Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire.

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.

Persistence and enrichment of dominant T cell clonotypes in expanded tumor-infiltrating lymphocytes of breast cancer.

BACKGROUND: Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown promising results in cancer treatment, including breast cancer. However, clonal dynamics and clinical significance of TIL expansion ex vivo remain poorly understood. METHODS: We investigated T cell receptor (TCR) repertoire changes in expanded TILs from 19 patients with breast cancer. We compared TCR repertoire of TILs at different stages of expansion, including initial (2W TILs) and rapid expansion (REP TILs), and their overlap with formalin fixed paraffin embedded (FFPE) and peripheral blood. Additionally, we examined differences in TCR repertoire between CD4+ and CD8+ REP TILs. RESULTS: In descending order of proportion, average of 60% of the top 10% clonotypes of FFPE was retained in 2W TIL (60% in TRB, 64.7% in TRA). Among the overlapped clonotypes between 2W TILs and REP TILs, 69.9% was placed in top 30% of 2W TIL. The proportion of clonotypes in 2W TIL and REP TIL showed a significant positive correlation. CD4+ and CD8+ T cells show similar results in diversity and CDR3 length. CONCLUSIONS: Our study traces the changes in TILs repertoire from FFPE to 2W TIL and REP TIL and confirmed that clonotypes with high frequencies in TILs have a high likelihood of maintaining their priority throughout culture process.

MR1-restricted T cell clonotypes are associated with "resistance" to Mycobacterium tuberculosis infection.

T cells are required for protective immunity against Mycobacterium tuberculosis. We recently described a cohort of Ugandan household contacts of tuberculosis cases who appear to "resist" M. tuberculosis infection (resisters; RSTRs) and showed that these individuals harbor IFN-γ-independent T cell responses to M. tuberculosis-specific peptide antigens. However, T cells also recognize nonprotein antigens via antigen-presenting systems that are independent of genetic background, known as donor-unrestricted T cells (DURTs). We used tetramer staining and flow cytometry to characterize the association between DURTs and "resistance" to M. tuberculosis infection. Peripheral blood frequencies of most DURT subsets were comparable between RSTRs and latently infected controls (LTBIs). However, we observed a 1.65-fold increase in frequency of MR1-restricted T (MR1T) cells among RSTRs in comparison with LTBIs. Single-cell RNA sequencing of 18,251 MR1T cells sorted from 8 donors revealed 5,150 clonotypes that expressed a common transcriptional program, the majority of which were private. Sequencing of the T cell receptor α/T cell receptor δ (TCRα/δ) repertoire revealed several DURT clonotypes were expanded among RSTRs, including 2 MR1T clonotypes that recognized mycobacteria-infected cells in a TCR-dependent manner. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between mycobacteria-reactive MR1T clonotypes and resistance to M. tuberculosis infection.

Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

Recurrent exposures to a pathogenic antigen remodel the CD8+ T cell compartment and generate a functional memory repertoire that is polyclonal and complex. At the clonotype level, the response to the conserved influenza antigen, M158-66 has been well characterized in healthy individuals, but not in patients receiving immunosuppressive therapy or with aberrant immunity, such as those with juvenile idiopathic arthritis (JIA). Here we show that patients with JIA have a reduced number of M158-66 specific RS/RA clonotypes, indicating decreased clonal richness and, as a result, have lower repertoire diversity. By using a rank-frequency approach to analyze the distribution of the repertoire, we found several characteristics of the JIA T cell repertoire to be akin to repertoires seen in healthy adults, including an amplified RS/RA-specific antigen response, representing greater clonal unevenness. Unlike mature repertoires, however, there is more fluctuation in clonotype distribution, less clonotype stability, and more variable IFNy response of the M158-66 specific RS/RA clonotypes in JIA. This indicates that functional clonal expansion is altered in patients with JIA on immunosuppressive therapies. We propose that the response to the influenza M158-66 epitope described here is a general phenomenon for JIA patients receiving immunosuppressive therapy, and that the changes in clonal richness and unevenness indicate a retarded and uneven generation of a mature immune response.

Epidemiología y mecanismos de resistencia a carbapenemas en Pseudomonas aeruginosa: papel de los clones de alto riesgo en la multirresistencia / Epidemiology and carbapenem resistance mechanisms in Pseudomonas aeruginosa: Role of high-risk clones in multidrug resistance

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Relación entre resistencia y virulencia en bacterias de interés clínico / Relationship between resistance and virulence in bacteria of clinical interest

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Comportamiento fractal del repertorio T específico contra el alergeno Poa p9 / Fractal behavior of T specify repertory against Poa p9 alergeno

Antecedentes: basados en una analogía lingüística aplicada al repertorio inmune B y T específicos, el sistema inmune se puede caracterizar por su grado de complejidad como se hace con los lenguajes naturales. Material y métodos: este estudio es la aplicación de una ley matemática al repertorio inmune. Se aplicó la ley de Zipf-Mandelbrot, observada en los lenguajes naturales, al estudio del repertorio T específico contra el alergeno Poa p9. Resultados: se observó un comportamiento a escala de la ley en el repertorio específico para el alergeno obtenido de un paciente alérgico en presencia y ausencia de interferón á, y en los clones de células Th. Los repertorios T en presencia y ausencia de interferón á se comportan fractalmente, con una dimensión fractal de 0.661165 y 0.923895 respectivamente. Conclusión: el grado de complejidad del repertorio T contra el alergeno Poa p9 es una medida matemática objetiva y reproducible del repertorio inmune, la dimensión fractal es un parámetro matemático apropiado para caracterizar la fisiología del sistema inmune. Este comportamiento fractal puede tener implicaciones generales para la inmunología

Clonal variation within a mucosal isolate derived from a patient with Leishmania (Viannia) braziliensis infection

No transcurso de um periodo de 5 anos foram estudados 3 isolados de um paciente com leishmaniose mucosa recidivante causada pela Leishmania (Viannia) braziliensis e 7 clones de um desses isolados. Este estudo foi feito pela analise dos serodemas e zimodemas. Os resultados indicaram a ocorrencia de variacoes fenotipicas clonais. Oito marcadores isoenzimaticos demonstraram diferencas nos padroes eletroforeticos em Acetato de Celulose (AC), bem como em camada fina de amido. Da mesma forma foram constatadas diferencas em um painel de anticorpos monoclonais especificos e subespecificos. Nossas observacoes indicam ainda que a leishmania (Viannia) braziliensis esta composta por subpopulacoes de parasitas com caracteristicas bioquimicas e antigenicas peculiares.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation

Anticuerpos monoclonales y clones de linfocitos T contra antigenos HLA: producción y caracterización / Monoclonal antibodies and T-cell clones against HLA antigens: production and characterization

Inmunizando ratones Balb/c con líneas linfoblastoides humanas se produjeron y caracterizaron 21 anticuerpos monoclonales de los cuales 12 parecen estar reconociendo un determinante HLA polimórfo; 4 de ellos reconecen un epítope común a los antígenos Clase I y otro reconece moléculas HLA Clase II. Estos resultados se confirmaron en ensayos de ELISA, citotoxicidad, inmunofluorescencia e inmunopreciptación y la clase y subclase de las inmunoglobulinas se determinó por inmunodifusión de Ouchterlony. Se generaron además 22 clones de células T aloreactivas, por dilución limitante de linfocitos de un donante estimulados en cultivo mixto con linfocitos alogeneicos de sangre periférica; 5 de estos clones se caracterizaron detalladamente. Evaluando su capacidad para proliferar ante un panel de líneas linfoblastoides humanas, se encontró que reconecen las especificidades HLA-A9, A23, A29-31, Bw62 y DR7. Los resultados obtenidos parecen indicar que la generación de clones de células T humanas aloreactivas es mucho más eficiente que la producción de anticuerpos monoclonales múridos en cuanto a la definición de variantes polimórficas de los antígenos HLA

Producción y análisis de proteínas recombinantes obtenidas de la cepa lisogénica Ro-531

La expresión del cDNA que codifica al antígeno Ro, fue evaluada usando la clona Ro=531 gt11 cuyos productos de traducción fueron inmunorreconocidos por un suero anti-Ro. La producción de proteína Ro recombinante, fue inducida en la cepa lisogénica E. coli Y1089. La antigenicidad de la proteína fue probada por Western blot y por ELISA. En la primera prueba, 15 de los 20 sueros anti-Ro positivos presentaron fuerte reactividad a la proteína de funsión Ro y 4 de los 20 sueros controles, mostraron reactividad débil. Por la técnica de ELISA, se observó una reacción más específica, ya que 15 de los 20 sueros anti-Ro positivos exhibieron afinidad por la proteína Ro recombinante y ninguno de los controles presentó falsos positivos