Immune cell kinetics and antibody response in COVID-19 patients with low-count monoclonal B-cell lymphocytosis.

Low-count monoclonal B-cell lymphocytosis (MBLlo ) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID-19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID-19 of MBLlo versus non-MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID-19 patients (74 MBLlo and 262 non-MBL), who had not been vaccinated against SARS-CoV-2, over a period of 43 weeks since the onset of infection, using high-sensitivity flow cytometry. Plasma levels of anti-SARS-CoV-2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID-19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non-MBL patients, MBLlo COVID-19 patients presented higher neutrophil counts, together with decreased pre-GC B-cell, dendritic cell, and innate-like T-cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti-SARS-CoV-2-specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID-19 patients share immune profiles previously described for patients with severe SARS-CoV-2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non-MBL patients.

Overrepresentation of influenza A(H1N1)pdm09 virus among severe influenza cases in the 2011/12 season in four European countries.

In France, Ireland, Spain and the United Kingdom, the influenza season 2011/12 started in the final weeks of 2011 and has been dominated by influenza A(H3) viruses with minimal circulation of influenza A(H1N1) pdm09 and B viruses. A relatively greater proportion, however, of influenza A(H1N1)pdm09 viruses were reported in hospitalised laboratory-confirmed influenza cases in four countries. Compared to the season 2010/11, the proportion of subtype A(H3) among hospitalised cases has increased, associated with a larger proportion of cases in the youngest and oldest age groups.

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαß-Large Granular Lymphocytic Leukemia.

Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor ß chain (TRBC1) expression for assessing Tαß-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαß cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1- ratios vs. total Tαß cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1- Tαß-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1- Tαß-LGL ranged between 0.36 and 571 cells/µL (3.2-91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/µL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVß families, the CD28- effector-memory and terminal-effector polyclonal Tαß cells ranged between 0 and 25 TRBC1+ or TRBC1- cells/µL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1- cells/µL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαß-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαß cells. However, in the absence of lymphocytosis or in the case of TαßCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαß-LGL-expressing individual TCRVß families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell Clonality: Standardization of Sample Preparation and Diagnostic Implementation.

A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor ß-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαß-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαß-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1- monoclonal and polyclonal Tαß-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1- ratios within different Tαß-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVß families, whereas highly-variable TRBC1+/TRBC1- ratios were observed in more mature vs. naïve Tαß-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαß-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαß-cells of ≤10-4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαß+ leukemia/lymphoma patients.

Specificity studies on cytolytic T lymphocytes directed against murine leukemia virus-induced tumors. Analysis of monoclonal cytolytic T lymphocytes.

The specificities of cloned cytolytic T lymphocytes (CTL) were studied for the analysis of CTL populations generated against murine leukemia viruses (MuLV) in H-2 congenic BALB/c (H-2d) and BALB.B (H-2b) mice. In particular, CTL generated in response to tumors induced by Gross MuLV and Friend MuLV were studied; these tumors expressed virus-induced antigens that do not cross-react and that can be distinguished from each other. The systematic study of 92 CTL clones clearly indicated that MuLV-immune CTL were highly heterogeneous with respect to both the intensities of target cell lysis that they mediated and to their specificity of recognition of MuLV-induced tumor target cells. Various categories of CTL clones were identified, ranging from CTL clones tht were tightly H-2 restricted and specific for the immunizing tumor to CTL clones that displayed no discernible patterns of specificity and that attacked a large number of different target cells. In addition, the surface markers of these cloned CTL were defined, and the best conditions for their prolonged maintenance in culture were determined. The present data indicate that future efforts in the definition of target antigens recognized by tumor-specific CTL should be performed with monoclonal lymphocytes.

Viral specificity of H-2-restricted T killer cells directed against syngeneic tumors induced by Gross, Friend, or Rauscher leukemia virus.

Cytolytic T lymphocytes (CTL) were generated against murine tumors induced by Gross, Friend, or Rauscher leukemia virus (LV) in syngeneic mixed leukocyte-tumor cell cultures. Analogous to the patterns of specificity observed with antibodies to LV-induced cell surface antigens, CTL could be classified into two major groups of specificity. Tumor cells induced by Friend, Moloney, or Rauscher virus and positive for the FMR antigen were killed by syngeneic CTL immune to any one of these three LV; the same CTL, however, were incapable of killing syngeneic tumor cells induced by Gross LV. The converse was true for Gross LV-specific CTL: these CTL were specific for syngeneic tumor cells expressing the Gross virus-associated cell-surface antigen (GCSA), and not the FMR antigen. The H-2 specificities of the two groups of LV-immune CTL were also compared, because in both cases, CTL were restricted in their killing activity to H-2-identical tumor target cells. When CTL from single strains of mice were generated against syngeneic FMR- or GCSA-positive tumor cells, differences were observed with respect both to the requirement for the expression of compatible H-2K or H-2D specificities, and to the intensity of the CTL response in congenic mice of the H-2b, H-2d, and H-2k haplotypes.

Tumors induced by murine sarcoma virus contain precursor cells capable of generating tumor-specific cytolytic T lymphocytes.

Leukocyte fractions extracted from the tumor mass and the lymphoid organs of C57BL/6 (B6) mice carrying murine sarcoma virus-induced tumors contained primed cytolytic T-lymphocyte (CTL) precursor cells, in addition to active cytotoxic T cells. These leukocyte fractions gave a secondary response when stimulated in vitro with syngeneic tumor cells, generating large numbers of specific CTL. The activity of these CTL (H-2b) was apparently H-2-restricted, because it was ineffective on tumor targets bearing strongly cross-reacting tumor-specific antigens but with the H-2d haplotype. Furthermore, only H-2b cells bearing the Friend, Moloney, Rauscher-associated antigen, such as Rauscher leukemia virus-induced RBL-5 cells and Friend leukemia virus-induced HFL/b cells, were lysed efficiently. B male GV cells (H-2b cells induced by Gross leukemia virus) were not affected by the same CTL. We propose the existence of a dynamic state involving the migration of primed CTL precursor cells between the lymphoid organs and the tumor mass, as well as the differentiation of these precursor cells within the tumor mass into highly specific CTL.

Studies of cloned Friend erythroleukemia tumor cells. Modulation of the tumor-specific cytologic T lymphocyte response by infectious Friend virus production in vitro.

The HFL/b tumor cell line, induced by Friend erythroleukemia virus in BALB.B mice, was used to study the relation between virus production or nonproduction and the antigens recognized by Friend virus-specific cytolytic T lymphocytes (CTL). Analysis of clones and subclones of these tumor cells revealed a high degree of heterogeneity with respect to the production and release into culture fluids of infectious Friend virus in vitro, ranging from high levels to low or undetectable levels of virus production. Although no major differences could be detected among the antibody-defined serotypes of the various clones, the susceptibility of cells of individual HFL/b clones to attack by Friend virus-specific CTL varied widely, and those clones which produced large amounts of infectious virus provided the most sensitive target cells. It was also apparent that production of infectious Friend virus was inhibitory to CTL generation in syngeneic mixed leukocyte-tumor cell cultures. Friend erythroleukemia virus-producing cells thus appeared to interact in a complex manner with the host CTL response by modulating their production of infectious Friend virus.

Quantitative variations in the expression of H-2 antigens on murine leukemia virus-induced tumor cells can affect the H-2 restriction patterns of tumor-specific cytolytic T lymphocytes.

Comparative quantitative experiments were designed to study the expression of H-2Kd and H-2Dd antigens on three different leukemia cell lines induced by Gross murine leukemia virus (MuLV)in BALB/c (H-2d) mice. The H-2 restriction patterns of syngeneic cytolytic T lymphocytes (CTL) directed against Gross MuLV-induced tumors were correlated with these quantitations of H-2Kd and H-2Dd antigens, Our results obtained by quantitative absorption of monospecific antisera indicated that the three BALB/c tumor cell lines expressed different amounts of H-2Kd and H-2Dd antigens, with H-2Dd antigen showing the greatest variability in expression because it ranged from barely detectable levels to one-eighth the amount of H-2Dd antigen expressed on normal BALB/c spleen cells. The H-2 restriction patterns of Gross MuLV-specific CTL were directly affected by these quantitative modulations in the expression of H-2Kd and H-2Dd antigens, as revealed by three independent approaches: (a) inhibition of CTL activity by monospecific anti-H-2 sera in the absence of complement; (b)competitive inhibition of CTL-mediated cytotoxicity by the addition of excess tumor cells into the reaction mixture; and (c) analysis of CTL specificities using cloned CTL populations. Our results thus indicate that H-2 restriction of tumor-specific CTL activity can be directed at the target cell level by variations in the expression of H-2 antigens.

Fine mapping of epitopes by intradomain Kd/Dd recombinants.

11 intradomain recombinants between H-2Kd and H-2Dd were produced using an original technique based on in vivo recombination in Escherichia coli. After transfection into mouse L cells, all these recombinants were expressed at high levels on the cell surface. The specificities of 77 mAbs were examined on these cell lines. mAbs could be organized in 12 groups. In each group, a small number of amino acids participating in the recognized epitope(s) were identified. In a few instances, noncontinuous epitopes comprising amino acids belonging to different domains of the antigen were found. The data thus obtained are compatible with those produced in previous exon-shuffling experiments, but permit a much more precise definition of recognized epitope(s).

Experience and lessons from surveillance and studies of the 2009 pandemic in Europe.

Surveillance and studies in a pandemic is a complex topic including four distinct components: (1) early detection and investigation; (2) comprehensive early assessment; (3) monitoring; and (4) rapid investigation of the effectiveness and impact of countermeasures, including monitoring the safety of pharmaceutical countermeasures. In the 2009 pandemic, the prime early detection and investigation took place in the Americas, but Europe needed to undertake the other three components while remaining vigilant to new phenomenon such as the emergence of antiviral resistance and important viral mutation. Laboratory-based surveillance was essential and also integral to epidemiological and clinical surveillance. Early assessment was especially vital because of the many important strategic parameters of the pandemic that could not be anticipated (the 'known unknowns'). Such assessment did not need to be undertaken in every country, and was done by the earliest affected European countries, particularly those with stronger surveillance. This was more successful than requiring countries to forward primary data for central analysis. However, it sometimes proved difficult to get even those analyses from European counties, and information from Southern hemisphere countries and North America proved equally valuable. These analyses informed which public health and clinical measures were most likely to be successful, and were summarized in a European risk assessment that was updated repeatedly. The estimate of the severity of the pandemic by the World Health Organization (WHO), and more detailed description by the European Centre for Disease Prevention and Control in the risk assessment along with revised planning assumptions were essential, as most national European plans envisaged triggering more disruptive interventions in the event of a severe pandemic. Setting up new surveillance systems in the midst of the pandemic and getting information from them was generally less successful. All European countries needed to perform monitoring (Component 3) for the proper management of their own healthcare systems and other services. The information that central authorities might like to have for monitoring was legion, and some countries found it difficult to limit this to what was essential for decisions and key communications. Monitoring should have been tested for feasibility in influenza seasons, but also needed to consider what surveillance systems will change or cease to deliver during a pandemic. International monitoring (reporting upwards to WHO and European authorities) had to be kept simple as many countries found it difficult to provide routine information to international bodies as well as undertaking internal processes. Investigation of the effectiveness of countermeasures (and the safety of pharmaceutical countermeasures) (Component 4) is another process that only needs to be undertaken in some countries. Safety monitoring proved especially important because of concerns over the safety of vaccines and antivirals. It is unlikely that it will become clear whether and which public health measures have been successful during the pandemic itself. Piloting of methods of estimating influenza vaccine effectiveness (part of Component 4) in Europe was underway in 2008. It was concluded that for future pandemics, authorities should plan how they will undertake Components 2-4, resourcing them realistically and devising new ways of sharing analyses.

Start of the influenza season 2008-9 in Europe - increasing influenza activity moving from West to East dominated by A(H3N2).

The influenza season 2008-9 started in week 49 of 2008 and is so far characterised by influenza virus type A subtype H3N2. Isolates of this subtype that were tested proved susceptible to neuraminidase inhibitors, but resistant to M2 inhibitors. The circulating A(H3N2) viruses are antigenically similar to the component in the current northern hemisphere influenza vaccine.

Cytotoxic T lymphocytes against HIV.

HIV-1 infection has clearly been shown to induce a vigorous CTL response in infected people, and this response is present at a time when immune function otherwise is globally impaired. HIV-1-specific CTL are detectable both in peripheral blood and tissues of infected people, and are aimed at multiple viral proteins. The precise epitopes recognized by these CTL are now being defined, and the establishment of CTL clones should facilitate further functional analysis of these cells. However, the central question as to the clinical relevance of HIV-1-specific CTL remains. By analogy with animal model systems of virus infection, it is reasonable to postulate that HIV-1-specific CTL serve a protective role as a host defense. In this regard, in vitro data indicate that HIV-1-specific CTL can suppress viral replication, and longitudinal clinical studies indicate that the vigorous CTL activity seen in the early stages of infection declines with disease progression. Alternatively, the presence of HIV-1-specific CTL in tissues such as the lung and brain have to at least raise the possibility that these cells may be contributing to the pathologic consequences of infection. In addition, the relative protective effects of virus-specific CTL compared to other effector mechanisms such as ADCC and neutralizing antibodies remain to be determined. Nevertheless, recent data in the SIV vaccine model give reason for encouragement that a state of protective immunity can be achieved in AIDS-like illness caused by retroviruses. The search continues presently not only for the parameters which define protective immunity in HIV-1 infection, but also for the ideal HIV-1 immunogens to be used for vaccination of human populations.

Evidence for a cytotoxic T-lymphocyte alveolitis in human immunodeficiency virus-infected patients.

A T8 lymphocyte alveolitis occurs in HIV-positive patients, even in the absence of any lung infections or tumors. Using the monoclonal antibody (MAb) D44, the CD8+ T cells can be further subdivided into two functional subsets of cytotoxic T lymphocytes (CTL; CD8+, D44+) and suppressor T cells (CD8+, D44-). A dual fluorescence analysis of alveolar and peripheral lymphocytes has been used in HIV-positive patients without lung infections or tumors to reveal a dramatic increase in alveolar T8 lymphocytes (83%), compared to peripheral values (52%), which was mainly composed (89%) of CD8+ D44+ CTLs. Functional studies confirmed the cytolytic activity of these phenotypically defined alveolar CTLs on autologous alveolar macrophages used as target cells, excluding a natural killer-like activity. An immuno-enzyme analysis concomitantly revealed the co-expression of the p18 HIV antigen and the CD4 molecule on the autologous alveolar macrophages. These data suggest that CTL alveolitis occurs during HIV infection and is directed against HIV-infected alveolar macrophages which are presumably the targets of the locally recruited lung CTLs.

HIV-1 env, nef, and gag-specific T-cell immunity in mice: conserved epitopes in nef p27 and gag p25 proteins.

Cellular immunogenicity of env gp160, nef p27, and gag p55 proteins of human immunodeficiency virus type 1 (HIV-1) was studied in mice immunized with vaccinia virus recombinants. Proliferative responses of spleen cells were comparable against env gp160, nef p27, and gag p25 recombinant proteins. No specific activity was observed against gag p18 protein. Env, nef, and gag-specific T-cell lines were generated by repeated stimulation of immune spleen cells with recombinant HIV-1 proteins. They were CD4 positive, proliferative, and also cytotoxic against HIV-transfected target cells. Specificity of the T-cell response against nef and gag protein was analyzed with synthetic peptides. Peptides nef 15, nef 16, and gag AM-30 were, respectively, reactive in nef- and gag-specific proliferative and cytolytic assays. The three peptides described have a relatively conserved amino acid sequence among HIV isolates and appear broadly immunoreactive among species.

HIV-1 infection of lung alveolar fibroblasts and macrophages in humans.

We have studied the infected cell populations in the lungs of four human immunodeficiency virus type 1 (HIV-1) seropositive patients suffering from lymphocytic alveolitis or lymphocytic interstitial pneumonitis. Adherent cells were obtained by bronchoalveolar lavage (BAL) and were analyzed by various technical approaches. The cells considered here were alveolar macrophages and fibroblasts, and could be clearly identified morphologically and by the expression of specific cell-surface markers using monoclonal antibodies. The presence of HIV-1 in both of these cell types was established by serological, virological, and molecular procedures. Our results show that alveolar macrophages and fibroblasts are naturally infected in the lungs of HIV+ patients. Both cell types express the CD4 receptor molecule, in contrast to skin fibroblasts which are negative. Alveolar macrophages and fibroblasts thus may act as eventual HIV-1 reservoirs in vivo, and are probably involved in the induction of inflammatory reactions because they are targets for CD8 cytotoxic T lymphocytes (CTL).

Human immunodeficiency virus-related lymphocytic alveolitis.

We observed 276 HIV-infected patients to determine the frequency, degree, and clinical presentation of the lymphocytic alveolitis in different stages of HIV disease, and also to identify the lymphocyte subsets involved. In 154 patients with proved lung infections or tumors (group A), bronchoalveolar lavage fluid showed lymphocytosis in 78 percent of cases. In 122 subjects (31 AIDS and 91 HIV-infected non-AIDS patients) without evidence of lung tumor or infection (group B), lymphocytic alveolitis was seen in 72 percent of cases. In 61 of 88 (69 percent) group B lymphocytic patients, we observed respiratory symptoms or diffuse interstitial opacities; however, we also observed such alveolitis in 27 of 46 (59 percent) group B patients free of respiratory symptoms and abnormality of chest x-ray film. This alveolitis was seen not only in AIDS or ARC patients but also at earlier stages of HIV infection. T-lymphocyte analysis showed a large majority (40 to 93 percent) of CD8 positive lymphocytes in the 37 patients tested. A dual fluorescence analysis revealed, in 18 subjects, that those cells were phenotypically cytotoxic (CD8 + D44 +). These findings suggest that, regardless of HIV-infection stages and of opportunistic lung infections, a CD8-positive T-lymphocyte alveolitis may be present in HIV-infected patients and could be responsible for cough, dyspnea, interstitial pneumonitis, and abnormalities of pulmonary function tests.

Neuralgia del trigémino: estudio funcional mediante tensor de difusión de alta densidad como herramienta diagnóstica / Trigeminal Neuralgia: functional study using high density diffusion tensor imaging as a diagnostic tool

Objetivo: El objetivo del presente artículo es demostrar la alteración de la fracción de anisotropía (FA) en la neuralgia esencial del trigémino (NET). Materiales y métodos: Se evaluaron 10 pacientes con diagnóstico de neuralgia esencial del trigémino mediante secuencias de tensor de difusión de alta densidad e imágenes anatómicas 3D en un resonador de alto campo 3 Tesla. En todos los casos se localizaron los nervios. Las imágenes obtenidas se posprocesaron para realizar la tractografía y medir la FA en 20 nervios. Resultados: Se correlacionaron los hallazgos patológicos entre la medición de la FA y la clínica de los pacientes. De los 10 casos, 6 presentaron compresión neurovascular de lado con neuralgia y un valor de FA descendido con respecto al contralateral en rango normal; mientras que 2 mostraron compresión neurovascular bilateral, pero solo descenso del valor de FA del lado afectado clínicamente. En los otros 2 pacientes no se determinó compresión neurovascular, aunque en el lado con manifestación clínica neurálgica la FA se encontraba descendida. Conclusión: La realización de la difusión anisotrópica de alta densidad y la medición de la FA pueden ser una herramienta en la evaluación de la neuralgia esencial del trigémino, ya que es un método reproducible y seguro que permite estudiar la función del nervio.

Objective: The objective of this article is to demonstrate the alteration of the anisotropy factor (FA) in essential trigeminal neuralgia. Materials and methods: Ten patients with essential trigeminal neuralgia were studied with sequences of high density diffusion tensor and anatomic 3D images with a high-field 3 Tesla resonator. The nerves were located in all cases studied. The obtained images were post-processed to perform tractography and FA was measured in 20 nerves. Results: There was correlation of pathological findings between measuring FA and clinical presentation of the patients. Six of the ten patients studied with neurovascular compression at the neuralgia side had decreased FA values compared to contralateral normal range. Two of the ten patients showed bilateral neurovascular compression, but only abnormal values of FA were found at the clinically affected side. In the remaining two patients no neurovascular compression was determined; however the FA was lower at the clinical manifestation neuralgic side. Conclusión: The performing of high density anisotropic diffusion and the measurement of the FA may be useful in the evaluation of Trigeminal neuralgia, since it is a safe and reproducible method that allows the study of nerve function.