Identification of a tumor-specific allo-HLA-restricted γδTCR.

γδT cells are key players in cancer immune surveillance because of their ability to recognize malignant transformed cells, which makes them promising therapeutic tools in the treatment of cancer. However, the biological mechanisms of how γδT-cell receptors (TCRs) interact with their ligands are poorly understood. Within this context, we describe the novel allo-HLA-restricted and CD8α-dependent Vγ5Vδ1TCR. In contrast to the previous assumption of the general allo-HLA reactivity of a minor fraction of γδTCRs, we show that classic anti-HLA-directed, γδTCR-mediated reactivity can selectively act on hematological and solid tumor cells, while not harming healthy tissues in vitro and in vivo. We identified the molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 as the essential determinant for recognition and describe the critical role of CD8 as a coreceptor. We conclude that alloreactive γδT-cell repertoires provide therapeutic opportunities, either within the context of haplotransplantation or as individual γδTCRs for genetic engineering of tumor-reactive T cells.

Comportamiento de las células nucleadas en los distintos tipos de derrame pleural / Behaviour of nucleated cells in various types of pleural effusion

Introducción. El conocimiento del comportamiento de los componentes celulares del líquido pleural puede ayudar a enfocar el diagnóstico diferencial de un derrame pleural. El objetivo es evaluar su composición en los distintos tipos de derrames y valorar si proporciona información clínica relevante. Pacientes y métodos. Estudio observacional, transversal y retrospectivo en el que se analiza el componente celular de derrames pleurales de diversa etiología. Los derrames se clasificaron como neutrofílicos, linfocíticos (≥50% de cada uno de ellos), eosinofílicos (≥10%) o mesoteliales (>5%) y se agruparon en 6 categorías diagnósticas. Resultados. Se estudiaron 1.467 pacientes (354 insuficiencia cardiaca; 59 otros trasudados; 349 paraneumónicos; 133 tuberculosos; 397 neoplásicos y 175 otros exudados). El predominio celular fue linfocítico en la insuficiencia cardiaca (44,4%), paraneumónicos no complicados (29,2%), tuberculosis (88%) y neoplasias (49,6%); neutrofílico en los paraneumónicos (57%) y neoplásicos (9,6%); eosinofílico en las neoplasias (6,3%) y mesotelial en las tuberculosis (12%). Las etiologías más frecuentes con un recuento linfocitario ≥80% fueron tuberculosis (35,1%) y neoplasias (23,3%). Los parámetros con mayor capacidad discriminante fueron: leucocitos (trasudados: AUC 0,835) y porcentaje de neutrófilos (empiemas: AUC 0,906 y paraneumónicos complicados + empiemas: AUC 0,907). Conclusiones. Los recuentos de células nucleadas ayudan a enfocar la etiología del derrame pleural, ya que cada etiología suele tener un predominio celular característico. El porcentaje de células nucleadas en el líquido pleural no puede descartar tuberculosis si existe un recuento elevado de células mesoteliales, ni un derrame paraneumónico ante un predominio linfocítico, o malignidad con un recuento de linfocitos ≥80% (AU)

Introduction. To know the behavior of cellular components of pleural fluid can help focus the differential diagnosis of a pleural effusion. Our objective was to assess their composition in different types of pleural effusions and assess whether it provides relevant clinical information. Patients and methods. Observational, cross-sectional and retrospective study in which the cellular components of pleural effusions of different etiology were analyzed. Pleural effusions were classified as neutrophilic, lymphocytic (≥50% of each one of them), eosinophilic (≥10%) or mesothelial (>5%) and were grouped into six diagnostic categories. Results. 1.467 patients were studied (354 heart failure; 59 other transudates; 349 paraneumonic; 133 tuberculous; 397 malignant and 175 other exudates). The predominance cell was lymphocytic in heart failure (44,4%), uncomplicated parapneumonic (29,2%), tuberculosis (88%) and malignant (49,6%); neutrophilic in parapneumonic (57%) and malignant (9,6%); eosinophilic in malignant (6,3%) and mesotelial in tuberculosis (12%). The most frequent etiologies with lymphocyte count ≥80% were tuberculosis (35,1%) and malignant (23,3%). Parameters with higher discriminating accuracy were: leukocytes (transudates: AUC 0,835) and percentage of neutrophils (empyemas: AUC 0,906 and complicated parapneumonic+empyemas: AUC 0,907). Conclusions. Nucleated cell counts will help focus the etiology of pleural effusions, since each etiology often have a characteristic cell predominance. The percentage of nucleated cells in pleural fluid not ruled out tuberculosis if there is a high count of mesothelial cells, nor a parapneumonic effusion with lymphocytic predominance, or malignancy with ≥80% lymphocytes (AU)

Behaviour of nucleated cells in various types of pleural effusion. / Comportamiento de las células nucleadas en los distintos tipos de derrame pleural.

INTRODUCTION: To know the behavior of cellular components of pleural fluid can help focus the differential diagnosis of a pleural effusion. Our objective was to assess their composition in different types of pleural effusions and assess whether it provides relevant clinical information. PATIENTS AND METHODS: Observational, cross-sectional and retrospective study in which the cellular components of pleural effusions of different etiology were analyzed. Pleural effusions were classified as neutrophilic, lymphocytic (≥50% of each one of them), eosinophilic (≥10%) or mesothelial (>5%) and were grouped into six diagnostic categories RESULTS: 1.467 patients were studied (354 heart failure; 59 other transudates; 349 paraneumonic; 133 tuberculous; 397 malignant and 175 other exudates). The predominance cell was lymphocytic in heart failure (44,4%), uncomplicated parapneumonic (29,2%), tuberculosis (88%) and malignant (49,6%); neutrophilic in parapneumonic (57%) and malignant (9,6%); eosinophilic in malignant (6,3%) and mesotelial in tuberculosis (12%). The most frequent etiologies with lymphocyte count ≥80% were tuberculosis (35,1%) and malignant (23,3%). Parameters with higher discriminating accuracy were: leukocytes (transudates: AUC 0,835) and percentage of neutrophils (empyemas: AUC 0,906 and complicated parapneumonic+empyemas: AUC 0,907). CONCLUSIONS: Nucleated cell counts will help focus the etiology of pleural effusions, since each etiology often have a characteristic cell predominance. The percentage of nucleated cells in pleural fluid not ruled out tuberculosis if there is a high count of mesothelial cells, nor a parapneumonic effusion with lymphocytic predominance, or malignancy with ≥80% lymphocytes.

Xpert® MTB/RIF in pleural fluid for the diagnosis of tuberculosis.

An automated nucleic acid amplification assay that simultaneously identifies Mycobacterium tuberculosis and rifampicin resistance, the Xpert® MTB/RIF test, has undergone extensive evaluation in sputum samples. Our aim was to define its diagnostic accuracy when performed on pleural fluid specimens. In 67 patients with pleural effusions, of whom half had tuberculous pleuritis, Xpert yielded 15% sensitivity and 100% specificity in the detection of tuberculosis (TB). Positive Xpert results tended to be more common in patients with microbiologically confirmed TB. Due to its low sensitivity, Xpert testing of pleural fluids has a limited role in the work-up of pleural effusions.

Diagnostic value of interleukin-12 p40 in tuberculous pleural effusions.

The diagnosis of tuberculous pleural effusion (TBPE) is frequently problematic. Several markers of TBPE in pleural fluid have been evaluated, with different results. Pleural effusions from 96 patients were classified on the basis of definitive diagnosis as tuberculous (n = 39), neoplastic (n = 42) or parapneumonic (n = 15). Adenosine deaminase (ADA), ADA isoform ADA-2, interferon (IFN)-gamma, CD3(+)/DR(+) T-lymphocytes and interleukin (IL)-12 p40 were determined in all 96 effusions. The efficiency of IL-12 p40 for diagnosis of TBPEs was evaluated, in comparison with those of the other parameters, by comparing the areas under their receiver operating characteristics. With the threshold value of 550 pg.mL(-1), IL-12 p40 had a sensitivity of 92.3% (36 out of 39) and specificity of 70.2% (17 false positives). The misclassification rate of IL-12 p40 was significantly greater than those of ADA-2 and ADA. Among TBPEs, ADA correlated significantly with ADA-2, and IFN-gamma with ADA and IL-12 p40. Although tuberculous pleural effusions show values of interleukin-12 p40 that are significantly higher than neoplastic and parapneumonic fluids, this parameter is less efficient than adenosine deaminase, adenosine deaminase isoform 2 and interferon-gamma. Its routine determination is, accordingly, not justified.

Modulación negativa del TCR por un mecanismo de todo-o-nada / All-or-none downregulation of the T cell antigen receptor

El contacto del TCR con MHC/antígeno resulta en su modulación y desaparición de la superficie celular. Recientemente hemos descrito que este proceso ocurre por al menos dos mecanismos: uno es dependiente de transmisión de señales, predomina a bajas concentraciones de antígeno y resulta en la modulación en trans de moléculas de TCR no contactadas. El otro requiere contacto directo y es independiente de transmisión de señales. En este artículo describimos que el TCR es modulado en una forma discontinua, es decir las células estimuladas oscilan de un estado no-modulado a otro completamente modulado sin transición aparente por estados intermedios, cuando las células T son estimuladas con altas dosis de antígeno o de anticuerpos anti-TCR. El fenómeno se reproduce cuando un receptor quimérico, que contiene la parte extracelular y transmembránica de CD8 y el tallo citoplásmico de CD3 , es entre cruzado con anticuerpos inmovilizados a un substrato. Este proceso de modulación de "todo-o-nada" no requiere de transmisión de señales o de polimerización del citoesqueleto de actina. El análisis por microscopía confocal muestra que el anticuerpo estimulante es tomado del substrato y concentrado junto con la quimera en un polo de la célula donde se constituye un sitio de nucleación para la formación de vesículas endocíticas. El efecto de "todo-o-nada" puede explicarse por la concentración lenta del TCR o de la quimera, seguido de la internalización rápida de los receptores agregados (AU)

Triggering the TCR complex causes the downregulation of nonengaged receptors by a signal transduction-dependent mechanism.

Downregulation of the TCR complex is believed to be intimately tied to T cell activation, allowing serial triggering of receptors and desensitization of stimulated cells. We studied transfected and transgenic T cells expressing CD3zeta chimeras to demonstrate that ligand engagement of the TCR or chimeras causes comodulation of nonengaged receptors. Comodulation required protein tyrosine kinase activity but not trans-phosphorylation of nonengaged receptors. The TCR appears to be downregulated by at least two mechanisms. One mechanism requires direct engagement, independent of signaling. The second requires signaling and downregulates nontriggered receptors. These results shed new light on the process of TCR downregulation and indicate that the number of downregulated TCRs cannot be assumed to equal the number of engaged receptors.

Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3gamma.

Mature CD4(+) and CD8(+) T lymphocytes are believed to build and express essentially identical surface alphabeta T-cell receptor-CD3 (TCR.CD3) complexes. However, TCR.CD3 expression has been shown to be more impaired in CD8(+) cells than in CD4(+) cells when CD3gamma is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR.CD3 complex in human CD3gamma-deficient CD8(+) and CD4(+) T cells. The results indicated that the membrane TCR.CD3 complex of CD8(+) T lymphocytes was conformationally different from that of CD4(+) lymphocytes in the absence of CD3gamma. In addition, CD8(+), but not CD4(+), CD3gamma-deficient T lymphocytes were shown to contain abnormally glycosylated TCRbeta proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCRalpha). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4(+) and CD8(+) T lymphocytes in the intracellular control of alphabetaTCR. CD3 assembly, maturation, or transport that are revealed when CD3gamma is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.

Receptor engagement transiently diverts the T cell receptor heterodimer from a constitutive degradation pathway.

In the absence of ligand, the T cell receptor (TCR)/CD3 complex is continuously internalized and recycled to the cell surface, whereas receptor engagement results in its down-regulation. The present study shows that the TCR and CD3 components follow different fates accompanying their constitutive internalization. Although the CD3 moiety is recycled to the cell surface, the TCR heterodimer is degraded and replaced by newly synthesized chains. Since the TCR heterodimer cannot reach the cell membrane on its own, we propose a model in which recycling CD3 is transported along a retrograde pathway to the endoplasmic reticulum, where it associates with newly made TCR. Interestingly, engagement of the TCR.CD3 complex by superantigen resulted not only in the down-regulation of the TCR and CD3 components but also caused a transient stabilization of the TCR heterodimer. This suggests that TCR engagement diverts the TCR heterodimer from a degradation to a recycling pathway. Contrary to CD3, the intracellular fate of the TCR heterodimer is thus regulated, providing a mechanism for rapidly replacing nonfunctional TCR during intrathymic development of T cells.

Tuberculous pleurisy: a study of 254 patients.

OBJECTIVES: To determine the age at which tuberculous pleural effusions occur, the radiological and biochemical characteristics of the effusions, the sensitivities of the various diagnostic tests, and the utility of combining clinical, radiological, and analytic data in diagnosis. METHODS: We studied the case histories of 254 patients in whom tuberculous pleural effusions were diagnosed with certainty between January 1, 1989, and June 30, 1997, in a Spanish university hospital in a region with a high incidence of tuberculosis. RESULTS: The mean (+/-SD) age of the patients was 34.1+/-18.1 years, and 62.2% were younger than 35 years. The effusion was on the right side in 55.9% of patients, on the left side in 42.5% of patients, and on both sides in 1.6% of patients. In 81.5% of patients, less than two thirds of the hemithorax was affected. Associated pulmonary lesions were detected in 18.9% of patients, of whom 14.6% exhibited cavitation. In 93.3% of the effusions, more than 50% of leukocytes were lymphocytes, and almost all had the biologic characteristics of exudates (98.8% had high total protein contents, 94.9% had high cholesterol levels, and 82.3% had high lactate dehydrogenase levels). All but 1 effusion (99.6%) had an adenosine deaminase (ADA) concentration higher than 47 U/L, 96.8% (123/127) of the effusions had high ADA2 levels, and 89% (73/82) of the effusions had high interferon gamma levels. Adenosine deaminase 2 contributed 72.2%+/-12.5% (mean +/- SD) of total ADA activity. Total ADA activity was significantly correlated with ADA2 (r = 0.83) and with interferon gamma (r = 0.30) levels. Definitive diagnosis was based on the observation of caseous granulomas in pleural biopsy tissue samples in 79.8% of patients, on the results of biopsy cultures in 11.7% of patients, and on pleural effusion cultures in the remaining 8.5% of patients. Results of the tuberculin skin test were positive in only 66.5% of patients. CONCLUSIONS: In these patients, lymphocyte-rich exudative pleural effusions occurred, on average, at a young age, with no preference for either the right or the left side; normally affected no more than two thirds of the hemithorax; and were generally unaccompanied by pulmonary infiltrates. High ADA concentration was a highly sensitive diagnostic sign and was caused by a rise in ADA2 concentration. The most sensitive criterion based on pleural biopsy was the observation of caseous granulomas, and culture of biopsy material further increased overall sensitivity. Negative skin test results were no guarantee of the effusion being nontuberculous. This, together with the low mean age of the patients and the low frequency of associated pulmonary lesions, suggests that tuberculous pleural effusion is a primary form of tuberculosis in this region.