CD4 and CD8 T cell response to the rHSP60 from Klebsiella pneumoniae in peripheral blood mononuclear cells from patients with ankylosing spondylitis

Respuesta de linfocitos T CD4 y CD8 contra la rHSP60 de Klebsiella pneumoniae en células mononucleares de sangre periférica de pacientes con espondilitis anquilosante

Francisco Zambrano–Zaragoza,*** Ethel García–Latorre,* María Lilia Domínguez–López,* Mario Eugenio Cancino–Díaz,* Rubén Burgos–Vargas,*** Luis Jiménez–Zamudio*

* Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional.

** Unidad Académica de Ciencias e Ingenierías, Universidad Autónoma de Nayarit.

Correspondence and reprint request:
Ethel García–Latorre Ph D
Departamento de Inmunología, ENCB, IPN
Carpió y Plan de Ayala s/n Colonia Santo Tomás
11340, México, D.F.
Tel.: 5255 5729–6300, Ext. 62365. Fax: 5255 5342–3331
E–mail: elatorre@encb.ipn.mx E–mail: ethelagarcia@hotmail.com

ABSTRACT

Objective. To determine the processing pathways used by peripheral blood mononuclear cells (PBMC) and present the rHSP60Kp, and the T cell subpopulations involved in the response, in patients with ankylosing spondylitis (AS)

Methods. The lymphoproliferative response to the rHSP60Kp in PBMC from 14 HLA–B27 + AS patients and 15 B27 healthy controls was assessed by ³H–TdR incorporation. The processing pathways for the rHSP60Kp were analyzed by ³H–TdR incorporation in fresh PBMC from patients using homologous PBMC preincubated with the antigen and specific inhibitors: chloroquine, N–acetyl–L–leucil–L–leucil–L–nor–leucinal (LLnL) or brefeldin A (BFA), fixed with p–formaldehyde (fixed APC). The CD4+/CD8+ T cell subpopulation activated with the antigen was determined by three colours flow cytometry in PBMC from patients.

Results. Eight out of fourteen patients showed positive lymphoproliferative responses to the rHSP60Kp while none of the healthy controls responded (p < 0.012). In five patients S.I. was above 4.0. In these patients lymphoproliferation was lower when chloroquine and LLnL was used and it became negative with BFA, indicating that both pathways are used. CD4+ and CD8+ T cells populations expressed CD69 when activated by the rHSP60Kp.

Conclusions. Our results suggest that CD4 and CD8 T cells participate in the response to the rHSP60Kp in B27+ AS patients.

Key words. Ankylosing spondylitis. HLA–B27. Antigen processing. HSP60. Klebsiella pneumoniae.

Objetivo. Determinar las vías utilizadas por las células mononucleares de sangre periférica (CMSP) de pacientes con espondilitis anquilosante para procesar a la rHSPGO de Klebsiella pneumoniae (rHSPGOKp) y las subpoblaciones de linfocitos T involucrados en la activación.

Métodos. Se determinó la respuesta linfoproliferativa, por incorporación de ³H–TdR en CMSP, en presencia de la rHSPGOKp, en 14 pacientes con EA HLA–B27+y en 15 sujetos sanos HLA–B27–. La ruta de procesamiento y presentación de la rHSPGOKp se determinó por incorporación de ³H–TdR en las CMSP de los pacientes utilizando como células presentadoras a las CMSP homologas, preíncubadas con el antígeno y los inhibidores específicos: cloroquína, brefeldína A y N–acetil–L–leucil–L–leucil–L–nor–leucinal (LLnL), y fijadas con p–formaldehído. Se evaluaron las subpoblaciones de linfocitos T CD4+ y CD8+ que expresaron CD69, frente al antígeno, por citometría de flujo.

Resultados. Ocho de los 14 pacientes y ninguno de los sujetos sanos, tuvo respuesta linfoproliferativa positiva (IE > 3.0) contra la rHSPGOKp (p < 0.012). En cinco de los pacientes el I.E. fue superior a 4.0. En estos pacientes la linfoproliferación disminuyó cuando se utilizó cloroquína y LLnL, y se hizo negativa cuando se utilizó BFA, lo que indica que ambas vías son empleadas. Las subpoblaciones de linfocitos T (CD4+ y CD8+) expresaron CD69 frente al antigeno.

Conclusiones. Nuestros resultados sugieren que ambas poblaciones de linfocitos T: CD4+ y CD8+ participan en la respuesta a la rHSPGOKp.

Palabras clave. Espondilitis anquilosante. HLA–B27. Procesamiento de antígeno. HSP. Klebsiella pneumoniae.

INTRODUCTION

Ankylosing spondylitis (AS) is a chronic inflammatory disease of unknown aetiology in which immunogenetic and environmental factors are involved.¹ Immunogenetic factors are clearly represented by AS strong association to HLA–B27 (90% of AS patients carry the HLA–B27 gene) and the environment by the possible role of Klebsiella sp. and some other bacteria in AS pathogenesis.^2–9 We have previously reported both lymphoproliferative and IgG responses to electroeluted HSP60 from Klebsiella pneumoniae in HLA–B27 positive AS patients.^10–14 We also reported in a theoretical study that there is a nonapeptide in the HSP60Kp protein (residues 117–125) with high affinity for the B27 molecule.¹² Therefore, CD8 T cells could participate in the cellular response to this antigen and in AS pathogenesis.¹⁵

HLA–B27 physiologically binds antigenic peptides and thus presents them to CD8+ T cells. Single HLA–B27 restricted cytotoxic T lymphocytes have been described in AS patients with specificity for bacterial and viral peptides that show cross–reactivity with self antigens. Additionally, CD8 T cells have been reported to maintain inflammatory process even after the eradication of the microbial pathogen.^15,16

However, to support this idea it is important to know if the rHSP60Kp is able to activate CD4 + and/ or CD8⁺ T cells, and therefore it is necessary to demonstrate if this molecule could be processed by both the endocytic and cytosolic pathways. The aim of this work was to explore the processing pathways for the rHSP60Kp in AS patients and to determine the T cell subpopulations activated by the antigen.

MATERIAL AND METHODS

Patients and controls

Fourteen B27+ patients with AS, independently of the time of onset, diagnosed according to current diagnostic criteria²⁷ at the "Servicio de Reumatologia" from "Hospital General de Mexico", were included. All of them were receiving non–steroidal anti–inflammatory drugs. Fifteen healthy subjects, none of them B27+, were included as control group. After written consent was given by each patient or control subject, they were bled by venipuncture and PBMC were obtained by using Lymphoprep (Nycomed, Oslo, Norway).

Purification of rHSP60Kp

Recombinant HSP60Kp (rHSP60Kp) was obtained from E. coli transformed with the plasmid containing the corresponding gene, as we have previously reported,¹² and purified in the Ni–NTA column system as described by the supplier (QIAGEN Inc. Valencia CA, USA). Purity was analyzed by gel electrophoresis under reducing conditions (12% SDS–PAGE) and concentration was determined by the Bradford method.²⁸

In order to select subjects responding to the rHSP60Kp, PBMC from patients and healthy subjects were obtained using Lymphoprep (Nycomed, Oslo, Norway). Viability was determined by trypan blue (Sigma Chemical Company, St. Louis, MO, USA) exclusion. 10⁵ viable PBMC, resuspended in 200 µL of AIM–V medium (Gibco, Life Technologies. Rockville, MD. USA), were plated in each well of a 96 well round bottom polystyrene microplate (Nunc, Roskilde, Denmark); 2 µg of rHSP60Kp or Concanavalin A (Con A, Sigma Chemical Co., St. Louis, MO, USA) as activation control, were added. Assays were done in triplicate. Plates were incubated for 5 days under 5% CO₂ atmosphere. After incubation, the wells were pulsed with ³H–TdR at 1 pCi/well concentration (Amersham International, Bucks, UK) for the last 7 h. After harvesting onto glass filters, ³H–TdR incorporation was measured by liquid scintillation spectroscopy.¹³ Conditions were selected from a dose response curve previously determined.

Results were expressed as stimulation index (SI). SI > 3.0 was considered positive¹³ (Stimulation Index = cpm of cells with antigen/cpm of cells without antigen).

Determination of the processing pathway for rHSP60Kp

First, in order to validate the experimental design we used varidase (Wyeth, S.A. de C.V., México), an antigen reported to be processed only by the endocytic pathway, in three intradermally positive healthy subjects, that in PBMC had a positive lymphoproliferative response to this antigen.^29–31

To elucidate the routes by which the rHSP60Kp is processed, 10⁶ PBMC from each one of the five high responding AS patients were incubated in a final volume of 500 pL of AIM–V medium with 6 pg of rHSP60Kp and one of the specific inhibitors at the following final concentration selected from a dose response curve: 10 pg/mL brefeldin A (BFA), 10 pg/mL chloroquine (clathrin mediated transport and endosomic inhibitors, respectively) (34) or 1.0 pg/mL of N–acetyl–L–leucil–L–leucil–L–nor–leucinal (LLnL, a proteasome inhibitor)³² all of them purchased from Sigma Chemical Co., St. Louis, MO, USA. PBMC were incubated 5 hours at 37°C in 5% CO₂ then centrifuged to remove the supernatant and fixed with 0.5% p–formaldehyde (Sigma Chemical Co., St. Louis, MO, USA) in PBS for 15 minutes at room temperature (RT). Paraformaldehyde excess was blocked by additional 20 minutes incubation with 0.1 M glycine (Sigma Chemical Co., St. Louis, MO, USA). Cells were washed twice with MEM (Sigma Chemical Co., St. Louis, MO, USA) and resuspended in AIM–V medium to be used as fixed antigen presenting cells (Fixed APC). PBMC incubated with antigen but without inhibitors and PBMC incubated with neither antigen nor inhibitors, but fixed as described above, were used as positive and negative controls, respectively.

To elucidate the processing pathways used by PBMC from AS patients for the rHSP60Kp, lymphoproliferation assays were done, briefly: 10⁵ Fixed APC were added to an equal number of homologous Fresh PBMC, in 96 well round bottom polystyrene microplates and incubated 6 days at 37 °C in 5% CO₂. As a control for toxicity of each one of the Fixed APC, the mixture of fresh PBMC and Fixed APC was stimulated with Con A. Proliferation was determined by ³H–TdR, as described above. Results of Fresh PBMC stimulated by Fixed APC with inhibitors were expressed in stimulation index and compared with the positive control.

CD4/CD8 subpopulations response to rHSP60Kp

Statistical analysis

The non–parametric Fisher exact test was used to compare the lymphoproliferative response to the rHSP60Kp between PBMC from AS patients and from healthy subjects. The Mann–Whitney U test was used to compare the response to Con A between PBMC from AS patients and healthy subjects, as well as, to compare the lymphoproliferative response to Con A between fresh PBMC from AS patients stimulated with Fixed APC and fresh PBMC from AS patients stimulated by Con A. In all cases the Sigma stats v2.03 software was used.

RESULTS

Proliferative response to rHSP60Kp

Eight out of the 14 patients studied had a positive lymphoproliferative response to the rHSP60Kp (S.I. > 3.0) and none of the healthy subjects responded (p = 0.012). In the group of patients five had a S.I. high above 4.0 so that they were chosen for these inhibition studies. All patients and healthy subjects responded to Con A as expected (Table 1).

rHSP60Kp Processing Pathway Determination

In the five selected patients, the lymphoproliferative response of Fresh PBMC stimulated by Fixed APC incubated with rHSP60Kp was above 4.0. The S.I. with chloroquine and LLnL were lower than S.I. without inhibitors (Figures 2A and 2B). When BFA was used, lymphoproliferation became negative (SI < 3.0) (Figure 2C).

No differences between the responses to Con A of Fresh PBMC from AS patients incubated in the presence or absence of different homologous Fixed APC was observed, p = 0.383.

T cell subpopulations activated with the rHSP60Kp

In order to confirm the results suggested by the processing pathway determinations, T cell subpopulations activated with the rHSP60Kp or with Con A as a control, were determined by measuring the CD69 expression by three colours flow cytometry in four of the five patients studied. In figure 3, a representative activation of PBMC from one AS patient expressing CD69 by both CD4 and CD8 T cell subpopulations in the presence of rHSP60Kp, and Con A are shown. Averages of percentage of CD4 and CD8 T cells activated with Con A or with rHSP60Kp in all patients analyzed are graphed in Figure 4a and 4b, respectively.

DISCUSSION

The aim of this work was to determine the processing pathway for the rHSP60Kp used by PBMC from AS patients and then propose the participation of one or both of the T cells subpopulations in the response. First of all, we confirmed our previous results¹³ that PBMC from the majority of HLA–B27 + AS patients' lymphoproliferate in the presence of the recombinant protein in contrast to healthy subjects which do not recognize it (Table 1). It would have been important to analyze HLA B27+ healthy subjects as well as HLA–B27–patients but in the duration of the study (almost two years) it was not possible to get them. It is important to point out that the frequency of B27 in the Mexican population is between 3–5% and that in the group of patients at the Hospital General not more than 8% are B27– (personal communication). Throughout the experiments Con A was used as a control of viability and functionality of the cells. As expected, the mitogenic response, as well as to antigen, was very variable as described by others. That is why, as a consensus, positive results are always reported as S.I. ³ 3.0, independently of the absolute values.^13,35–37

To determine the processing pathways for the rHSP60Kp, we validated our experimental design by using varidase as antigen. PBMC from three intradermally positive healthy subjects were incubated with antigen with or without inhibitors and then fixed to be used as APC for homologous Fresh PBMC. As expected, varidase is processed only by the endocytic pathway.^29,30 As shown in figure 1, lymphoproliferation was positive (S.I. > 3.0) when Fresh PBMC were stimulated by homologous Fixed APC incubated with antigen only, while the response was negative (S.I. < 3.0) when Fresh PBMC were stimulated by homologous Fixed APC incubated with antigen and either chloroquine or BFA. Chloroquine increases the pH within the acidic vacuolar compartments and inhibits the maturation of class II molecules by preventing the hydrolysis of the li chain. BFA inhibits the exocytosis of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus; therefore, it inhibits the endocytic and cytosolic pathways.³² In addition, Fresh PBMC stimulated with homologous Fixed APC incubated with antigen and LLnL did not diminish the lymphoproliferative response (Figure 1). LLnL is an inhibitor of calpain and cathepsins and blocks the proteolytic sites of proteasome, thus decreasing the supply of degraded peptides transported into the endoplasmic reticulum lumen to assembly with nascent MHC class I molecules.^32,38 These results led us to use the same experimental design to analyze the processing pathways for rHSP60Kp in PBMC from AS patients.

Despite the fact that eight out of fourteen AS patients had a positive lymphoproliferative response to the rHSP60Kp, in only five of them the processing pathways used by PBMC to process the rHSP60Kp could be analyzed, because they had a SI above 4.0 when Fixed APC incubated with antigen in the absence of inhibitors were used to stimulate homologous Fresh PBMC. When Fresh PBMC were stimulated by Fixed APC incubated in the presence of antigen and either chloroquine or LLnL, the S.I. were lowered, suggesting that both pathways are used to process the rHSP60Kp (Figure 2a and 2b). It has been reported that heat shock proteins could be presented by cross–presentation to CD8 T cells²⁰ and that in healthy subjects, this cross presentation could be involved in tolerance induction of CD8 T cells, but in the case of patients with autoimmune pathology, it could be involved in the induction of the disease.²⁰ As expected, when Fixed APC were incubated in the presence of antigen and BFA, lympho–proliferation became negative in all patients analyzed (Figure 2c).

One important control in our experimental design was to demonstrate that fixed APC were not toxic for Fresh PBMC. In order to test this, Fresh PBMC were stimulated by Con A in the presence of each one of the Fixed APC obtained by incubation with antigen, with or without inhibitors. Fresh PBMC were able to proliferate in response to Con A in the presence of the different Fixed APC. Therefore, the system was not toxic and negative S.I. could be taken as specific inhibition.

Unfortunately, all healthy subjects analyzed did not show lymphoproliferative response to the rHSP60Kp, however, this fact could support the association of this antigen with the disease.

We found that both CD4 and CD8 T cells are activated by the rHSP60Kp in PBMC from lymphoproliferative responding AS patients, as measured by the expression of CD69^33,34 after exposure to the rHSP60Kp protein (Figure 4). Although the expression of CD69 as an early activation marker does not necessarily correlate with proliferation, and it has been demonstrated that it is less sensitive than thymidine uptake, it has been used by many authors to identify T cell subpopulations involved in mitogen or antigen responses.^37,39 The percentages of CD69 rHSP60Kp positive cells were low but in the range accepted to be considered positive by others.⁴⁰ Responses to Con A were very variable, as expected from the results obtained in the proliferation assays. Our results suggest that both T cell subpopulations are involved in the response to rHSP60Kp which is important considering that in AS pathogenesis CD8 T cells could be involved.^2,15,25

The results obtained by flow cytometry reinforce those obtained in the processing pathways determinations. That is, CD8 T cells recognize antigenic peptides from the rHSP60Kp. The lymphoproliferation observed could be explained by the activation of both CD4 and CD8 T cells. The antibody responses reported before, by our group, could also be explained in terms of the specific activation and cooperation of CD4 cells.^10,11,14

These results can help us understand the role of the HSP60Kp in AS pathogenesis, because of the association of this protein with the disease and with the response by CD4 and CD8 T cells.

J. F. Zambrano–Zaragoza is a fellow of PROMEP from Universidad Autónoma de Nayarit and a graduate student in the CQB program of the ENCB, IPN.

L. Jiménez–Zamudio, M.L. Domínguez–López, M. E. Cancino–Diaz and E. Garcia–Latorre are fellows of COFAA and EDI, IPN.

This work was supported in part by a grant from CGPI, IPN 20010904.

REFERENCES

1. Burgos–Vargas R, Granados–Arrióla J. Ankylosing spondylitis and related diseases in the Mexican Mestizo. Spine–State of the Art Rev 1990; 4: 665–77.        [ Links ]

2. D'Amato M, Fiorillo MT, Carcassi C, Mathieu A, Zuccarelli A, Bitti PP, Tosi R, Sorretino R. Relevance of residue 116 of HLA–B27 in determining susceptibility to ankylosing spondylitis. Eur J Immunol 1995; 25: 3199–201.        [ Links ]

3. Khan MA. Update: the twenty subtypes of HLA–B27. Curr Opin Rheumatol 2000; 12: 235–8.        [ Links ]

4. Saraux A, Guedes C, Allain J, Vails I, Baron D, Youinou P, Le Goff P. HLA–B27 in French patients with rheumatoid arthritis. Scand J Rheumatol 1997; 26: 269–71.        [ Links ]

5. Avakian H, Welsh J, Ebringer A, Entwistle CC. Ankylosing spondylitis, HLA–B27 and Klebsiella pneumoniae II. Cross–reactive studies with human tissue typing sera. Br J Exp Path 1980; 61: 92–6.        [ Links ]

6. Collado A, Gratacos J, Ebringer A, Rashid T, Marti A, Sammarti R, Munoz–Gomez J. Serum IgA anti–Klebsiella pneumoniae in ankylosing spondylitis patients from Catalonia. Scand J Rheumatol 1994; 23: 119–23.        [ Links ]

7. Hermann E, Sukke B, Droste U, Büschenfelde KM. Klebsiella pneumoniae reactive T cells in blood and synovial fluid of patients with ankylosing spondylitis. Arthritis Rheum 1995; 38: 1277–82.        [ Links ]

8. Ebringer RW, Cawdell DR, Cowling P, Ebringer A. Sequential studies in ankylosing spondylitis: association of Klebsiella pneumoniae with active disease. Ann Rheum Dis 1978; 37: 146–51.        [ Links ]

9. Welsh J, Avakian H, Cowling P, Ebringer A, Wooley P, Panayi G, Ebringer R. Ankylosing spondylitis, HLA–B27 and Klebsiella pneumoniae I: cross–reactive studies with rabbit antisera. Br J Exp Path 1980; 61: 85–91.        [ Links ]

10. Parra–Campos V, Escobar–Gutiérrez A, Cancino–Díaz M, Domínguez–López ML, Burgos–Vargas R, Granados–Arreóla J, Garcia–Latorre E. Antibody response to nitrogenase–positive and–negative Klebsiella pneumoniae strains in juvenile–onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase. Rev Latamer Microbiol 1996; 38: 121–7.        [ Links ]

11. Cancino–Díaz ME, Pérez–Salazar JE, Domínguez–López ML. Escobar–Gutiérrez A, Granados–Arreóla J, Jiménez–Zamudio L, Burgos–Vargas R, Garcia–Latorre E. Antibody response to Klebsiella pneumoniae 60 kDa protein in familial and sporadic ankylosing spondylitis: role of HLA–B27 and protein characterization as a Gro–EL HSP60. J Rheumatol 1998; 25: 1756–64.        [ Links ]

12. Cancino–Diaz M, Curiel–Quesada E, García–Latorre E, Jimé–nez–Zamudio L. Cloning and sequencing of the gene that codes for the HSP60 of Klebsiella pneumoniae, related to ankylosing spondylitis. Microb Path 1998; 25: 23–32        [ Links ]

13. Domínguez–López ML, Cancino–Díaz ME, Jiménez–Zamudio L, Granados–Arreóla J, Burgos–Vargas R, García–Latorre E. Cellular immune response to Klebsiella pneumoniae antigens in HLA–B27 positive ankylosing spondylitis patients. J Rheumatol 2000; 27: 1453–60.        [ Links ]

14. Domínguez–López ML, Burgos–Vargas R, Galicia–Serrano H, Bonilla–Sánchez MT, Rangel–Acosta HH, Cancino–Díaz ME, Jiménez–Zamudio L, Granados J, García–Latorre E. IgG antibodies to enterobacteria 60 kDa heat shock proteins in the sera of HLA–B27 positive ankylosing spondylitis patients. Scand J Rheumatol 2002; 31: 260–5.        [ Links ]

15. Fiorillo MT, Maragna M, Butler R, Dupuis ML, Sorrentino R. CD8+ T–cell autoreactivity to an HLA–B27–restricted self–epitope corrrelates with Ankylosing spondylitis. J Clin Invest 2000; 106: 47–53.        [ Links ]

16. Schirmer M, Golberger C, Würzner R, Duftner C, Pfeiffer KP, Clausen J, Naumayr G, Falkenbach A. Circulating cytotoxic CD8+CD28– T cells in ankylosing spondylitis. Arthritis Res 2002; 4: 71–6.        [ Links ]

17.  Lehner PJ, Cresswell P. Processing and delivery of peptides presented by MHC class I molecules. Curr Opin Immunol 1996; 8: 59–67.        [ Links ]

18. Pamer EG. Antigen presentation in the immune response to infectious diseases. Clin Infect Dis 1999; 28: 714–6.        [ Links ]

19. Carbonetti NH, Irish TJ, Chen CH, O'Connell CB, Hadley GA, McNamara U, Tuskan, RG, Lewis GK. Intracellular delivery of a cytolytic T–lymphocyte epitope peptide by pertussis toxin to major histocompatibility complex class I without involvement of the cytosolic class I antigen processing pathway. Infect Immun 1999; 67: 602–7.        [ Links ]

20. Health WR, Carbone FR. Cross–presentation in viral immunity and self tolerance. Nature Rev 2001; 1: 127–34.        [ Links ]

21. Jensen PE, Weber DA, Thayer WP, Westerman LE, Dao CT. Peptide exchange in MHC molecules. Immunol Rev 1999; 172: 229–38.        [ Links ]

22. Reimann J, Kaufmann HE. Alternative antigen processing pathways in antiinfective immunity. Curr Op Immunol 1997; 9: 462–9.        [ Links ]

23. Reimann J, Schirmbeck R. Alternative pathways for processing exogenous antigens that can generate peptides for MHC class I–restricted presentation. Immunol Rev 1999; 172: 131–52.        [ Links ]

24. Wick MJ, Ljunggren HG. Processing of bacterial antigens for peptide presentation on MHC class I molecules. Immunol Rev 1999; 172: 153–62.        [ Links ]

25. Zhou X, Franksson LL, Lederer E, Ljunggren HG, Jondal M. Characterization of TAP–independent and brefeldin A–resistant presentation of sendai virus antigen to CD8+ cytotoxic T lymphocytes. Scand J Immunol 1995; 42: 66–75.        [ Links ]

26. Kovacsovics–Bankowski M, Rock KL. A phagosome–to–cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 1995; 267: 243–6.        [ Links ]

27. Bennet PH, Burch TA. New York Symposium on population studies in the rheumatic diseases: new diagnostic criteria. Bull Rheum Dis 1967; 17: 453–8.        [ Links ]

28. Bradford M. A rapid and sensitive method for the quantitation of micrograms quantities of proteins utilizing the principle of protein dye binding. Anal Biochem 1976; 72: 248.        [ Links ]

29. Braine HG, Elfenbeing GJ, Mellits ED. Peripheral blood lymphocyte numbers, lymphocyte proliferative responses in vitro, and serum immunoglobulins in regular hemapheresis donors. J Clin Apheresis 1985; 2: 213–8.        [ Links ]

30. Donalson SL, Ranney RR, Tew JG. Evidence of mitogenic activity in periodontitis–associated bacteria. Infect Immun 1983; 42: 487–95.        [ Links ]

31. Feurle GE, Dörken B, Schopf E, Lenhard V. HLA–B27 and defects in the T cell system in Whipple's disease. Eur J Clin Invest 1979; 9: 385–9.        [ Links ]

32. Men Y, Audran R, Thomasin C, Eberl G, Demotz S, Merkle HP, Gander B Corradin G. MHC class I– and class II–restricted processing and presentation of microencapsulated antigens. Vaccine 1999; 17: 1047–56.        [ Links ]

33. Afeltra A, Galeazzi M, Ferri G, Amoroso A, De Pita O, Porzio F, Bonomo L. Expression of CD69 antigen on synovial fluid T cells in patients with rheumatoid arthritis and other chronic synovitis. Ann Rheum Dis 1993; 52: 457–60.        [ Links ]

34. Hernández–García C, Fernández–Gutiérrez B, Morado C, Bañares A, Jover J. The CD69 activation pathway in rheumatoid arthritis synovial fluid T cells. Arthritis Rheum 1996; 39: 1277–86.        [ Links ]

35. Stone MA, Payne U, Schentag C, Rahman P, Pacheco–Tena C, Inman RD. Comparative immune responses to candidate arthritogenic bacteria do not confirm a dominant role for Klebsiella pneumoniae in the pathogenesis of familial ankylosing spondylitis. Rheumatol 2004; 43: 148–55.        [ Links ]

36. Parga Lozano C, Marrugo Cano J, Hernández Bonfante L. Respuesta proliferativa de células mononucleares de sangre periférica frente al alérgeno recombinante BtM del ácaro del polvo casero Blomia tropicalis. Allergol et Immunopathol 2004; 32: 247–51.        [ Links ]

37. Hutchinson P, Divola LA, Holdsworth R. Mitogen–induced T–cell CD69 expression is a less sensitive measure of T cell function than ( ³H)–thymidine uptake. Cytometry 1999; 38: 244–9.        [ Links ]

38. Lich JD, Elliot JF, Blum JS. Cytoplasmic processing is a prerequisite for presentation of endogenous antigen by major histocompatibility complex class II proteins. J Exp Med 2000; 191: 1513–23.        [ Links ]

39. Rutella S, Rumi C, Lucia MB, Barberi T, Puggioni PL, Lai M, Romano A, Cauda R, Leone G. Induction of CD69 antigen on normal CD4+ and CD8+ lymphocyte subsets and its relationship with phenotype of responding T cells. Cytometry 1999; 38: 95–101.        [ Links ]

40. Augustin B, Kotnik V, Skoberne M, Malovrh T, Skralovnick–Stern A, Tercelj M. CD 69 expression on CD4+ T lymphocyte after in vitro stimulation with tuberculin is an indicator of immune sensitization against Mycobacterium tuberculosis antigens. Clin Diagn Lab Immunol 2005; 12: 101–6.        [ Links ] ]]> 1 1990 4 665-77 2 1995 25 3199-201 3 2000 12 235-8 4 1997 26 269-71 5 1980 61 92-6 6 1994 23 119-23 7 1995 38 1277-82 8 1978 37 146-51 9 1980 61 85-91 10 1996 38 121-7 11 12 1998 25 23-32 13 2000 27 1453-60 14 2002 31 260-5 15 2000 106 47-53 16 2002 4 71-6 17 1996 8 59-67 18 1999 28 714-6 19 1999 67 602-7 20 2001 1 127-34 21 1999 172 229-38 22 1997 9 462-9 23 1999 172 131-52 24 1999 172 153-62 25 1995 42 66-75 26 1995 267 243-6 27 1967 17 453-8 28 1976 72 248 29 1985 2 213-8 30 1983 42 487-95 31 1979 9 385-9 32 1999 17 1047-56 33 1993 52 457-60 34 1996 39 1277-86 35 2004 43 148-55 36 2004 32 247-51 37 1999 38 244-9 38 2000 191 1513-23 39 1999 38 95-101 40 2005 12 101-6