RESUMEN

BACKGROUND/AIM: The epidermal growth factor receptor (EGFR) is over-expressed in several types of cancer, and monoclonal antibody therapy has been the strategy that has shown the best results. This study focused on the construction of a humanized single chain antibody (huscFv) directed against EGFR (HER1). MATERIALS AND METHODS: The CDR grafting method was used to incorporate murine complementarity determining regions (CDRs) of cetuximab into human sequences. A dot blot assay was used to examine the affinity of the huscFv secreted by HEK293T for EGFR. The inhibitory effect on the viability of A549 cells was evaluated using the WST-1 assay. RESULTS: The incorporation of murine CDRs of cetuximab into human sequences increased the degree of humanness by 16.4%. The increase in the humanization of scFv did not affect the affinity for EGFR. Metformin had a dose-dependent effect, with an IC50 of 46 mM, and in combination with huscFv, the cell viability decreased by 45% compared to the 15% demonstrated by huscFv alone. CONCLUSION: The CDR grafting technique is efficient for the humanization of scFv, maintaining its affinity for EGFR and demonstrating its inhibitory effect when combined with metformin in A549 cells.

Asunto(s)

Cetuximab , Receptores ErbB , Metformina , Anticuerpos de Cadena Única , Animales , Humanos , Ratones , Células A549/efectos de los fármacos , Anticuerpos Monoclonales Humanizados/farmacología , Supervivencia Celular/efectos de los fármacos , Cetuximab/farmacología , Regiones Determinantes de Complementariedad/inmunología , Receptores ErbB/inmunología , Receptores ErbB/antagonistas & inhibidores , Células HEK293 , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/inmunología , Metformina/farmacología , Anticuerpos de Cadena Única/farmacología , Anticuerpos de Cadena Única/inmunología

RESUMEN

Horse serum antibodies have been used for greater than a century for the treatment and prophylaxis of infectious diseases and envenomations. Little is known, however, about the immunogenetic diversity that produces horse serum antibodies. Here, we employed next-generation sequencing for a first-in-kind comprehensive analysis of the equine B-cell repertoire. Nearly 45,000 and 30,000 clonotypes were obtained for the heavy-chain (IGH) and lambda light-chain (IGL) loci, respectively. We observed skewed use of the common subgroups IGHV2 (92.49%) and IGLV8 (82.50%), consistent with previous reports, but also novel use of the rare genes IGHV6S1 and IGLV4S2. CDR-H3 amino acid composition revealed different amino acid patterns at positions 106 and 116 compared to human, rabbit, and mouse, suggesting that an extended conformation predominates among horse CDR-H3 loops. Our analysis provides new insights regarding the mechanisms employed to generate antibody diversity in the horse, and could be applicable to the optimized design of synthetic antibodies intended for future therapeutic use.

Asunto(s)

Regiones Determinantes de Complementariedad/genética , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Caballos/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Animales , Regiones Determinantes de Complementariedad/inmunología , Caballos/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/inmunología , Ratones , Conejos

RESUMEN

INTRODUCTION: Chronic hepatitis B (CHB) is still a public health problem and its mechanism remains unclear. In this study, we detect the skewness of T cell receptor beta chain variable gene (TCR Vß) in peripheral blood lymphocytes (PBL) and the liver infiltrating lymphocytes (LIL) of patients with CHB; and hope to provide information for further research on the pathogenic mechanism of CHB. MATERIAL AND METHODS: Fifteen patients with CHB, ten healthy volunteers and three patients with liver cysts were recruited as the subjects. The usage of TCR Vß of PBL and LIL were measured and compared; the associations of the TCR Vß usage of PBL with some hematological indices, including human leukocyte antigen (HLA) alleles, percents of CD4+ and CD8+ T cells, sera levels of HBV-DNA and IFN-γ, were analyzed. RESULTS: In PBL, Vß12 and Vß13.1 were the highest predominant usage genes which usage frequencies were all 46.7%; Vß23 was the key limited usage gene (40.0%). In LIL, the mainly predominant and limited usage gene was Vß13.1 (73.3%) and Vß23 (46.7%), respectively. About half of the patients with CHB with HLA-DR9 or HLA-DR12 showed the predominant usage of Vß5.2 or Vß13.2. In patients with CHB, the percentage of CD4+ T cells was 33.41 ± 5.39 %, that of CD8+ T cells was 28.67 ± 6.77 %; the concentration of IFN-γ was 182.52 ± 44.16 pg/mL. Compared to the healthy controls, there were significant differences for these data (P < 0.05). Neither ALT nor HBV-DNA was relative to the usage of TCR Vß. CONCLUSIONS: PBL and LIL share the common sknewness of TCR Vß genes, which probably relates to some hematological indices. However, the roles of such similarities and associations in the development of CHB need further study.

Asunto(s)

Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Hepatitis B Crónica/genética , Hepatitis B Crónica/inmunología , Región Variable de Inmunoglobulina/genética , Hígado/inmunología , Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Adulto , Estudios de Casos y Controles , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Femenino , Subtipos Serológicos HLA-DR/genética , Subtipos Serológicos HLA-DR/inmunología , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/virología , Interacciones Huésped-Patógeno , Humanos , Región Variable de Inmunoglobulina/inmunología , Hígado/virología , Linfocitos/virología , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología

RESUMEN

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the VHCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca2+ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.

Asunto(s)

Regiones Determinantes de Complementariedad/genética , Proteínas HSP90 de Choque Térmico/genética , Melanoma Experimental/tratamiento farmacológico , Péptidos/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Adhesión Celular/genética , Adhesión Celular/inmunología , Movimiento Celular/genética , Regiones Determinantes de Complementariedad/inmunología , Proteínas HSP90 de Choque Térmico/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Invasividad Neoplásica/genética , Neuropéptidos/genética , Péptidos/administración & dosificación , Péptidos/inmunología , Receptores Acoplados a Proteínas G/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/inmunología , Proteína de Unión al GTP rac1/genética

RESUMEN

OBJECTIVE: To compare the differences of immunological characteristics between newborn and adults, we performed high-throughput sequencing to reveal the diversity of umbilical cord blood and adult peripheral blood at both T-cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) levels. STUDY DESIGN: High-throughput sequencing was performed to analyze the expression of TRB-CDR3 and IGH-CDR3 in circulating T and B cells isolated from 20 healthy adults, 56 pregnant women, and 40 newborns. RESULTS: Our results revealed different immunological characteristics between newborn and adults, such as distinctive complementarity determining region 3 (CDR3) lengths, usage bias of variable and joining segments, random nucleotide addition, a large number of unique CDR3 peptides, and a greater repertoire diversity. Moreover, each newborn had a distinctive TRB-/IGH-CDR3 repertoire that was independent of the maternal immune status. CONCLUSIONS: This study presents comprehensive, unrestricted profiles of the TRB/IGH-CDR3 repertoire of newborns, pregnant women, and healthy adults at a sequence-level resolution. Our data may contribute to a better understanding of the immune system of newborns and benefit the efficient application of umbilical cord blood transplantation in future.

Asunto(s)

Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Sangre Fetal , Secuenciación de Nucleótidos de Alto Rendimiento , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Análisis de Secuencia de ADN , Adulto , Regiones Determinantes de Complementariedad/sangre , Femenino , Humanos , Cadenas Pesadas de Inmunoglobulina/sangre , Recién Nacido , Embarazo , Receptores de Antígenos de Linfocitos T alfa-beta/sangre

RESUMEN

Short synthetic peptides corresponding to sequences of complementarity-determining regions (CDRs) from different immunoglobulin families have been shown to induce antimicrobial, antiviral and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). Presently, we studied the in vitro and in vivo antitumor activity of synthetic peptides derived from conserved CDR sequences of different immunoglobulins against human tumor cell lines and murine B16F10-Nex2 melanoma aiming at the discovery of candidate molecules for cancer therapy. Four light- and heavy-chain CDR peptide sequences from different antibodies (C36-L1, HA9-H2, 1-H2 and Mg16-H2) showed cytotoxic activity against murine melanoma and a panel of human tumor cell lineages in vitro. Importantly, they also exerted anti-metastatic activity using a syngeneic melanoma model in mice. Other peptides (D07-H3, MN20v1, MS2-H3) were also protective against metastatic melanoma, without showing significant cytotoxicity against tumor cells in vitro. In this case, we suggest that these peptides may act as immune adjuvants in vivo. As observed, peptides induced nitric oxide production in bone-marrow macrophages showing that innate immune cells can also be modulated by these CDR peptides. The present screening supports the search in immunoglobulins of rather frequent CDR sequences that are endowed with specific antitumor properties and may be candidates to be developed as anti-cancer drugs.

Asunto(s)

Antineoplásicos/química , Antineoplásicos/farmacología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/farmacología , Inmunoglobulinas/química , Melanoma/tratamiento farmacológico , Péptidos/farmacología , Animales , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regiones Determinantes de Complementariedad/inmunología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunoglobulinas/inmunología , Inmunomodulación , Células MCF-7 , Melanoma/inmunología , Melanoma/patología , Ratones , Péptidos/síntesis química , Péptidos/química , Relación Estructura-Actividad

RESUMEN

Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4(+) T cells from chagasic patients preferentially express T cell receptor (TCR) ß-chain variable region (Vß) 5. The aim of this work was to determine whether T cells expressing this particular Vß region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vß5·1 expressing CD4(+) T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jß region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4(+)Vß5·1(+) cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Enfermedad de Chagas/inmunología , Regiones Determinantes de Complementariedad/inmunología , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Linfocitos T CD4-Positivos/metabolismo , Enfermedad de Chagas/genética , Enfermedad de Chagas/metabolismo , Regiones Determinantes de Complementariedad/química , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Prueba de Histocompatibilidad , Humanos , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo

RESUMEN

Gangliosides are sialic acid-containing glycosphingolipids present in the plasma membrane of most mammalian cells. In humans, the expression of the N-glycolylated (Neu5Gc) variant of the sialic acid has been associated with malignant transformation, constituting therefore an attractive target for cancer immunotherapy. P3 monoclonal antibody (mAb) recognizes Neu5Gc-containing gangliosides, as well as sulfatides. Heavy chain CDR3 (H-CDR3) arginine residues have been shown to be crucial for ganglioside recognition, but less important for anti-idiotypic antibody binding. Here, we describe the effect on antibody reactivity of different mutations involving a single H-CDR3 acid residue. Substitution of glutamate 99 (Kabat numbering) by arginine, aspartate or serine residues resulted in no differences in anti-idiotype binding. However, the first mutation caused increased reactivity with the antigen, including a cytotoxic effect of the antibody on ganglioside-expressing cells previously unseen for the wild type antibody. Another antibody that recognizes N-glycolyl-GM3 ganglioside (GM3(Neu5Gc)), but not other glycolipids, named 14F7, exhibits also an arginine-enriched H-CDR3 and a complement-independent cell death activity. Unlike 14F7 mAb, the cytotoxicity of the P3 E(99)→R mutant antibody did not exclusively depend on ganglioside expression on tumor cells.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Gangliósido G(M2)/inmunología , Gangliósido G(M3)/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Mutación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Idiotipos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Datos de Secuencia Molecular , Unión Proteica/inmunología

RESUMEN

The aim of this study was to investigate HIV-1 molecular diversity and the epidemiological profile of HIV-1-infected patients from Ribeirão Preto, Brazil. A nested PCR followed by sequencing of a 302-base pair fragment of the env gene (C2-V3 region) was performed in samples from HIV-1-positive patients. A total of 45 sequences were aligned with final manual adjustments. The phylogenetic analyses showed a higher prevalence of HIV-1 subtype B in the studied population (97.8%) with only one sample yielding an F1 subtype. The viral genotyping prediction showed that CCR5 tropism was the most prevalent in the studied cohort. Geno2pheno analysis showed that R5 and CXCR4 prediction were 69% and 31%, respectively. There was no statistical significance, either in viral load or in CD4(+) T cell count when R5 and X4 prediction groups were compared. Moreover, the GPGR tetramer was the most common V3 loop core motif identified in the HIV-1 strains studied (34.1%) followed by GWGR, identified in 18.1% of the samples. The high level of B subtype in this Brazilian population reinforces the nature of the HIV epidemic in Brazil, and corroborates previous data obtained in the Brazilian HIV-infected population.

Asunto(s)

Linfocitos T CD4-Positivos/virología , Regiones Determinantes de Complementariedad/genética , Genes env , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/epidemiología , VIH-1/genética , Adolescente , Adulto , Secuencia de Bases , Brasil/epidemiología , Linfocitos T CD4-Positivos/inmunología , Estudios de Cohortes , Regiones Determinantes de Complementariedad/inmunología , Epidemias , Femenino , Genotipo , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/inmunología , VIH-1/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Receptores CCR5/genética , Receptores CCR5/inmunología , Receptores CXCR/genética , Receptores CXCR/inmunología , Carga Viral

RESUMEN

Detailed information on the immunological relevance of α-type anti-idiotypic antibodies is lacking after more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-human chimeric version of the B7 MAb, a polyreactive α-type anti-idiotypic antibody, generated against an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispecificity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this antibody might have immunoregulatory properties, and therefore we investigated the possible interaction of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition assays using flow cytometry indicated that this antibody is capable of binding the Fc γ receptor II (FcγRII). The recognition of FcγRII-expressing K562, Raji and Daudi human cell lines, together with the capability of inhibiting the binding of an anti-human FcγRII antibody to these cells, suggest that B7Y33 interacts with both the FcγRIIa and FcγRIIb isoforms. We evaluated the contribution to the binding of different surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the construction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the "HCDR4", located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by performing key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispecificity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that this phenomenon occurs in a non-classical way with the participation of both the variable and constant regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to two FcγRIIb molecules through the Fc and Fv regions, or simultaneously to FcγRIIb and another unknown antigen on B cells. The FcγRIIb has recently received great attention as an attractive target for therapies directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell malignancies.

Asunto(s)

Anticuerpos Antiidiotipos/inmunología , Especificidad de Anticuerpos/inmunología , Linfocitos B/inmunología , Receptores de IgG/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/genética , Línea Celular , Separación Celular , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Receptores de IgG/genética

RESUMEN

Our previous work showed that transporter associated with antigen processing 1 (TAP1)-/- (H-2b) mice rejected grafts from H-2b mice which display a normal density of class I major histocompatibility complex (MHC) molecules at the cell surface. Our results indicated that H-2b molecules themselves may be a target in this kind of rejection and that CD4+ T cells play a major role in this autoreactive process. Our data also suggested that TAP1-/- mice, in addition to the well-recognized phenotype of class I and CD8+ T-cell deficiency, present a functional alteration in their autoreactive CD4+ T-cell repertoires. In this model of inflammatory autoreactivity to modified self, we have analysed T-cell receptor (TCR) V-beta-J-beta (BV-BJ) usage by complementarity determining region 3 (CDR3) length spectratyping in splenocytes from naïve TAP1-/- mice and transplanted TAP1-/- mice that rejected B6 heart grafts or responded to synthetic self H-2Kb peptides. Importantly, oligoclonal T-cell expansions shared by different animals were detected in the peripheral T-cell repertoire of transplanted TAP1-/- mice. Such public expansions were also induced in vitro by H-2Kb peptides, suggesting that dominant class I peptides can induce preferential expansions of restricted T-cell populations during rejection. Some of these public T-cell expansions were also detected in transplanted mice even before in vitro stimulation with peptides, indicating that post-transplantation expansion of these populations had occurred in vivo. The functional activity of these T-cell populations awaits elucidation, as do the underlying mechanisms involved in the inflammatory autoreactive process, in TAP1-/- mice.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Autoantígenos , Linfocitos T CD4-Positivos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Rechazo de Injerto/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Animales , Presentación de Antígeno , Autoinmunidad , Proliferación Celular , Citotoxicidad Inmunológica , Antígenos H-2/inmunología , Trasplante de Corazón , Hibridación Fluorescente in Situ , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales

RESUMEN

The mechanisms underlying long-term acceptance of kidney allografts in humans under minimal or no maintenance immunosuppression are poorly understood. We analyzed the T-cell receptor (TCR) repertoires in circulating T cells of patients with long-term (> or = 9 years) renal allograft survival with (LTS-IS) and without immunosuppression (LTS-NoIS). T cells of LTS patients exhibited strongly altered TCR Vss usage, including an increased frequency of oligoclonality and a decreased frequency of polyclonality. All 3 LTS-NoIS and 12 of 16 LTS-IS patients demonstrated oligoclonality in at least three or more TCR V beta families, and the frequency of oligoclonality in these patients was significantly higher as compared to patients with well-functioning grafts at 3 years (p < 0.005 both), an uncomplicated course during the first year (p < 0.0001, both), acute rejection (p < 0.0001, both), chronic allograft nephropathy at 7 (p < 0.0001, both) or 13 years (p < 0.0001, both), dialysis patients (p < 0.0001, both) or healthy controls (p < 0.0001, both). In contrast to LTS patients, all other studied patient groups exhibited a polyclonal TCR repertoire. Our data indicate that TCR alteration is a common feature of long-term allograft outcome, which might be explained by clonal deletion, exhaustion of alloreactive T cells or predominant expression of particular T-cell subpopulations, such as regulatory T cells.

Asunto(s)

Regiones Determinantes de Complementariedad/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Riñón , Receptores de Antígenos de Linfocitos T/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Diálisis Renal , Factores de Tiempo , Trasplante Homólogo

RESUMEN

Using the same mouse strain and two Trypanosoma cruzi sub-populations (CA-I and RA) it is possible to induce pathology in different target tissues: skeletal muscle (CA-I) or sciatic nerve and spinal cord (RA). On the other hand, T cells are directly involved in tissue injury in a strain-dependent way, resembling the abnormalities of chronic Chagas' disease. In the present work, we examined the TCRBV repertoire and the CDR3 sequence polymorphism of T cells infiltrating spinal cord, sciatic nerve and skeletal muscle in chronically infected mice. The TCRBV9 segment was systematically over-represented in the target tissues for each T. cruzi strain: sciatic nerve and spinal cord in RA and skeletal muscle in CA-I-infected mice. The analysis of CDR3 sequence polymorphism in the same tissues showed a high proportion of identical TCRBV9 clones in RA-infected mice: 66.6% of the TCRBV9 clones found in sciatic nerve and spinal cord expressed one out of four major CDR3 rearrangements. Sequence identity was shared among clones from sciatic nerve and spinal cord, tissues that are also damaged by passive transfer of CD8 + TL. Those observations are consistent with an antigen driven T-cell expansion sequestered at the inflammation site and demonstrate -- for the first time -- the presence of an oligoclonal repertoire in the antigen recognition site of over-represented T cells in nervous system tissues in chronic Chagas' disease.

Asunto(s)

Enfermedad de Chagas/inmunología , Enfermedades Neuromusculares/parasitología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/parasitología , Trypanosoma cruzi/inmunología , Secuencia de Aminoácidos , Animales , Enfermedad de Chagas/parasitología , Células Clonales , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Músculo Esquelético/inmunología , Músculo Esquelético/parasitología , Enfermedades Neuromusculares/inmunología , Polimorfismo Genético , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nervio Ciático/inmunología , Nervio Ciático/parasitología , Médula Espinal/inmunología , Médula Espinal/parasitología , Linfocitos T/inmunología , Trypanosoma cruzi/genética

RESUMEN

Several approaches have been developed to reduce the human immune response to nonhuman antibodies. However, chimeric antibodies and humanized antibodies often have decreased binding affinity. We described a new approach for reducing the immunogenicity of chimeric antibodies while maintaining the affinity. This approach seeks to prevent the recognition of murine immunogenic peptides from the antibody variable region by human lymphocytes. Putative immunogenic epitopes in the variable region are identified and subjected to site directed mutagenesis to make them human and/or to break the amphipathic motifs. The R3 antibody, which blocks the epidermal growth factor (EGF) receptor, was used as a model system to test this approach. Four segments containing possible amphipathic epitopes were found in the heavy variable domain using the program AMPHI. Six amino acids within two of these segments were substituted by the corresponding residues from a homologous human sequence. No mutations were made in the murine light variable domain. Experiments in monkeys suggested that the "detope" R3 antibody was less immunogenic than its chimeric analogue. A search for possible amphipathic epitopes in the Kabat database revealed the presence of conserved patterns in the different families of variable region sequences, suggesting that the proposed method may be of general applicability.

Asunto(s)

Anticuerpos/genética , Anticuerpos/farmacología , Epítopos/inmunología , Ingeniería de Proteínas , Algoritmos , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Chlorocebus aethiops , Regiones Determinantes de Complementariedad/inmunología , Epítopos/química , Receptores ErbB/inmunología , Humanos , Inmunización , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Homología de Secuencia de Aminoácido