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1.
Int Immunol ; 18(7): 1139-45, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16751231

RESUMEN

Recombinant soluble multimeric forms of MHC class I molecules loaded with antigenic peptides (pMHC) have turned out to be particularly useful to detect and isolate specific T cells. These applications both rely on the oligomerization of pMHC monomers in order to compensate for their inherent low affinity for the TCR. In this study, we have evaluated the precise contribution of CD8-pMHC interaction on the specificity and sorting efficiency of pMHC multimers according to their degree of oligomerization. To this end several wild-type versus mutated pMHC complexes (A*0201, B*0701, B*0801, B*3501) carrying point mutations known to reduce (245V mutants) or to abrogate (227,8KA mutants) CD8-pMHC interaction and showing various degrees of valency have been used. We show that irrespective of the HLA allele tested, 245V mutation strongly improves the binding specificity and immunomagnetic sorting efficiency of pMHC multimers. Our results also indicate that the contribution of CD8 to the binding of pMHC multimers to specific CD8+ T cells is inversely correlated to the degree of pMHC oligomerization. Consequently, efficient staining or specific sorting of high-affinity T cells (i.e. CD8 independent) can only be achieved using 227,8KA pMHC complexes with low-order oligomerization.


Asunto(s)
Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Alelos , Sustitución de Aminoácidos , Antígenos CD8/química , Linfocitos T CD8-positivos/química , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/inmunología , Péptidos/química , Péptidos/genética , Unión Proteica/genética , Receptores de Antígenos de Linfocitos T/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología
3.
BMC Immunol ; 6: 17, 2005 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-16026627

RESUMEN

BACKGROUND: Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors. RESULTS: Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p

Asunto(s)
Presentación de Antígeno , Antígenos Virales/inmunología , Criopreservación , Citomegalovirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Antígeno HLA-A2/análisis , Leucocitos Mononucleares/inmunología , Fosfoproteínas/inmunología , Proteínas de la Matriz Viral/inmunología , Biomarcadores/análisis , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/inmunología , Antígeno HLA-A2/inmunología , Humanos , Inmunidad Celular , Laboratorios , Leucocitos Mononucleares/química , Fragmentos de Péptidos/inmunología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Método Simple Ciego , Manejo de Especímenes , Vacunación
4.
J Virol ; 79(13): 8131-41, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15956558

RESUMEN

Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Delta3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01- animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01- animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Productos del Gen env/inmunología , Depleción Linfocítica , Proteínas Oncogénicas de Retroviridae/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/sangre , Inmunofenotipificación , Macaca mulatta , Eliminación de Secuencia , Virus de la Inmunodeficiencia de los Simios/fisiología , Vacunas Virales , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología
5.
Immunol Lett ; 98(2): 208-15, 2005 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-15860220

RESUMEN

MHC tetramers are used to directly enumerate and visualize the antigen-specific T lymphocyte population of interest by flow cytometry, regardless of the T lymphocyte's functional capacity. Assay sensitivity can be hindered by non-specific binding activity, which is due to the inherent interactions of CD8 and MHC. Point mutations within the alpha3 loop of the HLA MHC class I heavy chain have been shown to reduce or abrogate MHC/CD8 interactions and also alleviate non-specific binding. This report compares the effects of two well-described mutations on the binding capacity and functional capacity of MHC tetramers in the H-2 MHC murine system. Tetramers folded with MHC mutated at either residue 227 or 245 of the class I heavy chain were compared to wild-type tetramer in binding studies using various antigen-specific, TCR-positive lymphocytes and cell lines. These experiments showed that the binding of wild-type and residue 245-mutated tetramer were comparable on CTL cultures, OT-1 splenocytes, and hybridomas. Both wild-type and 245-mutated tetramers' binding capacity was observed to be equally dependent on CD8 expression. Residue 227-mutated tetramer consistently bound antigen-specific CTL less efficiently, but in the absence of CD8 all three tetramers had similar binding capacity. In functional studies, 227-mutated tetramer had the greatest capacity to stimulate cytokine production in the absence of exogenous antigen addition. These experiments demonstrate that reduction of a tetramer's high avidity interaction with CD8 will not necessarily decrease the ability to stimulate the effector functions of activated T cells.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos/inmunología , Mutación/genética , Alanina/genética , Alanina/metabolismo , Animales , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Antígenos CD8/metabolismo , Células Cultivadas , Antígenos de Histocompatibilidad Clase I/química , Hibridomas/inmunología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Bazo/inmunología , Linfocitos T Citotóxicos/inmunología
6.
Nat Biotechnol ; 22(11): 1429-34, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15502816

RESUMEN

In this study we extend tetramerization technology to T-cell receptors (TCRs). We identified TCR alpha beta pairs in the absence of accessory molecules, ensuring isolation of high-affinity TCRs that maintain stable binding characteristics after tetramerization. Subtle changes in cognate peptide levels bound to the class I molecule were accurately reflected by parallel changes in the mean fluorescence intensity of cells that bound TCR tetramers, allowing us to accurately assess the binding affinity of a panel of peptides to major histocompatibility complex (MHC) class I. Using a TCR tetramer specific for the Mamu-A(*)01 allele, we identified animals expressing this restricting class I allele from a large cohort of outbred rhesus macaques. TCR tetramers should facilitate analysis of the MHC-peptide interface and, more generally, the design of immunotherapeutics and vaccines.


Asunto(s)
Inmunoensayo de Polarización Fluorescente/métodos , Genes MHC Clase I/inmunología , Ingeniería de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Animales , Complejo Antígeno-Anticuerpo/análisis , Reacciones Antígeno-Anticuerpo/inmunología , Células Cultivadas , Dimerización , Genes MHC Clase I/genética , Macaca mulatta , Complejos Multiproteicos/genética , Complejos Multiproteicos/inmunología , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/genética
7.
Hum Immunol ; 65(5): 507-13, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15172451

RESUMEN

Tracking antigen specific T cells with major histocompatibility complex (MHC) tetramers has provided us with insights into the dynamics of the adaptive immune system and holds great promise to aid in patient management and drug and vaccine development. Progress has been made primarily using MHC class I tetramers to monitor CD8(+) T cells, whereas corresponding efforts to stain CD4(+) T cells with class II tetramers have not been as successful. Two major reasons have been proposed for this lack of progress: (1). The frequency of antigen-specific CD4(+) T cells is lower than the frequency of CD8(+) T cells and (2). some, but not all, antigen- specific CD4(+) T cells can bind tetramer because of low functional avidity. In this study, we asked if CD4(+) T cells specific for common human viruses (e.g., influenza and Epstein-Barr) can be detected in healthy individuals previously exposed to them. We were able to clearly detect specific CD4(+) T cells in all donors after in vitro expansion of peripheral blood mononuclear cells. Furthermore, we observe a clear separation of tetramer negative and tetramer positive CD4(+) T cells in most samples similar to patterns commonly seen with class I tetramers. The data indicate that MHC class II tetramers can be used reliably for the identification of CD4(+) T cells specific for ubiquitous infectious agents in normal donors.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Fragmentos de Péptidos/inmunología , Adulto , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Fluoresceínas/química , Antígenos HLA-DR/química , Antígenos HLA-DR/inmunología , Hemaglutininas Virales/química , Hemaglutininas Virales/inmunología , Hemaglutininas Virales/farmacología , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/farmacología , Humanos , Interleucina-2/farmacología , Leucocitos Mononucleares/química , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Succinimidas/química
8.
Virology ; 301(2): 365-73, 2002 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-12359438

RESUMEN

The precise measurement of epitope-specific cytotoxic T lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected or vaccinated rhesus monkeys has been important in the evaluation of potential HIV vaccine strategies. This quantitation of CTL has been limited to date by the identification of only one dominant SIV/SHIV epitope in these monkeys. We have recently defined a Nef CTL epitope p199RY (YTSGPGIRY) that is recognized by CD8(+) T lymphocytes from all SIV/SHIV-infected Mamu-A*02(+) rhesus monkeys that have been evaluated. We now measure the frequency of p199RY-specific CD8(+) T lymphocytes in the peripheral blood of these monkeys with quantitative precision, using MHC class I/peptide tetramer staining and peptide-stimulated interferon-gamma Elispot assays. These epitope-specific CD8(+) T lymphocytes are present at a very high frequency and represent a significant proportion of the entire SIV- or SHIV-specific CD8(+) T lymphocyte population in SIV/SHIV-infected Mamu-A*02(+) rhesus monkeys. Knowledge of this dominant CTL epitope should prove valuable in the evaluation of HIV vaccine strategies using this animal model.


Asunto(s)
Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Epítopos Inmunodominantes/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Reguladoras y Accesorias Virales/inmunología , Animales , Biomarcadores , Complejo CD3 , Linfocitos T CD8-positivos/inmunología , Productos del Gen gag/inmunología , VIH/inmunología , Humanos , India , Macaca mulatta , Péptidos/inmunología
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