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1.
Biomaterials ; 118: 16-26, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27940380

RESUMO

Biomimetic materials that target the immune system and generate an anti-tumor responses hold promise in augmenting cancer immunotherapy. These synthetic materials can be engineered and optimized for their biodegradability, physical parameters such as shape and size, and controlled release of immune-modulators. As these new platforms enter the playing field, it is imperative to understand their interaction with existing immunotherapies since single-targeted approaches have limited efficacy. Here, we investigate the synergy between a PLGA-based artificial antigen presenting cell (aAPC) and a checkpoint blockade molecule, anti-PD1 monoclonal antibody (mAb). The combination of antigen-specific aAPC-based activation and anti-PD-1 mAb checkpoint blockade induced the greatest IFN-γ secretion by CD8+ T cells in vitro. Combination treatment also acted synergistically in an in vivo murine melanoma model to result in delayed tumor growth and extended survival, while either treatment alone had no effect. This was shown mechanistically to be due to decreased PD-1 expression and increased antigen-specific proliferation of CD8+ T cells within the tumor microenvironment and spleen. Thus, biomaterial-based therapy can synergize with other immunotherapies and motivates the translation of biomimetic combinatorial treatments.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Células Apresentadoras de Antígenos/imunologia , Células Artificiais/imunologia , Materiais Biomiméticos/uso terapêutico , Melanoma/imunologia , Melanoma/terapia , Implantes Absorvíveis , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Implantes de Medicamento/administração & dosagem , Sinergismo Farmacológico , Ácido Láctico/química , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Resultado do Tratamento
2.
J Phys Chem B ; 117(49): 15774-85, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-23941277

RESUMO

Dispersed IR three-pulse photon echoes due to the antisymmetric (ν3) stretch mode of N2O dissolved in H2O and 1-octanol at room temperature are reported and analyzed. The experimentally determined transition frequency-frequency correlation function (FFCF) in these two solvents is explained in terms of inertial solvent contributions, hydrogen bond network fluctuations, and, for octanol, the motions of the alkyl chains. The H2O hydrogen bond fluctuations result in 1.5 ps FFCF decay, in agreement with relaxation rates determined from photon echo based measurements of other aqueous solutions including salt solutions. In octanol, hydrogen bond fluctuations decay on a slower time scale of 3.3 ps and alkyl chain motions result in an inhomogeneous broadening contribution to the ν3 absorption spectrum that decays on a 35 ps time scale. Rotational reorientation of N2O is nearly 3 times faster in octanol as compared to water. Although the vibrational ν3 N2O absorption line shapes in water and octanol are similar, the line widths result from different coherence loss mechanisms. A hot band contribution in the N2O in octanol solution is found to have a significant effect on the echo spectrum due to its correspondingly stronger transition moment than that of the fundamental transition. The dephasing dynamics of the N2O ν3 stretch mode is of interest as a probe in ultrafast studies of complex or nanoconfined systems with both hydrophobic and hydrophilic regions such as phospholipids, nucleic acids, and proteins. These results demonstrate the value of the N2O molecule to act as a reporter of equilibrium fluctuations in such complex systems particularly due to its solubility characteristics and long vibrational lifetime.


Assuntos
Óxido Nitroso/química , Octanóis/química , Água/química , Azidas/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fótons , Espectrofotometria Infravermelho , Fatores de Tempo
3.
J Phys Chem A ; 115(16): 3917-23, 2011 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-21241060

RESUMO

Highly nonlinear pump fluence dependence was observed in the ultrafast one-color pump-probe responses excited by 38 fs pulses resonant with the E(22) transition in a room-temperature solution of (6,5) carbon nanotubes. The differential probe transmission (ΔT/T) at the peak of the pump-probe response (τ = 20 fs) was measured for pump fluences from ∼10(13) to 10(17) photons/pulse cm(2). The onset of saturation is observed at ∼2 × 10(15) photons/pulse cm(2) (∼8 × 10(5) excitons/cm). At pump fluences >4 × 10(16) photons/pulse cm(2) (∼1.6 × 10(6) excitons/cm), ΔT/T decreases as the pump fluence increases. Analogous signal saturation behavior was observed for all measured probe delays. Despite the high exciton density at saturation, no change in the E(22) population decay rate was observed at short times (<300 fs). The pump probe signal was modeled by a third-order perturbation theory treatment that includes the effects of inhomogeneous broadening. The observed ΔT/T signal is well-fit by a pump-fluence-dependent dephasing rate linearly dependent on the number of excitons created by the pump pulse. Therefore, the observed nonlinear pump intensity dependence is attributed to the effects of quasi-elastic exciton-exciton interactions on the dephasing rates of single carbon nanotubes. The low fluence total dephasing time is 36 fs, corresponding to a homogeneous width of 36 meV (290 cm(-1)), and the derived E(22) inhomogeneous width is 68 meV (545 cm(-1)). These results are contrasted with photon-echo-derived parameters for the E(11) transition.


Assuntos
Elétrons , Nanotubos de Carbono/química , Termodinâmica , Fatores de Tempo
4.
J Proteome Res ; 9(11): 5629-37, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-20836567

RESUMO

We present a novel cellular microarray assay using soluble peptide-loaded HLA A2-Ig dimer complexes that optimizes the avidity of peptide-HLA binding by preserving the molecular flexibility of the dimer complex while attaining much higher concentrations of the complex relative to cognate T-cell receptors. A seminal advance in assay development is made by separating the molecular T-cell receptor recognition event from the binding interactions that lead to antigen-specific cell capture on the microarray. This advance enables the quantitative determination of antigen-specific frequencies in heterogeneous T-cell populations without enumerating the number of cells captured on the microarray. The specificity of cell capture, sensitivity to low antigen-specific frequencies, and quantitation of antigenic T-cell specificities are established using CD8 T-cell populations with prepared antigen-specific CTL frequencies and heterogeneous T cells isolated from peripheral blood. The results demonstrate several advantages for high-throughput broad-based, quantitative assessments of low-frequency antigen specificities. The assay enables the use of cellular microarrays to determine the stability and flux of antigen-specific T-cell responses within and across populations.


Assuntos
Epitopos de Linfócito T/análise , Especificidade do Receptor de Antígeno de Linfócitos T , Análise Serial de Tecidos/métodos , Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-A2 , Ensaios de Triagem em Larga Escala , Humanos , Análise em Microsséries , Receptores de Antígenos de Linfócitos T , Sensibilidade e Especificidade , Linfócitos T Citotóxicos , Análise Serial de Tecidos/normas
5.
Scand J Immunol ; 68(1): 30-42, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18533931

RESUMO

Lipid overload, associated with metabolic disorders, occurs when fatty acids accumulate in non-adipose tissues. Cells of these tissues use major histocompatibility complex (MHC) class I molecules to present antigen to T cells in order to eliminate pathogens. As obesity is associated with impaired immune responses, we tested the hypothesis that the early stages of lipid overload with saturated fatty acids (SFA) alters MHC class I antigen presentation. Antigen presenting cells (APC) were treated with either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet. PA-treatment lowered APC lysis by activated cytotoxic T lymphocytes and inhibited APC ability to stimulate naïve T cells. Inhibition of immune responses with PA was due to a significant reduction in MHC class I surface expression, inhibition in the rate of APC-T-cell conjugation, and lowering of plasma membrane F-actin levels. OA-treatment had no effect on antigen presentation and upon exposure with PA, prevented the phenotypic effects of PA. OA-treatment conferred protection against changes in antigen presentation by accumulating fatty acids into triglyceride-rich lipid droplets of APC. Our findings establish for the first time a link between the early stages of lipid overload and antigen presentation and suggest that dietary SFA could impair immunity by affecting MHC I-mediated antigen presentation; this could be prevented, paradoxically, by accumulation of triglycerides rich in monounsaturated fatty acids.


Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Ácidos Graxos/farmacologia , Antígenos de Histocompatibilidade Classe I , Animais , Apresentação de Antígeno/imunologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Microscopia Confocal , Ácido Oleico/farmacologia , Ácido Palmítico/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
7.
J Helminthol ; 79(2): 177-8, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15946400

RESUMO

Echinostoma caproni tail loss was studied in vitro in the presence of the toxicant copper sulphate (CuSO4) in concentrations ranging from 1 to 10 000 mg l(-1) in standardized artificial spring water (pH 7.4, osmolarity 34 mOsm kg(-1) H2O, Ca(2+) 20 mg l(-1)) at 23 degrees C. Tail loss was also studied in the absence of toxicants during in vivo encystment of the cercariae in juvenile Biomphalaria glabrata. As the concentration of CuSO4 increased, the percentage of cercarial tail loss increased. By 2 h in 10 000 mg l(-1), 1000 mg l(-1) and 100 mg l(-1) CuSO4, 50%, 23% and 13%, respectively, of the cercariae had lost their tails. In the in vivo studies, by 1 h PI, 59+/-5% of cercariae had lost their tails and only 4+/-1% of the cercariae were actively swimming in the multi-well dishes. At 3 h PI, 72+/-3% of the cercariae began to form cysts within the snails.


Assuntos
Biomphalaria/parasitologia , Sulfato de Cobre/toxicidade , Echinostoma/fisiologia , Animais , Relação Dose-Resposta a Droga , Echinostoma/efeitos dos fármacos , Técnicas In Vitro , Estágios do Ciclo de Vida , Cauda/efeitos dos fármacos
8.
J Helminthol ; 78(4): 371-2, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15575997

RESUMO

The effects of various concentrations of copper sulphate were studied on in vitro encystment of Echinostoma caproni in a Locke's-artificial spring water (ASW) (1:1) medium. Cercariae were killed in 10,000 mg l(-1) CuSO4 in Locke's-ASW (1:1) within 24 h and extruded cystogenous material to produce an abnormal cyst wall. The 'emergency response' of encystment to high concentrations of copper reported for Parorchis acanthus cercariae did not occur in E. caproni. Concentrations of 1000 mg l(-1) and 100 mg l(-1) CuSO4 in Locke's-ASW (1:1) also killed the cercariae without encystment by 48 h. A concentration of 10 mg l(-1) CuSO4 in Locke's-ASW (1:1) allowed for normal in vitro encystment within 48 h and these cysts were capable of excystation in a trypsin-bile salts medium.


Assuntos
Anti-Helmínticos/farmacologia , Sulfato de Cobre/farmacologia , Echinostoma/efeitos dos fármacos , Animais , Echinostoma/crescimento & desenvolvimento , Echinostoma/ultraestrutura , Estágios do Ciclo de Vida/efeitos dos fármacos , Parasitologia/métodos
9.
J Helminthol ; 78(3): 277-9, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15469634

RESUMO

The effects of snail size on encystment of Echinostoma caproni cercariae in neonatal and juvenile Biomphalaria glabrata (NMRI strain) snails were studied. Encystment in neonatal (0.7-1.1 mm shell diameter) and juvenile (2-3 mm shell diameter) snails was compared 24 h post-infection (PI) following individual exposure of snails of each size to 1, 5, 10, 25 and 50 cercariae. Significantly more cysts were recovered from juveniles exposed to 1, 5, 10 and 50 cercariae than from neonatals with comparable exposure. Size of B. glabrata was a major factor in determining cyst burden in this planorbid. Survival of infected versus uninfected neonatals and juveniles was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 3, 6 and 7 days PI when compared to the uninfected controls. There was no significant decrease in the survival of juveniles exposed to 10 cercariae compared to uninfected controls at any time point. Snail size was a factor in mortality associated with echinostome cercarial penetration and encystment.


Assuntos
Biomphalaria/parasitologia , Echinostoma/fisiologia , Animais , Tamanho Corporal , Larva
10.
J Immunol ; 167(5): 2555-60, 2001 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-11509595

RESUMO

Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR zeta-chain and recruitment and phosphorylation of zeta-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.


Assuntos
Antígenos H-2/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Citotoxicidade Imunológica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Antígenos H-2/química , Humanos , Tolerância Imunológica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T Citotóxicos/metabolismo , Imunologia de Transplantes , Proteína-Tirosina Quinase ZAP-70
11.
J Exp Med ; 193(10): 1113-21, 2001 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-11369783

RESUMO

Lymphoproliferative diseases are characterized by massive accumulation of CD4(-)CD8(-)B220(+) (double-negative [DN]) T cells in peripheral organs. Although evidence indicates these cells are derived from mature autoreactive alpha/beta T cells, the significance of coreceptor downregulation is not known. In this study, we examined the role CD4 coreceptor plays in the survival of repeatedly stimulated T cells. CD4(+/+) and CD4(-/-) T cells from AND T cell receptor (TCR) transgenic mice exhibited similar phenotypes after antigenic stimulation, but the CD4(-/-) T cells survived in much larger numbers than the CD4(+/+) cells upon primary and secondary major histocompatibility complex (MHC)/peptide stimulation. Enhanced survival of CD4(-/-) T cells was due to decreased apoptosis rather than enhanced proliferation. Similarly, circumvention of the CD4/MHC interaction by using a surrogate TCR ligand that does not engage CD4 led to significant enhancement of CD4(+/+) cells than when stimulated with MHC/peptide. Finally, we generated DN B220(+) T cells using an in vitro model system and showed they were more tolerant to chronic stimulation than CD4(+/+) cells. Together, these results indicate that coreceptor engagement controls expansion of normal T cells. In the absence of coreceptor, T cells survive chronic stimulation and express B220 as seen in autoimmune lymphoproliferative diseases.


Assuntos
Autoimunidade/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores Imunológicos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Apoptose , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sobrevivência Celular , Antígenos Comuns de Leucócito/imunologia , Transtornos Linfoproliferativos/etiologia , Camundongos , Camundongos Transgênicos , Subpopulações de Linfócitos T/citologia
12.
Immunity ; 14(2): 135-43, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11239446

RESUMO

While activated T cells are known to have enhanced biological responses to antigen stimulation, the biophysical basis of this increased sensitivity remains unknown. Here, we show that, on activated T cells, the TCR avidity for peptide-MHC complexes is 20- to 50-fold higher than the TCR avidity of naive T cells. This increased avidity for peptide-MHC depends on TCR reorganization and is sensitive to the cholesterol content of the T cell membrane. Analysis of the binding data indicates the enhanced avidity is due to increases in cross-linking of TCR on activated T cells. Activation-induced membrane (AIM) changes in TCR avidity represent a previously unrecognized means of increasing the sensitivity of activated T cells to small amounts of antigen in the periphery.


Assuntos
Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Animais , Membrana Celular/imunologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Reagentes de Ligações Cruzadas , Dimerização , Antígenos H-2/química , Antígenos H-2/metabolismo , Técnicas In Vitro , Cinética , Lipídeos de Membrana/metabolismo , Camundongos , Modelos Moleculares , Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Receptores de Antígenos de Linfócitos T/química , Linfócitos T/metabolismo
13.
Blood ; 97(6): 1776-86, 2001 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11238120

RESUMO

The pp65(495-503) cytotoxic T-lymphocyte (CTL) epitope from cytomegalovirus (CMV) is universally recognized among CMV+ individuals who express an allele of the human leukocyte antigen A (HLA-A*0201). The relative binding affinity of the epitope to HLA-A*0201 is moderate, and its increased activity might prove beneficial in its use as a CTL epitope vaccine. A new approach to enhance the activity of T-cell epitopes is the use of positional scanning synthetic combinatorial libraries (PS-SCLs). Using a nonamer PS-SCL, the pp65(495-503) epitope was modified after screening a CMV-specific T-cell clone (TCC) (3-3F4) from which the native peptide sequence was derived. Two peptides with amino acid substitutions at P1, P3, P7, and P8 are between 10(3) and 10(4) more active than the native epitope. Although the native CTL epitope terminates as a free acid, both tetrasubstituted peptides only function as CTL epitopes when the carboxyl terminus is amidated. Selective substitution of the native sequence based on PS-SCL screening results identified 3 amidated monosubstituted and disubstituted peptides that are better recognized than the native epitope by TCCs from a cohort expressing HLA-A*0201. In vitro stimulation of peripheral blood mononuclear cells with each of the peptide epitope analogs stimulated memory CTLs, which recognized CMV-infected targets among a high percentage of CMV+ individuals. Binding studies of peptide analogs with HLA-Ig (immunoglobulin) dimers and 2 different TCCs correlated with in vitro lysis results. These data suggest that increasing the activity of CTL epitopes while maintaining broad recognition is possible, which holds promise for vaccine development in infectious disease and cancer.


Assuntos
Técnicas de Química Combinatória/métodos , Epitopos/química , Linfócitos T Citotóxicos/imunologia , Antígenos Virais/imunologia , Citomegalovirus/imunologia , Epitopos/imunologia , Humanos , Complexo Principal de Histocompatibilidade , Estrutura Molecular , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Fosfoproteínas/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , Relação Estrutura-Atividade , Proteínas da Matriz Viral/imunologia
14.
Int Rev Immunol ; 20(5): 535-46, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11890611

RESUMO

Activation induced cell death (AICD) is a major physiologic pathway that regulates T cell homeostasis. In CD4 T cells, AICD is mediated mainly through Fas/FasL interactions. Although TCR occupancy triggers AICD, the contribution of its tightly associated CD4 coreceptor to the process that leads to AICD is not known. Here we show that CD4 molecule plays an essential regulatory role of TCR dependent AICD. Loss of CD4 rendered activated 5kc T cell hybridoma resistant to AICD. The resistance of CD4 negative 5kc T cells to AICD was due to selective inhibition of FasL expression and it could be reversed by addition of recombinant FasL. Furthermore, a direct functional link between CD4 and FasL was demonstrated by induction of FasL upon CD4 crosslinking in a TCR independent fashion. The importance of CD4 interaction with MHC/peptide complex in mediating AICD was also evident in normal T cells that could survive chronic stimulation with anti-CD3 but died after short period of proliferation after stimulation with MHC/peptide. Thus it appears that AICD is controlled by the CD4 molecule via regulation of FasL expression. These findings have important implications for our understanding of mechanisms of peripheral tolerance as well as pathogenesis of autoimmune diseases.


Assuntos
Apoptose/imunologia , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Animais , Antígenos/metabolismo , Proteína Ligante Fas , Antígenos de Histocompatibilidade/metabolismo , Humanos , Ativação Linfocitária , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptor fas/metabolismo
15.
Curr Protoc Immunol ; Chapter 17: Unit 17.2, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18432743

RESUMO

The lack of high affinity reagents has made distinguishing T cells on the basis of antigen specificity difficult to accomplish. This unit provides protocols that utilize innovations in molecular design to permit construction of soluble multivalent MHC complexes (MHC-Ig dimers) with high avidity for cognate T cell receptors. MHC-Ig dimers display stable binding properties when they interact with antigen-specific T cells thus allowing their use in the staining of antigen-specific T cells by flow cytometry. Methods for constructing and detecting these MHC-Ig dimers are included along with protocols for applying their use for the quantitation of antigen-specific T cells.


Assuntos
Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoglobulinas/imunologia , Animais , Antígenos de Superfície/imunologia , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/imunologia , Dimerização , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Imunoglobulinas/genética , Imunoglobulinas/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
16.
J Infect Dis ; 183(2): 197-205, 2001 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11120926

RESUMO

To discern the T cell subtype associated with T cell differentiation, the expression of CD45RA and CD27 was measured from total CD8(high) cells and from human T cell lymphotropic virus type I (HTLV-I) Tax11-19 peptide-specific CD8(+) cells in peripheral blood lymphocytes of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Phenotypically defined memory and/or effector cells (CD45RA(-)CD27(+), CD45RA(+)CD27(-), and CD45RA(-)CD27(-)) were increased in HAM/TSP CD8(+) cells, compared with those of HTLV-I-seronegative healthy control subjects. The percentage of human leukocyte antigen (HLA)-DR-positive cells was also increased in CD8(+) cells of HAM/TSP, compared with those in HLA-DR(+)CD8(+) cells of healthy control subjects. HTLV-I provirus load correlated with the frequency of Tax11-19-specific CD8(+) cells. The high frequency of memory and/or effector type HTLV-I Tax11-19-specific CD8(+) cells suggests that continuous restimulation driven by HTLV-I antigens in vivo may be associated with the pathogenesis of HAM/TSP.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Produtos do Gene tax/imunologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Memória Imunológica , Paraparesia Espástica Tropical/imunologia , Adulto , Linfócitos T CD8-Positivos/classificação , Testes Imunológicos de Citotoxicidade , DNA Viral/sangue , Feminino , Citometria de Fluxo , Produtos do Gene tax/química , Antígeno HLA-A2/análise , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Humanos , Imunoglobulina G/metabolismo , Antígenos Comuns de Leucócito/análise , Ativação Linfocitária , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/fisiopatologia , Paraparesia Espástica Tropical/virologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Perforina , Proteínas Citotóxicas Formadoras de Poros , Provírus , Linfócitos T Reguladores/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Carga Viral
17.
Immunity ; 13(4): 529-38, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11070171

RESUMO

T cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I-restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells.


Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias Colorretais/imunologia , Epitopos de Linfócito T/imunologia , Antígenos H-2/metabolismo , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Alanina/metabolismo , Substituição de Aminoácidos , Animais , Antígenos de Neoplasias/metabolismo , Células Clonais , Citotoxicidade Imunológica/imunologia , Epitopos de Linfócito T/metabolismo , Feminino , Antígeno de Histocompatibilidade H-2D , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/síntese química , Ressonância de Plasmônio de Superfície , Subpopulações de Linfócitos T/imunologia
18.
Immunol Invest ; 29(2): 163-9, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10854185

RESUMO

To summarize, two novel approaches are currently being examined that allow for identification of antigen-specific T cells. A biochemical approach to generating soluble multivalent MHC complexes has been to generate tetrameric MHC complexes linked to avidin. We have also generated a general approach for producing soluble divalent versions of class I and class II MHC molecules, using Ig as a molecular scaffold. The experimental system described here outlines a general approach of using multivalent high affinity ligands to study cell-cell interactions, driven by multivalent ligand-receptor interactions. Our work indicates that divalent chimeric molecules are high-avidity analogs of proteins useful in probing and selectively regulating cellular responses.


Assuntos
Antígenos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoglobulinas/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos de Superfície/imunologia , Dimerização , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe II/química , Humanos , Imunoglobulinas/química , Técnicas In Vitro , Estrutura Quaternária de Proteína
19.
Immunity ; 11(2): 231-40, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10485658

RESUMO

Lateral diffusion of GFP-tagged H2Ld molecules in the ER membrane reports on their interaction with the TAP complex during synthesis and peptide loading. Peptide-loaded H2Ld molecules diffuse rapidly, near the theoretical limit for proteins in a bilayer. However, these molecules are retained in the ER for some time after assembly. H2Ld molecules, associated with the TAP complex, diffuse slowly, as does GFP-tagged TAP1. This implies that the association of H2Ld molecules with the TAP complex is stable for at least several minutes. It also suggests that the TAP complex is very large, perhaps containing hundreds of proteins.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Retículo Endoplasmático/metabolismo , Antígenos H-2/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Animais , Difusão , Proteínas de Fluorescência Verde , Antígenos H-2/química , Antígeno de Histocompatibilidade H-2D , Proteínas Luminescentes/química , Camundongos , Camundongos Knockout
20.
AIDS Res Hum Retroviruses ; 15(11): 1021-34, 1999 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-10445814

RESUMO

Development of an effective vaccine against HIV-1 will likely require the induction of a broad array of immune responses, including virus-specific CTLs and neutralizing antibodies. One promising vaccine approach involves live recombinant canarypox (CP)-based vectors (ALVAC) containing multiple HIV-1 genes. In phase I clinical trials in HIV-1-seronegative volunteers, the cumulative rate of detection of HIV-1-specific CTLs has been as high as 60-70%. In the present study, the factors associated with CTL responsiveness were evaluated in a subset of vaccinees immunized with a CP vector expressing portions of the gag, pro, and env genes of HIV-1 (ALVAC-HIV). CTL responses were detected in one of seven examined. While the responding individual had both CD4+ and CD8+ CTLs directed at multiple HIV-1 antigens, this response was not detectable 1 year after the last vaccination. In-depth characterization of "CTL nonresponders" showed that nonresponsiveness was not associated with defects in antigen processing or presentation. A generalized defect in CTL responsiveness was ruled out by parallel assays to detect CMV-specific CTLs from these same volunteers. Furthermore, HIV-1-specific memory CTLs were not detectable by peptide stimulation or by a novel technique for flow cytometric visualization of Gag epitope-specific T lymphocytes while HIV-1-seropositive donors frequently had 0.1-3% of CD8+ cells stain positively for this epitope (SLYNTVATL). Taken together, these results suggest that the lack of detectable HIV-1 CTLs in these volunteers was not due to classic MHC-linked nonresponsiveness.


Assuntos
Vacinas contra a AIDS/imunologia , HIV-1 , Modelos Imunológicos , Linfócitos T Citotóxicos/imunologia , Algoritmos , Animais , Apresentação de Antígeno , Avipoxvirus , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , DNA Viral/administração & dosagem , Citometria de Fluxo , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/genética , Soronegatividade para HIV , HIV-1/imunologia , Humanos , Camundongos
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