0447), slanDCs (p<0.0001), myeloid DCs (mDCs, p<0.0001), and plasmacytoid DCs (pDCs, p=0.0014). Compared to the non-ARDS group, the ARDS group showed lower percentages of non-classical monocytes (p=0.0006), mDCs (p=0.0346), and pDCs (p=0.0492). Conclusions: The increase in sCD163 and sCD14 plasmatic levels, observed on hospital admission in COVID-19 subjects, especially in those who developed ARDS, and the correlations of these monocyte/macrophage activation markers with typical inflammatory markers of COVID-19 pneumonia, underline their potential use to assess the risk of progression of the disease. In an early stage of the disease, the assessment of sCD163 plasmatic levels could have clinical utility in predicting the severity of COVID-19 pneumonia.

Major reduction of NKT cells in patients with severe COVID-19 pneumonia.

BACKGROUND: NK cells seem to be mainly involved in COVID-19 pneumonia. Little is known about NKT cells which represent a bridge between innate and adaptive immunity. METHODS: We characterized peripheral blood T, NK and NKT cells in 45 patients with COVID-19 pneumonia (COVID-19 subjects) and 19 healthy donors (HDs). According to the severity of the disease, we stratified COVID-19 subjects into severe and non-severe groups. RESULTS: Compared to HDs, COVID-19 subjects showed higher percentages of NK CD57+ and CD56dim NK cells and lower percentages of NKT and CD56bright cells. In the severe group we found a significantly lower percentage of NKT cells. In a multiple logistic regression analysis, NKT cell was independently associated with the severity of the disease. CONCLUSIONS: The low percentage of NKT cells in peripheral blood of COVID-19 subjects and the independent association with the severity of the disease suggests a potential role of this subset.

The peripheral blood immune cell profile in a teriflunomide-treated multiple sclerosis patient with COVID-19 pneumonia.

A teriflunomide-treated multiple sclerosis patient with COVID-19 pneumonia was hospitalized and recovered in 15 days. The immunophenotyping analysis of peripheral blood cells was performed in two time points: the first was 1 month before (pre-infection) while the second was during COVID-19 pneumonia (infection). At the infection time point, no differences in the percentages of immune activation and immunesenescence of CD4+ and CD8+ T-cells were observed compared to the pre-infection time point. Our evaluation seems to confirm that teriflunomide controls T-cells immune activation and immunosenescence suggesting that teriflunomide should not be discontinued in MS patients with an active COVID-19 pneumonia.

JCPyV NCCR analysis in PML patients with different risk factors: exploring common rearrangements as essential changes for neuropathogenesis.

BACKGROUND: During severe immunosuppression or treatment with specific biological drugs, human polyomavirus JC (JCPyV) may establish a lytic infection in oligodendrocytes, leading to progressive multifocal leukoencephalopathy (PML). Beyond AIDS, which represents the most common predisposing condition, several biological drugs have been associated to the development of PML, such as natalizumab, fingolimod and dimethyl fumarate, which have been showed to increase the risk of PML in the multiple sclerosis (MS) population. JCPyV non-coding control region (NCCR) can be found in two different forms: a virulent neurotropic pathogenic form and a latent non-pathogenic form. The neurotropic forms contain a rearranged NCCR and are typically found in the cerebrospinal fluid, brain or blood of PML patients. CASE PRESENTATION: We sequenced and critically examined JCPyV NCCR from isolates detected in the cerebrospinal fluid of four newly diagnosed progressive multifocal leukoencephalopathy patients: two HIV-positive and two HIV-negative multiple sclerosis patients. More complex NCCR rearrangements were observed in the two HIV-positive patients compared to the HIV-negative multiple sclerosis patients with PML. CONCLUSIONS: The comparison of HIV-positive and HIV-negative MS patients with PML, allowed us to evidence the presence of a common pattern of JCPyV NCCR rearrangement, characterized by the deletion of the D-block, which could be one of the initial rearrangements of JCPyV NCCR needed for the development of PML.

Identification of GAD65 AA 114-122 reactive 'memory-like' NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers.

Type 1 diabetes is an autoimmune disease, in which pancreatic ß cells are destroyed by autoreactive T cells in genetically predisposed individuals. Serum beta cell autoantibody specificities have represented the mainstay for classifying diabetes as autoimmune-mediated and for stratifying risk in first-degree relatives. In recent years, approaches were attempted to solve the difficult issue of detecting rare antigen-specific autoreactive T cells and their significance to etiopathogenesis such as the use of the MHC multimer technology. This tool allowed the specific detection of increased percentages of GAD65 autoreactive T cells by means of HLA A*02:01 GAD65 AA 114-122 pentamers in newly diagnosed diabetics. Here we provide evidence that GAD65 AA 114-122 pentamers can depict a GAD65 AA114-122 peptide expandable population of functionally and phenotypically skewed, preliminary characterized CD3-CD8dullCD56+ 'memory-like' NK cells in PBMC of newly diagnosed diabetics. Our data suggest that the NK cell subset could bind the HLA class I GAD65 AA 114-122 pentamer through ILT2 inhibitory receptor. CD107a expression revealed increased degranulation of CD3-CD8dullCD56+ NK cells in GAD65 AA 114-122 and FLU peptide expanded peripheral blood mononuclear cells of diabetics following GAD65 AA 114-122 peptide HLA A*02:01 presentation in respect to the unpulsed condition. CD107a expression was enriched in ILT2 positive NK cells. As opposite to basal conditions where similar percentages of CD3-CD56+ILT2+ cells were detected in diabetics and controls, CD3-CD56+CD107a+ and CD3-CD56+ILT2+CD107a+ cells were significantly increased in T1D PBMC either GAD65 AA 114-122 or FLU peptides stimulated after co-culture with GAD65 AA 114-122 pulsed APCs. As control, healthy donor NK cells showed similar degranulation against both GAD65 AA 114-122 pulsed and unpulsed APCs. The pathogenetic significance of the CD3-CD8dullCD56+ 'memory-like NK cell subset' with increased response upon secondary challenge in diabetics remains to be elucidated.

Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes.

Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

Altered B cell homeostasis and Toll-like receptor 9-driven response in patients affected by autoimmune polyglandular syndrome Type 1: Altered B cell phenotype and dysregulation of the B cell function in APECED patients.

APECED is a T-cell mediated disease with increased frequencies of CD8+ effector and reduction of FoxP3+ T regulatory cells. Antibodies against affected organs and neutralizing to cytokines are found in the peripheral blood. The contribution of B cells to multiorgan autoimmunity in Aire-/- mice was reported opening perspectives on the utility of anti-B cell therapy. We aimed to analyse the B cell phenotype of APECED patients compared to age-matched controls. FACS analysis was conducted on PBMC in basal conditions and following CpG stimulation. Total B and switched memory (SM) B cells were reduced while IgM memory were increased in patients. In those having more than 15 years from the first clinical manifestation the defect included also mature and transitional B cells; total memory B cells were increased, while SM were unaffected. In patients with shorter disease duration, total B cells were unaltered while SM and IgM memory behaved as in the total group. A defective B cell proliferation was detected after 4day-stimulation. In conclusion APECED patients show, in addition to a significant alteration of the B cell phenotype, a dysregulation of the B cell function involving peripheral innate immune mechanisms particularly those with longer disease duration.

Phosphorylation of the AMPA receptor GluA1 subunit regulates memory load capacity.

Memory capacity (MC) refers to the number of elements one can maintain for a short retention interval. The molecular mechanisms underlying MC are unexplored. We have recently reported that mice as well as humans have a limited MC, which is reduced by hippocampal lesions. Here, we addressed the molecular mechanisms supporting MC. GluA1 AMPA-receptors (AMPA-R) mediate the majority of fast excitatory synaptic transmission in the brain and are critically involved in memory. Phosphorylation of GluA1 at serine residues S831 and S845 is promoted by CaMKII and PKA, respectively, and regulates AMPA-R function in memory duration. We hypothesized that AMPA-R phosphorylation may also be a key plastic process for supporting MC because it occurs in a few minutes, and potentiates AMPA-R ion channel function. Here, we show that knock-in mutant mice that specifically lack both of S845 and S831 phosphorylation sites on the GluA1 subunit had reduced MC in two different behavioral tasks specifically designed to assess MC in mice. This demonstrated a causal link between AMPA-R phosphorylation and MC. We then showed that information load regulates AMPA-R phosphorylation within the hippocampus, and that an overload condition associated with impaired memory is paralleled by a lack of AMPA-R phosphorylation. Accordingly, we showed that in conditions of high load, but not of low load, the pharmacological inhibition of the NMDA-CaMKII-PKA pathways within the hippocampus prevents memory as well as associated AMPA-R phosphorylation. These data provide the first identified molecular mechanism that regulates MC.

Expression of PD-1 Molecule on Regulatory T Lymphocytes in Patients with Insulin-Dependent Diabetes Mellitus.

Type 1 diabetes is caused by autoreactive T cells that destroy pancreatic beta cells. Animal models suggested that a CD4⁺CD25⁺ population has a regulatory function capable of preventing activation and effector functions of autoreactive T cells. However, the role of CD4⁺CD25high T cells in autoimmunity and their molecular mechanisms remain the subject of investigation. We therefore evaluated T regulatory cell frequencies and their PD-1 expression in the peripheral blood of long-standing diabetics under basal conditions and after CD3/CD28 stimulation. Under basal conditions, the percentages of T regulatory cells were significantly higher while that of T effector cells were significantly lower in patients than in controls. The ratio of regulatory to effector T cells was higher in patients than that in controls, suggesting that T regulatory cells were functional in patients. Percentages of total PD-1⁺, PD-1low and PD-1high expressing T regulatory cells did not change in patients and in controls. After stimulation, a defect in T regulatory cell proliferation was observed in diabetics and the percentages of total PD-1⁺, PD-1low and PD-1high expressing cells were lower in patients. Our data suggest a defective activation of T regulatory cells in long-standing diabetics due to a lower expression of PD-1 on their surface.

Phosphorylation of S845 GluA1 AMPA receptors modulates spatial memory and structural plasticity in the ventral striatum.

The function of AMPA receptors phosphorylation in synaptic plasticity has been dissected in many in vitro models but its role and dynamics on experience-dependent plasticity are still unclear. Here we studied the effects of AMPA receptor manipulations in the ventral striatum, where glutamatergic transmission is known to mediate spatial memory. We first demonstrate that intra-ventral striatal administrations of the AMPA receptors blocker, NBQX, dose dependently impair performance in the Morris water maze. We also report that spatial learning induced a time-limited increase in GluA1 phosphorylation in this same brain region. Finally, through focal, time-controlled ventral striatal administrations of an RNA aptamer interfering with GluA1-S845 phosphorylation, we demonstrate that phosphorylation at this site is a necessary requirement for spatial memory formation and for the synaptic remodeling underlying it. These results suggest that modulation of AMPA receptors by S845 phosphorylation could act as an essential starting signal leading to long-term stabilization of spatial memories.

Altered B cell homeostasis and toll-like receptor 9-driven response in type 1 diabetes carriers of the C1858T PTPN22 allelic variant: implications in the disease pathogenesis.

Type 1 diabetes is an autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Among the genetic variants associated with type 1 diabetes, the C1858T (Lyp) polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene alters the function of T cells but also of B cells in innate and adaptive immunity. The Lyp variant was shown to diminish interferon production and responses upon Toll-like receptor stimulation in macrophages and dendritic cells, possibly leading to uncontrolled infections as triggers of the diabetogenic process. The aim of this study was to unravel the yet uncharacterized effects that the variant could exert on the immune and autoimmune responses, particularly regarding the B cell phenotype, in the peripheral blood lymphocytes of diabetic patients and healthy controls in basal conditions and after unmethylated bacterial DNA CpG stimulation. The presence of the Lyp variant resulted in a significant increase in the percentage of transitional B cells in C/T carriers patients and controls compared to C/C patients and controls, in C/T carrier patients compared to C/C controls and in C/T carrier patients compared to C/C patients. A significant reduction in the memory B cells was also observed in the presence of the risk variant. After four days of CpG stimulation, there was a significant increase in the abundance of IgM+ memory B cells in C/T carrier diabetics than in C/C subjects and in the groups of C/T carrier individuals than in C/C individuals. IgM- memory B cells tended to differentiate more precociously into plasma cells than IgM+ memory B cells in heterozygous C/T subjects compared to the C/C subjects. The increased Toll-like receptor response that led to expanded T cell-independent IgM+ memory B cells should be further investigated to determine the putative contribution of innate immune responses in the disease pathogenesis.